Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)

Title of invention:
A METHOD FOR SCREENING HPV USING A SYSTEM AND KIT THEREOF

APPLICANT:
MYLAB DISCOVERY SOLUTIONS PRIVATE LIMITED
An Indian Entity having address as:
PLOT NO 99-B, LONAVALA INDUSTRIAL CO-OPERATIVE ESTATE LTD, NANGARGAON, LONAVALA, PUNE – 410401 MAHARASHTRA, INDIA

The following specification describes the invention and the manner in which it is to be performed.
CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY
The present application does not claim priority from any other patent application(s).
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 4, 442 Byte ASCII (Text) file named "HPV Sequence listing.txt," created on September 29, 2022.
TECHNICAL FIELD
The present disclosure relates to the field of Nucleic Acid Testing (NAT)/Biotechnology. The subject matter, in general relates to a method for self-sampling, simultaneously detecting one or more of the HPV subtypes from a test sample and indicating HPV screening results to user using a tracking system, as well as a kit thereof.
BACKGROUND
Human papilloma virus (HPV) is a small, non-enveloped, icosahedral DNA virus belonging to Papillomaviridae family that causes viral infection in humans through skin-to-skin contact, vaginal, anal, or oral sex. Currently, there are over 100 subtypes of HPV, more than 40 of which are transmitted through sexual contact as reported by national Institute of Health (NIH). These subtypes are drawn to the body's mucous membranes, such as the moist layers around the anus and genitals, wherein, they infect the thin, flat squamous cells that line the inner surface of these organs. However, not all the 40 sexually transmitted human papillomaviruses subtypes cause serious health problems.
Studies reveal that most women and men develop HPV infection at least once during their lifetime, even though they may not have any signs or symptoms of infection. HPV infection usually clears on its own with the body’s immune system response. However, in some women, especially those older than 30 years of age, HPV infection may persist over time, and may lead to changes in the cervix that could be cancerous.
While low risk, or “non-oncogenic” subtypes of HPV rarely cause precancerous lesions, they may still cause cellular changes. If certain low risk subtypes of HPV inhabit the body, they may cause genital warts, which are benign growths that can develop around the genitals, groin, and anus. Some of the examples of low-risk HPV subtypes are HPV6, HPV11, and HPV42.
However, persistent infections with high risk, or oncogenic subtypes of HPV may cause cervical cancer. High-risk HPV subtypes include HPV 31, 33, 45, 52, 58, and a few others of which subtypes 16 and 18 alone are responsible for about 70% of cervical cancers.
Therefore, cervical cancer screening is an essential part of a woman’s routine health care. The primary goal of screening is to identify precancerous lesions to prevent it from developing into aggressive invasive cancers. Routine cervical screenings have been shown to greatly reduce both, number of cervical cancer cases and deaths from the disease. For many years, cytology-based screening, known as the Pap test or Pap smear, was the only method of screening. Pre-cancerous lesions in the cervix are the first changes in the cells, which can be detected with a pap test and treated effectively. Visual inspection with acetic acid to detect precancerous cervical lesions was another method for cervical cancer inspection. However, due to lack of symptoms during early infection stage, these methods are not substantially effective to identify HPV invasion in the body.
To address this, nucleic acid testing (NAT), a highly sensitive and advanced technique that can detect viral infections even during their window periods (WP) was applied to HPV testing wherein, the viral nucleic acid sequences are targeted allowing earlier detection of the infection and decreasing the dissemination of pathogen within the community.
Currently, state of the art methods detects high risk HPV subtypes as well as low risk HPV subtypes from samples. However, these tests do not efficiently discriminate HPV 16 and HPV18, from other high-risk subtypes. Therefore, detection methods, are required to be in followed by a separate discrimination method to identify whether a patient is infected with HPV16 and HPV18, and furthermore, which of the HPV16 or HPV18, being the most common cause for almost 70% of all cervical cancers. Furthermore, although in some kits, HPV16 and HPV18 are discriminated from other high risk HPV subtypes, these kits conventionally need numerous reaction tubes and detection compositions, that reduce the feasibility of their simultaneous detection and discrimination.
Further, at the outset, many individuals are deterred from regular testing at a medical facility due to unavailability of such facility, financial burdens, time constraints, cultural issues, or personal modesty. As a result, many develop fatal cervical cancer that could otherwise be avoided with proper testing. Therefore, there is a need for a cervico-vaginal sample collection kit that is safe, simple, and as inexpensive as possible.
Despite the benefits associated with timely and accurate determination of a person's physiological status, in many instances, people do not seek to obtain a diagnosis of their status as to one or more health-related conditions. Such a failure to seek a diagnosis may be based on a fear that their confidentiality as to the health-related condition will not be fully preserved by the testing facility or a related party. Moreover, rapid HIV self-testing is high, but potential users remain concerned about correct use, interpretation of test results, and linkage to care.
In order to assist user in performing a medical self-test, there is a requirement to devise a system in the form of “apps” which may be executed on a user device such as a smart phone, tablet or a general-purpose computer, that can potentially help the user to channelize their diagnosis in the right direction.
Moreover, most commercially available kits for real-time PCR based detection of HPV involve liquid reagents that either need a cold supply chain of sub-zero temperatures such as -20°C for transport and storage or may be thermolabile and susceptible to loss of efficacy leading to plausibly false test outcomes.
Further, automation of the entire process from extraction to detection or even to reporting of results may reduce the manual errors and shorten the time required for end-to-end processing of the samples.
HPV vaccination plays a pivotal role in preventing more than 90% of HPV-attributable cancers. It is reported that since HPV vaccination was first recommended in 2006, infections with HPV types that cause most HPV cancers and genital warts have dropped 88% among teen girls and 81% among young adult women. Further, vaccination has also reduced the number of cases of precancers of the cervix in young women. Currently, three HPV vaccines—9-valent HPV vaccine (Gardasil 9, 9vHPV), quadrivalent HPV vaccine (Gardasil, 4vHPV), and bivalent HPV vaccine (Cervarix, 2vHPV)—have been licensed by the U.S. Food and Drug Administration (FDA). All three HPV vaccines protect against HPV types 16 and 18 that cause most HPV cancers. However, to be eligible to for the same, HPV screening is a pre-requisite. Therefore, in addition to addressing the above discussed problems, the inventors of the instant disclosure are motivated to carry hustle-free screening of one or more of HPV subtypes from a user’s test sample to encourage vaccination within the community.
SUMMARY
The present disclosure is directed to an in-vitro method for screening one or more HPV subtypes from a user’s test sample; characterized in that, said method comprises: collecting the test sample using a self-sampling device; transporting the collected test sample using a transport medium; carrying out simultaneous detection of one or more HPV subtypes from the collected test sample using real time PCR; and using a tracking system for indicating/communicating HPV screening results to the user.
In an exemplary embodiment, the instant disclosure relates to an in-vitro kit to screen one or more HPV subtypes from a user’s test sample; characterized in that, said kit comprises: self-sampling device for collecting the test sample; transport medium for transporting the collected test sample; real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample; and tracking system for communicating HPV screening results to the user.
This summary is not intended to identify all the essential features of the claimed subject matter, nor is it intended to use in determining or limiting the scope of the claimed subject matter.
BRIEF DESCRIPTION OF DRAWINGS
The detailed description of drawings is outlined with reference to the accompanying figures. In the figures, the left-most digit (s) of a reference number identifies the Figure in which the reference number first appears. The same numbers are used throughout the drawings to refer like features and components.
Fig. 100 demonstrates the flow chart of the tracking system aided sample collection and HPV detection
101 indicates Collection:
• User to undertake self-sampling using the self-sampling device
• Collected sample to be transported to Centre (if collected remotely (at home) or deposit it to center) via transport medium
102 indicates Detection:
• Centre to conduct Real Time PCR to detect one or more HPV subtypes from the collected test sample
• Categorize report in desired formats: (a) Presence or absence of HPV If present, (b) high or (a) low risk (detection and discrimination),
103 indicates Communication:
• Report is shared with the users via a tracking system which is linked with the user's details at the time of sample collection
DETAILED DESCRIPTION
Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” “alternate embodiment”, or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment”, “in an alternate embodiment”, or “in a related embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
Before the present methods, compositions, and kits are described, it is to be understood that this disclosure is not limited to the particular methods, compositions, and kits as described, as there can be multiple possible embodiments which are not expressly illustrated in the present disclosure but may still be practicable within the scope of the present disclosure.
Reference throughout specification to the term “screening” refers to detection and isolation of pathogen/pathogens or as-yet-unrecognised conditions from test samples.
Further, reference throughout the specification to “composition”, or “compositions” refers to a specific formulation or dosage form of an active/functional element or components either alone or with essential compatible components and carriers for the intended use or a product comprising the specified ingredients in the specified amounts or any product which results, directly or indirectly, from combination of the specified ingredients in the specified amount.
Additionally, reference throughout the specification to “multiplex assay” refers to an assay for simultaneous analysis of multiple analytes in a single experiment. Further, for the purpose of the present disclosure, the term “buffer” means a composition of reagents. Additionally, the term “pathogen”, or “pathogens” refer to organisms causing infection, lesions or abnormal cellular architecture of the uterine cervix including, but not limited to, viruses, bacteria, yeast, and fungi.
For the purpose of the instant disclosure, “HPV”, also referred to as “Human papillomavirus” is the most common sexually transmitted virus, as is conventionally known to a skilled person in the art. Further, for the purpose of the instant disclosure, the term “subtype” refers to a sub-classification of the same pathogen depending on its serotype, biotype, pathotype, or genotype, as is conventionally known to a skilled person in the art. It is also known to a skilled person in the art that there are more than 100 subtypes of HPV; out of which, 14 high-risk HPV subtypes and 3 low-risk HPV subtypes are extensively studied for their association to cervical cancer and common warts in humans.
The technical solutions offered by the present disclosure are clearly and completely described below. Examples in which specific reagents or conditions may not have been specified, have been conducted under conventional conditions or in a manner recommended by the manufacturer.
The present disclosure relates to an in-vitro method for screening one or more HPV subtypes from a user’s test sample; characterized in that, said method comprises: collecting the test sample using a self-sampling device; transporting the collected test sample using a transport medium; carrying out simultaneous detection of one or more HPV subtypes from the collected test sample using real time PCR; and using a tracking system for indicating/communicating HPV screening results to the user.
The present disclosure further relates to an in-vitro kit to screen one or more HPV subtypes from a user’s test sample; characterized in that, said kit comprises: self-sampling device for collecting the test sample; medium for transporting the collected test sample; real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample; and tracking system for communicating HPV screening results to the user.
The first aspect of the present disclosure is related to an in-vitro method for screening one or more HPV subtypes from a user’s test sample; characterized in that, said method comprises: collecting the test sample using a self-sampling device; transporting the collected test sample using a transport medium; carrying out simultaneous detection of one or more HPV subtypes from the collected test sample using real time PCR; and using a tracking system for indicating/communicating HPV screening results to the user.
One embodiment of the present disclosure is related to an in-vitro method for screening one or more HPV subtypes from a user’s test sample.
For the purpose of the instant disclosure, the term “screening” refers to qualitative testing or detecting the presence of an analyte in the test sample.
For the purpose of the instant disclosure, the HPV subtypes are at least one of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV6, HPV11, and HPV42.
Further, for the purpose of the instant disclosure, the term “user’ refers to any person carrying out any embodiment of the instant method and/or kit.
Further, for the purpose of the instant disclosure, the term “users” refers user, healthcare professional, doctors. any official or employee of a government agency or other organisation (whether in the public or private sector) that may prescribe, purchase, supply or administer medicinal products and any employee of a member whose primary occupation is that of a practising healthcare professional.
In a related embodiment, test sample is at least one of genital tract specimen suspected of carrying HPV DNA, such as, but not limited to, urinal, cervical, and vaginal samples.
Another embodiment of the present disclosure is related to collecting the test sample using a self-sampling device.
For the purpose of the instant disclosure, a self-sampling device used for collecting the test sample includes, but is not limited to swab, speculum, spatula, brush, lavage-based device, etc. as is known to a skilled person in the art.
In a related embodiment, the self-sampling device is contained within sterilised container.
Further embodiment of the present disclosure is related to transporting the collected test sample using a transport medium.
For the purpose of the instant disclosure, transport medium includes, but is not limited to, any solid, liquid, semi-solid medium conventionally known to a skilled person in the art.
In a related embodiment, the transport medium is contained within a sterilized container.
In yet another related embodiment, the transport medium is portable.
In a further embodiment, the transport medium is transported as a conventionally known transportable package.
In yet another embodiment, the transportable medium is transported using means that are conventionally known.
Another embodiment of the instant disclosure relates to carrying out simultaneous detection of one or more HPV subtypes from the collected test sample using real time PCR.
In an exemplary embodiment, the present disclosure further discloses a method as described in the foregoing paragraphs, wherein the simultaneous detection of one or more HPV subtypes from the collected test sample is carried out by: extracting HPV DNA from the collected test sample using extraction compositions; and contacting the extracted HPV DNA with one or more detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in a non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes using real time PCR.
For the purpose of the instant disclosure, it is clarified that in the present disclosure, target sequences pertain to the specific regions, wholly or partly within HPV DNA. For the purpose of the current disclosure, the said primer and probe sequences are specifically engineered to hybridise with the target sequences; wherein, the said target sequences may be amplified, detected, or otherwise analysed.
One embodiment of the present disclosure relates to the simultaneous detection of one or more HPV subtypes from the collected test sample.
For the purpose of the instant disclosure, ‘collected test sample” refers to the test sample that is collected using a self-sampling device as is disclosed in the above paragraphs.
The present disclosure may provide flexibility in processing a sample through various volume sizes. Sample size plays an integral role in determining the outcome of extraction method. A large sample size has advantages of providing better and accurate data by reducing sensitivity problems. On the other hand, smaller sample size prevents financial, transportation, storge, and time commitments. In a preferred embodiment, the collected test sample volume is ? 500 µL.
Another embodiment of the present disclosure relates to extracting HPV DNA from the collected test sample using extraction compositions.
In a related embodiment, the extraction of HPV DNA is carried out using one of conventionally known column-based, paramagnetic particles-based extraction or POC (Point of care) extraction.
In one embodiment, the extraction of HPV DNA is carried out using paramagnetic particles-based extraction; wherein the paramagnetic particle-based extraction is carried out using: lysis composition; lysis enhancer composition; one or more wash compositions; and elution compositions; preferably, in a single container.
In order to limit the risk of cross-contamination, truncate the overall turnaround time, limit multiple pipetting, reduce material wastage, and requirement of equipment and space associated with the extraction of nucleic acids, in a preferred embodiment, lysis, wash, and elution steps are carried out in a single container. A “container” is any vessel capable of holding the components or compositions of the method for carrying out the present disclosure. The advantage of using a single container lies with its versatility since it can carry out a sequence of processes without the need to break the containment. This is particularly useful when processing toxic, infectious, or highly potent samples like HPV.
In an embodiment, the lysis composition comprises lysis buffer and lysis enhancer buffer.
In a related embodiment, the lysis buffer may be selected from a group of Tris, Tris hydrochloride, Tris chloride, phosphate, carbonate, citrate and others known to a skilled person in the art. In a preferred embodiment, the lysis buffer is Tris hydrochloride.
In a further embodiment, the lysis buffer may further comprise detergent selected from sodium dodecyl sulphate, Triton-X, and Tween 20; preferably sodium dodecyl sulphate.
Additionally, the lysis buffer may comprise chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, and cyanoguanidine isosulphate. In a preferred embodiment, the lysis composition comprises guanidine thiocyanate at a concentration ranging from 2M to 6M.
In an embodiment, the lysis buffer may further comprise sodium salt selected from sodium citrate, sodium chloride, sodium acetate or combinations thereof that are known to a skilled person in the art.
In another embodiment, the lysis buffer may also comprise additives such as stabilizing/reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), glycerol and /or chelating agent such as ethylenediaminetetraacetic acid (EDTA), anti-microbials such as sodium azide, or combinations thereof that are known to a skilled person in the art.
In an embodiment, the lysis enhancer buffer comprises lysis enzymes selected from proteinase K, RNase, DNase. Additionally, the lysis enzyme may be stabilised using additives such as alkaline salts such as sodium, potassium, calcium and/or long-chain alcohols such as glycerol, sorbitol, xylitol. In a preferred embodiment, the lysis enhancer buffer comprises proteinase K as a liquid stabilized with calcium chloride and glycerol.
In another embodiment, the lysis composition may be used along with alcohol such as, but not limiting to, ethanol or isopropanol; preferably isopropanol.
In an embodiment, sample lysis is carried out at a temperature ranging from 20°C to 70°C, preferably at a temperature ranging from 50°C to 60°C for up to 15 minutes at a pH ranging from 7.0 to 14, preferably at a pH ranging from 7.0 to 10.
Extraction of HPV DNA may be carried out by employing silica gel solid supports such as membranes or columns; or paramagnetic particles as is known to a skilled person in the art. Use of paramagnetic particles eliminates the centrifugation steps normally employed by conventional methods. When magnetic field is applied to the sides of the vessel with sample, the paramagnetic particles aggregate without being denatured or damaged. In addition, extraction methods using paramagnetic particles have become a popular approach due to their high potential for automation. So, in a preferred embodiment, the extraction of HPV DNA is carried out using paramagnetic particles due to its above-mentioned advantages as well as its potential for automation and semi-automation.
Paramagnetic particles encompassing all its forms such as magnetic beads, paramagnetic nanoparticles, microparticles are constituted of one or several magnetic ores such as magnetite (Fe3O4) or maghemite (gamma Fe2O3), and may be coated with a matrix of polymers, silica, or hydroxyapatite with terminal functionality as is known to a skilled person in the art.
In a preferred embodiment, the lysis composition may additionally apply paramagnetic particles selected from magnetic beads, paramagnetic nanoparticles, microparticles that may be uncoated or coated with polymers, silica, or hydroxyapatite with terminal functionality.
In addition to sample lysis, in a preferred embodiment of the present invention, the method comprises additional steps of washing and/or eluting.
In an embodiment, the lysed sample is washed using a wash composition comprising alcohol such as, but is not limited to, ethanol and isopropanol. Alternatively, lysed sample is washed using a wash composition comprising at least two washes of alcohol such as, but is not limited to, ethanol and isopropanol. In a preferred embodiment, ethanol is used for washing the lysed sample; preferably 60% to 80% ethanol is used for washing the lysed sample.
In another embodiment, washed lysed sample is eluted using an elution composition to obtain the target sequences that can be directly applied for further processing or analyses.
In an embodiment, elution step involves elution composition comprising acidic solution selected from water having pH less than 7 such as nuclease free water, de-ionized water, DEPC autoclaved water, and acidic buffer, and more preferably nuclease-free water or DEPC treated water.
Various modifications to the embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments.
In an alternate embodiment, the extraction of HPV DNA is carried out using POC (Point of care) extraction; wherein, the POC extraction is carried out using POC extraction composition.
In a related embodiment, the said POC extraction composition comprises lysis composition.
In yet another related embodiment, the lysis composition comprises, but is not limited to, Tris, Triton, sucrose, and magnesium salt; particularly, tris hydrochloride, triton 100, sucrose, and magnesium chloride.
In a further embodiment, the POC extraction composition comprises proteinase K.
In an embodiment, extraction of HPV DNA by applying POC extraction composition is carried out by performing sample lysis and proteinase K inactivation.
In a preferred embodiment, POC extraction is carried out using POC extraction composition comprising but is not limited to Tris hydrochloride, Triton 100, Sucrose, magnesium chloride, and proteinase K. In a related embodiment, the POC extraction composition may be in a liquid form.
Alternatively, the POC extraction composition may be in a lyophilized form.
In yet another related embodiment, the POC (Point of care) extraction is carried out in less than 16 minutes.
In an exemplary embodiment, the extraction of HPV DNA is carried out using POC (Point of care) extraction composition; said composition comprising: tris hydrochloride, magnesium chloride, sucrose, Triton 100, and proteinase K; characterized in that, the POC extraction is completed in less than 16 minutes.
The present disclosure further relates to contacting the extracted HPV DNA with one or more detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in a non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes. The said primer sequences refer to nucleic acid sequences capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, and the said probe sequences refer to nucleic acids of unique sequence capable of hybridizing to a correctly amplified fragment. The term “hybridization” or “hybridize” used herein indicates refers the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing.
In an embodiment, the said detection compositions are lyophilized, as a whole or in part.
Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. A rehydration buffer may be applied in order to rehydrate the constitution and resume their application in the process. As known to a skilled person, lyophilization makes room temperature storage feasible with desirable stability, and reduces challenges to transport of the materials, thus maximising applications of the kit and ensuring wider reach. In a preferred embodiment, lyophilized method and kit components exhibit increased shelf life, preferably more than 24 months.
In an embodiment, the instant method may further comprise a sample collection media selected from, but not limited to, Tris Buffer (pH 7- 8), Phosphate Buffer Saline (pH 7.0-8.0), HEPES, magnesium and calcium salts (<10%), Bovine serum albumin, protease inhibitor (<1%) and Stabilizers, with or without alcohol; preferably, Phosphate buffer saline with BSA or TE buffer with salts.
Ethanol or methanol can be a part of sample collection based on their utility in the cytology.
In a related embodiment, one or more detection compositions comprise Detection compositions A-R.
In yet another related embodiment, one or more of the detection compositions comprises primer and probe sequences selected from SEQ ID NO: 1 to SEQ ID NO: 58.
In an embodiment, Detection composition A comprises primer and probe sequences for HPV16, HPV35, HPV31, HPV33, HPV58, and HPV52.
In a related embodiment, Detection composition A comprises SEQ ID NO: 1, 5, and 17.
In another embodiment, Detection composition B comprises primer and probe sequences for HPV 18, HPV 45, and HPV51.
In a related embodiment, Detection composition B comprises SEQ ID NO: 2, 6, and 18.
In yet another embodiment, Detection composition C comprises primer and probe sequences for HPV 68, HPV39, and HPV59.
In a related embodiment, Detection composition C comprises SEQ ID NO: 3, 7, and 19.
In one embodiment, Detection Compositions D and E comprises primer and probe sequences for HPV18.
In a related embodiment, Detection composition D comprises SEQ ID NO: 4, 8, and 20.
In yet another related embodiment, Detection composition E comprises SEQ ID NO: 9, 10, and 21.
In another embodiment, Detection compositions F and G comprise primer and probe sequences for HPV16.
In a related embodiment, Detection composition F comprises SEQ ID NO: 13, 14, and 22.
In yet another related embodiment, Detection composition G comprises SEQ ID NO: 15, 16, and 23.
In yet another embodiment, Detection composition H comprises primer and probe sequences for HPV52 and HPV53.
In a related embodiment, Detection composition H comprises SEQ ID NO: 26, 27, and 28.
In an embodiment, Detection composition I comprises primer and probe sequences for HPV16, HPV31, and HPV35.
In a related embodiment, Detection composition I comprises SEQ ID NO: 29, 30, and 31.
In another embodiment, Detection composition J comprises primer and probe sequences for HPV51, and HPV56.
In a related embodiment, Detection composition J comprises SEQ ID NO: 32, 33, and 34.
In yet another embodiment, Detection composition K comprises primer and probe sequences for HPV33, and HPV58.
In a related embodiment, Detection composition K comprises SEQ ID NO: 35, 37, and 37.
In a further embodiment, Detection composition L comprises primer and probe sequences for HPV6.
In a related embodiment, Detection composition L comprises SEQ ID NO: 38, 39, and 40.
In an embodiment, Detection composition M comprises primer and probe sequences for HPV11.
In a related embodiment, Detection composition M comprises SEQ ID NO: 41, 42, and 43.
In another embodiment, Detection composition N comprises primer and probe sequences for HPV42.
In a related embodiment, Detection composition N comprises SEQ ID NO: 44, 45, and 46.
In an embodiment, Detection composition O comprises primer and probe sequences for HPV 52.
In a related embodiment, Detection composition O comprises SEQ ID NO: 47, 48, and 49.
In another embodiment, Detection composition P comprises primer and probe sequences for HPV 58 and 33.
In a related embodiment, Detection composition P comprises SEQ ID NO: 50, 51, and 52.
In yet another embodiment, Detection composition Q comprises primer and probe sequences for HPV 31.
In a related embodiment, Detection composition Q comprises SEQ ID NO: 53, 54, and 55.
In further embodiment, Detection composition R comprises primer and probe sequences for HPV 45.
In a related embodiment, Detection composition R comprises SEQ ID NO: 56, 57, and 58.
In a preferred embodiment, one or more Detection compositions comprise:
Detection composition A comprising primer and probe sequences for HPV16, HPV35, HPV31, HPV33, HPV58, and HPV52;
Detection composition B comprising primer and probe sequences for HPV 18, HPV 45, and HPV51;
Detection composition C comprising primer and probe sequences for HPV 68, HPV39, and HPV59;
Detection composition D and E comprising primer and probe sequences for HPV18;
Detection composition F and G comprising primer and probe sequences for HPV16;
Detection composition H comprising primer and probe sequences for HPV52, and HPV53;
Detection composition I comprising primer and probe sequences for HPV 16, HPV 31, and HPV 35;
Detection composition J comprising primer and probe sequences for HPV 51, and HPV 56;
Detection composition K comprising primer and probe sequences for HPV 33, and HPV 58;
Detection composition L comprising primer and probe sequences for HPV 6;
Detection composition M comprising primer and probe sequences for HPV 11;
Detection composition N comprising primer and probe sequences for HPV 42;
Detection composition O comprising primer and probe sequences for HPV 52;
Detection composition P comprising primer and probe sequences for HPV 58 and 33;
Detection composition Q comprising primer and probe sequences for HPV 31;
Detection composition R comprising primer and probe sequences for HPV 45;
wherein, said primer and probe sequences are specific for one or more HPV subtypes; such that, said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously detect one or more HPV subtypes using real time PCR.
In order to increase testing feasibility, reduce material wastage, and requirement of equipment and space associated with PCR screenings, in a preferred embodiment, the detection compositions enable simultaneous detection of one or more HPV subtypes using up to two reaction tubes. For the purpose of the instant disclosure, a “reaction tube” is any container capable of holding the components or compositions for carrying out PCR screening; preferably, real time PCR screening.
In an embodiment, one or more detection compositions enable simultaneous detection of high-risk HPV subtypes in a single reaction tube; preferably, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68; and particularly, further enable simultaneous discrimination of HPV16 and HPV18 from the other high-risk HPV subtypes in the same reaction tube.
In another embodiment, one or more detection compositions enable simultaneous detection and discrimination of low-risk HPV subtypes in another reaction tube; particularly, of HPV6, HPV11, and HPV42.
In a further embodiment, one or more detection compositions enable simultaneous detection of 7 high-risk HPV subtypes in a single reaction tube; preferably, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58; and particularly, further enable simultaneous discrimination of HPV16 and HPV18 from the other high-risk HPV subtypes in the same reaction tube.
In yet another further embodiment, one or more detection compositions enable simultaneous detection of 9 HPV subtypes in a single reaction tube; preferably, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV6, and HPV11.
In a preferred embodiment, one or more detection compositions enable simultaneous detection and discrimination of low-risk HPV subtypes in one reaction tube; particularly, HPV6, HPV11, and HPV42, and one or more detection compositions enable simultaneous detection of high-risk HPV subtypes in other reaction tube; preferably, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68; and particularly, further discriminates HPV16 and HPV18 from HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68.
In one embodiment, a single reaction tube comprises one or more Detection compositions selected from A-K; said Detection compositions comprising:
forward primer sequences selected from SEQ 1D NO: 1, 2, 3, 4, 9, 11, 12, 13, 15, 26, 29, 32, and 35;
reverse primer sequences selected from SEQ ID NO: 5, 6, 7, 8, 10, 14, 16, 27, 30, 33, and 36; and
dual-labelled probe sequences selected from SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 31, 34, and 37;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify and detect at least one of the high-risk HPV subtypes from HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68.
In another embodiment, the Detection compositions described in the foregoing paragraphs further comprise:
forward primer sequences selected from SEQ ID NO: 1, 2, 4, 9, 13, 15, and 29;
reverse primer sequences selected from SEQ ID NO: 5, 6, 8, 10, 14, 16, and 30; and
dual-labelled probe sequences selected from SEQ ID NO: 17, 18, 20, 21, 22, 23, and 31;
to enable simultaneous discrimination of HPV16 and HPV18 in the same reaction tube.
In a further embodiment, a single reaction tube comprises one or more Detection compositions selected from L-N; said Detection compositions comprising:
• forward primer sequences selected from SEQ ID NO: 38, 41, and 44;
• reverse primer sequences selected from SEQ ID NO: 39, 42, and 45; and
• dual-labelled probe sequences selected from SEQ ID NO: 40, 43, and 46;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify, detect, and discriminate at least one of the low-risk HPV subtypes from HPV 6, HPV11, and HPV42.
In a related embodiment, a single reaction tube comprises one or more Detection compositions selected from D-G, and O-R; said Detection compositions comprising:
forward primer sequences selected from SEQ 1D NO: 4, 9, 13, 15, 47, 50, 53, and 56;
reverse primer sequences selected from SEQ ID NO: 8, 10, 14, 16, 48, 51, 54, and 57; and
dual-labelled probe sequences selected from SEQ ID NO: 20, 21, 22, 23, 49, 52, 55, and 58;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify and detect at least one of the 7 high-risk HPV subtypes from HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58.
In a preferred embodiment, the Detection compositions described in the foregoing paragraphs further comprise:
forward primer sequences selected from SEQ ID NO: 4, 9, 13, and 15;
reverse primer sequences selected from SEQ ID NO: 8, 10, 14, and 16; and
dual-labelled probe sequences selected from SEQ ID NO: 20, 21, 22, and 23;
to enable simultaneous discrimination of HPV16 and HPV18 in the same reaction tube.
In a further embodiment, a single reaction tube comprises one or more Detection compositions selected from D-G, L, M, and O-R; said Detection compositions comprising:
forward primer sequences selected from SEQ 1D NO: 4, 9, 13, 15, 38, 41, 47, 50, 53, and 56;
reverse primer sequences selected from SEQ ID NO: 8, 10, 14, 16, 39, 42, 48, 51, 54, and 57; and
dual-labelled probe sequences selected from SEQ ID NO: 20, 21, 22, 23, 40, 43, 49, 52, 55, and 58;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify and detect at least one of the 9 HPV subtypes from HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV6, HPV11, and HPV58.
In yet another preferred embodiment, the primer and probe sequences hybridize with target sequences in HPV DNA without cross-reacting or overlapping with each other or with any other nucleic acids present in the test sample. The non-cross reactive and non-overlapping nature helps in specifically amplifying the said specific target sequences, without interfering or reacting even with genomic DNA thus ensuring that only the pathogens to be screened are amplified, and eventually detected and discriminated. Furthermore, due to the non-cross reactive and non-overlapping nature of the primer and probe sequences comprised in the detection composition, multiple/one or more detection compositions can be applied together in up to two reaction tubes to carry out simultaneous detection of 15 high-risk HPV subtypes, and 3 low-risk HPV subtypes; while further discriminating HPV16, HPV18, and all the low-risk subtypes in the same PCR run.
Additionally, in some embodiments of the present disclosure, any one or combinations of primer/probe sequences described herein may contain chemically modified bases. Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments.
In an embodiment, the probe sequences described herein may be, but are not limited to, hydrolysis probe sequences or dual hybridization probe sequences. More preferably, the probe sequences in the present disclosure are hydrolysis probe sequences. In some embodiments of the present disclosure, the probe sequences are dual-labelled probe sequences; preferably dual labelled with at least one of the fluorescent reporter groups at their 5 ‘ends and a fluorescence quencher at their 3’ ends.
In a preferred embodiment, the probe sequences are dual labelled with at least one of the fluorescent reporter groups selected from FAM™, HEX™, ROX™, JOE™, CY3®, VIC®, TET™, Texas Red®, NED™, Alexa®, TAMRA™, CY5.5® and CY5®, and the fluorescence quencher is selected from BHQ®- 1, BHQ®- 2, BHQ®-3, MGB, DABCYL, and Iowa Black® dark quenchers.
It should be noted that, those skilled in the art may select other types of fluorescence reporters and quenchers according to need and are within the scope of the present disclosure.
Furthermore, an internal control (IC) containing endogenous or exogenous synthetic DNA target should be used to monitor the test sample recovery during extraction, amplification, and detection. An internal control is a nucleic acid sequence, unrelated to the target sequences, and may be selected from a list comprising ICs such as, but not limited to, buffer suspended endogenous IC and exogenous IC.
In an embodiment IC may be endogenous housekeeping gene or added exogenously during the sample preparation procedure to monitor extraction efficiency and PCR inhibition if any or may be added during PCR in the reaction mix as a RT-PCR inhibition control.
In a preferred embodiment, internal control (IC) is an endogenous housekeeping gene target such as RNAseP, GAPDH or beta globin.
As is known to a skilled person in the art, said IC helps in validating the sample collection efficacy, DNA isolation and PCR amplification/detection, as well as reporting false negative test.
A related embodiment may further comprise primer and probe sequences for the IC.
In one embodiment, the simultaneous detection of one or more HPV subtypes from the collected test sample is carried out using real time PCR.
In an exemplary embodiment, the simultaneous detection of one or more HPV subtypes from the collected test sample is carried out by: extracting HPV DNA from the collected test sample using extraction compositions; and
contacting the extracted HPV DNA with one or more detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in a non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes using real time PCR.
In one embodiment, the real-time PCR is carried out using PCR buffer components, and Taq Polymerase.
In a related embodiment, PCR buffer components, include, but are not limited to MgCl2; preferably in ?50 mM/reaction concentrations.
In yet another embodiment, PCR buffer components include conventionally known dNTPs, such as, dATP, dGTP, dCTP, and dTTP/dUTP; preferably in ?0.5 mM each/ reaction concentration.
In a further embodiment, PCR buffer components include one or more conventionally known nuclease inhibitors such as Dnase/Rnase inhibitor.
In yet another further embodiment, the concentration of Taq Polymerase is upto 5U.
In one embodiment, the real-time PCR buffer components, and Taq Polymerase are in liquid form, partially, or as a whole.
Alternatively, the real-time PCR buffer components, and Taq Polymerase are in lyophilized form, partially, or as a whole.
In a preferable embodiment, an excipient is further added for the lyophilized form.
In a related embodiment, the excipient is a saccharide; preferably, selected from, but not limited to sucrose, trehalose, and raffinose; and more preferably, the excipient is trehalose.
In a related embodiment, the real-time PCR is carried out within 70 minutes.
In a preferred embodiment, the real-time PCR is carried out using excipient selected from, but not limited to, sucrose, trehalose, raffinose, PCR buffer components including MgCl2, dNTPs, one or more conventionally known nuclease inhibitors, and Taq Polymerase within 70 minutes.
In yet another related embodiment, the real-time PCR is carried out using requisite PCR Mix comprising PCR enzymes comprising DNA polymerases selected from Taq, Fast Taq, Hot Start Fast Taq, Tfl, Pfu, or Tth DNA polymerase; preferably Hot Start Fast Taq polymerase.
In a preferred embodiment, the overall run time of the instant method is less than 3 hours; preferably ?2.5 hours.
In a further embodiment, the overall run time of the instant method for detecting and discriminating HPV 16 and HPV 18 is ? 40 minutes; preferably by applying POC extraction.
The present disclosure further relates to an in-vitro kit to screen one or more HPV subtypes from a user’s test sample; characterized in that, said kit comprises: self-sampling device for collecting the test sample; medium for transporting the collected test sample; real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample; and tracking system for communicating HPV screening results to the user. In the present disclosure, the term “kit”, “instant kit”, or “multiplex assay kit” refers to a consumable or cartridge comprising a set of articles, components, cartridges, or compositions in a packet required for a specific purpose.
One embodiment of the present disclosure is related to an in-vitro method for screening one or more HPV subtypes from a user’s test sample.
Further, for the purpose of the instant disclosure, the term “users” refers user, healthcare professional, doctors. any official or employee of a government agency or other organisation (whether in the public or private sector) that may prescribe, purchase, supply or administer medicinal products and any employee of a member whose primary occupation is that of a practising healthcare professional.
Another embodiment of the present disclosure is related to self-sampling device for collecting the test sample disclosed in the earlier embodiments.
Further embodiment of the present disclosure is related to transporting the collected test sample using a transport medium disclosed in the earlier embodiments.
Another embodiment of the instant disclosure relates to real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample.
In an exemplary embodiment, the present disclosure further discloses a kit as described in the foregoing paragraphs, wherein the real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample is carried out using: Extraction compositions for extracting HPV DNA from the collected test sample; and one or more Detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes.
One embodiment of the present disclosure relates to the real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample.
For the purpose of the instant disclosure, ‘collected test sample” refers to the test sample that is collected using a self-sampling device as is disclosed in the above paragraphs.
In a preferred embodiment, the collected test sample volume is selected from ? 500 µL.
One embodiment of the present disclosure discloses Extraction compositions for extracting HPV DNA from the collected test sample.
In a related embodiment, the extraction of HPV DNA is carried out using one of conventionally known column based, paramagnetic particles-based extraction or POC (Point of care) extraction as described in the above paragraphs.
Extraction of HPV DNA may be carried out by employing silica gel solid supports such as membranes or columns, or paramagnetic particles, as is known to a skilled person in the art.
In a preferred embodiment, the extraction of HPV DNA is carried out using paramagnetic particle-based extraction in a single container by applying: lysis composition; lysis enhancer composition; one or more wash compositions; and elution compositions, as is described in the above paragraphs.
Various modifications to the embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments.
In an alternate embodiment, the extraction of HPV DNA is carried out using POC (Point of care) extraction; wherein, the POC extraction is carried out using POC extraction composition.
In a related embodiment, the said POC extraction composition comprises lysis composition.
In yet another related embodiment, the lysis composition comprises, but is not limited to, Tris, Triton, sucrose, and magnesium salt; particularly, tris hydrochloride, triton 100, sucrose, and magnesium chloride.
In a further embodiment, the POC extraction composition comprises proteinase K.
In an embodiment, extraction of HPV DNA by applying POC extraction composition is carried out by performing sample lysis and proteinase K inactivation.
In a preferred embodiment, POC extraction is carried out using POC extraction composition comprising but is not limited to Tris hydrochloride, Triton 100, Sucrose, magnesium chloride, and proteinase K.
In a related embodiment, the POC extraction composition may be in a liquid form.
Alternatively, the POC extraction composition may be in a lyophilized form.
In yet another related embodiment, the POC (Point of care) extraction is carried out in less than 16 minutes.
In an exemplary embodiment, the extraction of HPV DNA is carried out using POC (Point of care) extraction; said composition comprising: tris hydrochloride, magnesium chloride, sucrose, Triton 100, and proteinase K; characterized in that, the POC extraction is completed in less than 16 minutes.
The present disclosure further relates to one or more Detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes.
In a related embodiment, the said Detection compositions are lyophilized, as a whole or in part.
In an embodiment, the instant kit may further comprise a sample collection media selected from, but not limited to, Tris Buffer (pH 7- 8), Phosphate Buffer Saline (pH 7.0-8.0), HEPES, magnesium and calcium salts (<10%), Bovine serum albumin, protease inhibitor (<1%) and Stabilizers, with or without alcohol; preferably, Phosphate buffer saline with BSA or TE buffer with salts.
Ethanol or methanol can be a part of sample collection based on their utility in the cytology.
In a preferred embodiment, one or more of the Detection compositions comprises primer and probe sequences selected from SEQ ID NO: 1 to SEQ ID NO: 58.
In yet another related embodiment, the kit comprises one or more Detection compositions selected from Detection compositions A-R disclosed in the earlier embodiments.
In a preferred embodiment, one or more Detection compositions comprise:
Detection composition A comprising primer and probe sequences for HPV16, HPV35, HPV31, HPV33, HPV58, and HPV52;
Detection composition B comprising primer and probe sequences for HPV 18, HPV 45, and HPV51;
Detection composition C comprising primer and probe sequences for HPV 68, HPV39, and HPV59;
Detection composition D and E comprising primer and probe sequences for HPV18;
Detection composition F and G comprising primer and probe sequences for HPV16;
Detection composition H comprising primer and probe sequences for HPV52, and HPV53;
Detection composition I comprising primer and probe sequences for HPV 16, HPV 31, and HPV 35;
Detection composition J comprising primer and probe sequences for HPV 51, and HPV 56;
Detection composition K comprising primer and probe sequences for HPV 33, and HPV 58;
Detection composition L comprising primer and probe sequences for HPV 6;
Detection composition M comprising primer and probe sequences for HPV 11;
Detection composition N comprising primer and probe sequences for HPV 42;
Detection composition O comprising primer and probe sequences for HPV 52;
Detection composition P comprising primer and probe sequences for HPV 58 and 33;
Detection composition Q comprising primer and probe sequences for HPV 31;
Detection composition R comprising primer and probe sequences for HPV 45;
wherein, said primer and probe sequences are specific for one or more HPV subtypes;
such that, said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously detect one or more HPV subtypes using real time PCR.
In yet another preferred embodiment, the Detection compositions enable simultaneous detection of one or more HPV subtypes using up to two reaction tubes.
In a preferred embodiment, one or more Detection compositions enable simultaneous detection and discrimination of low-risk HPV subtypes in one reaction tube; particularly, HPV6, HPV11, and HPV42, and one or more Detection compositions enable simultaneous detection of high-risk HPV subtypes in other reaction tube; preferably, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68; and particularly, further discriminates HPV16 and HPV18 from HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68 in the same reaction tube.
In a further embodiment, one or more detection compositions enable simultaneous detection of 7 high-risk HPV subtypes in a single reaction tube; preferably, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58; and particularly, further enable simultaneous discrimination of HPV16 and HPV18 from the other high-risk HPV subtypes in the same reaction tube.
In yet another further embodiment, one or more detection compositions enable simultaneous detection of 9 HPV subtypes in a single reaction tube; preferably, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV6, and HPV11.
In another embodiment, one or more Detection compositions enable simultaneous detection and discrimination of HPV6, HPV11, and HPV42 in another reaction tube.
In one embodiment, a single reaction tube comprises one or more Detection compositions selected from A-K; said Detection compositions comprising:
forward primer sequences selected from SEQ 1D NO: 1, 2, 3, 4, 9, 11, 12, 13, 15, 26, 29, 32, and 35;
reverse primer sequences selected from SEQ ID NO: 5, 6, 7, 8, 10, 14, 16, 27, 30, 33, and 36; and
dual-labelled probe sequences selected from SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 31, 34, and 37;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify and detect at least one of the high-risk HPV subtypes from HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68.
In another embodiment, the Detection compositions described in the foregoing paragraphs further comprise:
forward primer sequences selected from SEQ ID NO: 1, 2, 4, 9, 13, 15, and 29;
reverse primer sequences selected from SEQ ID NO: 5, 6, 8, 10, 14, 16, and 30; and
dual-labelled probe sequences selected from SEQ ID NO: 17, 18, 20, 21, 22, 23, and 31;
to enable simultaneous discrimination of HPV16 and HPV18 in the same reaction tube.
In a further embodiment, a single reaction tube comprises one or more Detection compositions selected from L-N; said Detection compositions comprising:
forward primer sequences selected from SEQ ID NO: 38, 41, and 44;
reverse primer sequences selected from SEQ ID NO: 39, 42, and 45; and
dual-labelled probe sequences selected from SEQ ID NO: 40, 43, and 46;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify, detect, and discriminate at least one of the low-risk HPV subtypes from HPV 6, HPV11, and HPV42.
In a related embodiment, a single reaction tube comprises one or more Detection compositions selected from D-G, and O-R; said Detection compositions comprising:
forward primer sequences selected from SEQ 1D NO: 4, 9, 13, 15, 47, 50, 53, and 56;
reverse primer sequences selected from SEQ ID NO: 8, 10, 14, 16, 48, 51, 54, and 57; and
dual-labelled probe sequences selected from SEQ ID NO: 20, 21, 22, 23, 49, 52, 55, and 58;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify and detect at least one of the 7 high-risk HPV subtypes from HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58.
In a preferred embodiment, the Detection compositions described in the foregoing paragraphs further comprise:
forward primer sequences selected from SEQ ID NO: 4, 9, 13, and 15;
reverse primer sequences selected from SEQ ID NO: 8, 10, 14, and 16; and
dual-labelled probe sequences selected from SEQ ID NO: 20, 21, 22, and 23;
to enable simultaneous discrimination of HPV16 and HPV18 in the same reaction tube.
In a further embodiment, a single reaction tube comprises one or more Detection compositions selected from D-G, L, M, and O-R; said Detection compositions comprising:
forward primer sequences selected from SEQ 1D NO: 4, 9, 13, 15, 38, 41, 47, 50, 53, and 56;
reverse primer sequences selected from SEQ ID NO: 8, 10, 14, 16, 39, 42, 48, 51, 54, and 57; and
dual-labelled probe sequences selected from SEQ ID NO: 20, 21, 22, 23, 40, 43, 49, 52, 55, and 58;
such that said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously amplify and detect at least one of the 9 HPV subtypes from HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV6, HPV11, and HPV58.
In a preferred embodiment, the primer and probe sequences hybridize with target sequences in HPV DNA without cross-reacting or overlapping with each other or with any other nucleic acids present in the test sample.
In yet another preferred embodiment, the probe sequences are dual labelled with at least one of the fluorescent reporter groups selected from FAM™, HEX™, ROX™, JOE™, CY3®, VIC®, TET™, Texas Red®, NED™, Alexa®, TAMRA™, CY5.5® and CY5®, and the fluorescence quencher is selected from BHQ®- 1, BHQ®- 2, BHQ®-3, MGB, DABCYL, and Iowa Black® dark quenchers.
It should be noted that, those skilled in the art may select other types of fluorescence reporters and quenchers according to need and are within the scope of the present disclosure.
In an embodiment IC may be endogenous housekeeping gene or added exogenously during the sample preparation procedure to monitor extraction efficiency and PCR inhibition if any or may be added during PCR in the reaction mix as a RT-PCR inhibition control.
In a preferred embodiment, internal control (IC) is an endogenous housekeeping gene target such as RNAseP, GAPDH or beta globin.
As is known to a skilled person in the art, said IC helps in validating the sample collection efficacy, DNA isolation and PCR amplification/detection, as well as reporting false negative test.
A related embodiment may further comprise primer and probe sequences for the IC.
In one embodiment, the simultaneous detection of one or more HPV subtypes from the collected test sample is carried out using real time PCR.
In an exemplary embodiment, real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample is carried out using: Extraction compositions for extracting HPV DNA from the collected test sample; and one or more Detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes.
In one embodiment, the real-time PCR is carried out using PCR buffer components, and Taq Polymerase.
In a related embodiment, PCR buffer components, include, but are not limited to MgCl2; preferably in ?50 mM/reaction concentrations.
In yet another embodiment, PCR buffer components include conventionally known dNTPs, such as, dATP, dGTP, dCTP, and dTTP/dUTP; preferably in ?0.5 mM each/ reaction concentration.
In a further embodiment, PCR buffer components include one or more conventionally known nuclease inhibitors such as Dnase/Rnase inhibitor.
In yet another further embodiment, the concentration of Taq Polymerase is upto 5U.
In one embodiment, the real-time PCR buffer components, and Taq Polymerase are in liquid form, partially, or as a whole.
Alternatively, the real-time PCR buffer components, and Taq Polymerase are in lyophilized form, partially, or as a whole.
In a preferable embodiment, an excipient is further added for the lyophilized form.
In a related embodiment, the excipient is a saccharide; preferably, selected from, but not limited to sucrose, trehalose, and raffinose; and more preferably, the excipient is trehalose.
In a related embodiment, the real-time PCR is carried out within 70 minutes.
In a preferred embodiment, the real-time PCR is carried out using excipient selected from, but not limited to, sucrose, trehalose, raffinose, PCR buffer components including MgCl2, dNTPs, one or more conventionally known nuclease inhibitors, and Taq Polymerase within 70 minutes.
In yet another related embodiment, the real-time PCR is carried out using requisite PCR Mix comprising PCR enzymes comprising DNA polymerases selected from Taq, Fast Taq, Hot Start Fast Taq, Tfl, Pfu, or Tth DNA polymerase; preferably Hot Start Fast Taq polymerase.
In a preferred embodiment, the overall run time of the instant method is less than 3 hours; preferably ?2.5 hours.
The present disclosure also relates to a tracking system, wherein said system comprises: Module/program for collecting user details; Module/program for automatically inputting user’s HPV screening result; Module/program for linking the user details and the user’s HPV screening result to the specific user; and Module/program for communicating HPV screening result to said user.
For the purpose of the instant disclosure, “tracking system” or “system” refers to, but is not limited to a module, apparatus, program, application, etc. that is applied for, but is not limited to communicating HPV screening results to the user, collecting user details, linking user details with the HPV screening results of the user, indicating further steps to the user, etc.
In one embodiment, the tracking system provides HPV screening results.
In a related embodiment, the HPV screening result is at least one of positive result or negative result.
In another embodiment, the tracking system directs user for carrying out further steps.
In a related embodiment, the system directs the user “to treatment” if the HPV screening result is a positive result.
In yet another related embodiment, the system directs the user “to vaccinate” if the HPV screening result is a negative result.
In another embodiment, the system further tracks the treatment of the user.
In yet another embodiment, the system further tracks the vaccination of the user.
One embodiment of the instant disclosure relates to an in-vitro method for screening one or more HPV subtypes from a user’s test sample; characterized in that, said method comprises: using a tracking system for indicating/communicating HPV screening results to the user.
Further embodiment of the instant disclosure relates to an in-vitro kit to screen one or more HPV subtypes from a user’s test sample; characterized in that, said kit comprises: tracking system for communicating HPV screening results to the user.
The method and kit compositions described in the foregoing embodiments are in liquid form or as a lyophilized form, as a whole or in part.
Alternatively, the method and kit compositions described in previous embodiments, are lyophilized, as a whole or in part.
In one embodiment, the use of the compositions, described in the previous embodiments, as a whole or in part is automated. As known to a skilled person, automated systems have been increasingly utilized to minimize the risk of human errors such as failure to follow the exact protocol, measuring mishaps, laboratorial accidents, contamination of materials due to over-handling, and miscalculations of data. Risk of human error could be disastrous, in a routine analytical process such as extraction of nucleic acids that may form the basis of health diagnosis and prognosis discussed hereinabove. Additionally, an automated system also highlights numerous advantages like higher production rates and increased productivity, efficient use of raw materials, better product quality, assurance of high reproducibility, improved safety, and established protocol. It also encourages versatility and reliability to procure better results.
In an embodiment, the kit of the present disclosure may be coupled with any PCR machine having at least four channels.
In another aspect, the kit packet also carries a user instructions manual.
Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. However, one of ordinary skill in the art will readily recognize that the present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded the widest scope consistent with the principles and features described herein.
The foregoing description shall be interpreted as illustrative and not in any limiting sense. A person of ordinary skill in the art would understand that certain modifications could come within the scope of this disclosure.
The features and properties of the present disclosure are described in further detail below with reference to examples.
Table A: List of Sequences for detecting HPV subtypes
SEQ ID NO Sequences (5’?3’) Bases
1 tttgttactg ttgttgacac tac 23
2 tttgttaccg tggtagatac cac 23
3 tttcttactg tagtggatac aac 23
4 tttgttactg tkgtwgatac ha 22
5 tgcaaatcat attcctcaac atg 23
6 tgtaaatcat attcctcccc at 22
7 tgtaattcat actcttccac gtg 23
8 tgyaaatcat aytcctcmac rtg 23
9 caactgacct atactgttat gagc 24
10 gtctggtttt gcttgtcca 19
11 cagaggaata tgaattacaa tttg 24
12 ccaatattcc agtcctccag ta 22
13 ttcggttgtg cgtacaaagc 20
14 cccattaaca ggtcttccaa a 21
15 agaggccagt gccattcgt 19
16 tttctctgcg tcgttggagt 20
17 acatataaaa gtgctaactt ta 22
18 agtaccaatt taac 14
19 agcactaacc tttc 14
20 cctgggcaat atg 13
21 cctacgacat ggg 13
22 cacacgtaga cattc 15
23 cctgtcgtgc tcggttgcag c 21
24 acagctcaga ggagga 16
25 tgtctgctga ggttatggcc tatttacata 30
26 aaatggtgca gtgggcat 18
27 ctgtctacat ctgctaactg tgcata 26
28 atgatagtga catagca 17
29 gtccagatta agtttgcacg 20
30 tattttgtcc tgacacacat tta 23
31 aggacaagga aaacgatgga ga 22
32 ggtgcaatgg gcatttg 17
33 ccacaatctt ttacatattt tgc 23
34 tatgcacaat tagcagat 18
35 aaccaactga cctatactgc tatg 24
36 ctggttgtgc ttgtccatc 19
37 aaccaactga cctatactgc tat 23
38 tggggtaatc aactgtttgt tactgtggta 30
39 gcatgtactc tttataatca gaattggtgt atgtg 35
40 gacattatgt gcatccgtaa ctac 24
41 tggggaaacc acttgtttgt tactgtg 27
42 cgcatgtatt ccttataatc tgaattagtg tatgt 35
43 gacactatgt gcatctgtgt cta 23
44 aacccagaga cacagcgcat 20
45 ccactaatac caacgcccaa a 21
46 cctgtgtggg gctagaagta ggtc 24
47 cctgcacgaa ttgtgtgagg t 21
48 gcacataata cacacgccat atgg 24
49 tggaagaatc ggtgcatgaa ataaggctgc 30
50 aaaccacgga cattgcatga 20
51 catatacctc agatcgctgc aaag 24
52 gcattggaga caact 15
53 cctcggaaat tgcatgaac 19
54 gacctttgca gtagacacaa ttc 23
55 gctcggcatt ggaaataccc tacga 25
56 gaatacatca ctacaagacg tatctattg 29
57 cataggctat acagtctcta tacactatac at 32
58 caacattgga acgcacagag gtatatcaat 30

y - C/T m - A/C r - A/G k - G/T w - A/T h - A/C/T
Example 1
Extraction of HPV DNA using Extraction compositions
The extraction of HPV DNA can be carried out using conventional methods known to a skilled person such as silica membrane or paramagnetic particle-based extraction.
In this example, HPV DNA was extracted from ?500µl of cervical or urine sample using paramagnetic particle-based extraction.
A negative extraction control was used as a test control.
The lysis composition was prepared by mixing the reagents shown in Table 1. The lysis enhancer buffer was prepared was prepared by dissolving freeze-dried proteinase K powder (up to 20mg/ml) in a solution containing up to 1.5mM calcium chloride and up to =40% glycerol.
Sample lysis was carried out by mixing the sample with the lysis/binding composition (Table 2) in the sample tube and incubating at 56°C for up to 25 minutes with continuous shaking at up to 900 rpm.
Table 1: Components of lysis buffer
Reagent Stock Concentration pH
Tris HCl 1M 6.0 -6.8
Sodium acetate =3M 9-9.4
Guanidine thiocyanate (freshly prepared) >4 M NA
EDTA 0.5 M 8-8.5
DTT =10% 4-5
SDS =5% 7-8
Sodium azide 1% NA
DEPC treated water for volume make up 5-7
Final pH 7.0-8.0
Table 2: Lysis/binding composition for 200 µl sample volume
Sr. No. Reagents Volume (µl) /Rx
1 Lysis Buffer (see Table 1) Up to 250
2 Lysis enhancer buffer Up to 50
3 Paramagnetic particles Up to 50
4 Internal Control (IC) Up to 25
5 Isopropanol 400-600 µl
Following the incubation, magnetic separation of the lysis composition in the sample tube was performed and the supernatant was discarded.
The DNA bound paramagnetic particles in the sample tube were washed in two steps using Wash buffers 1 and 2 prepared by mixing the reagents as shown in Tables 3 and Table 4 and further reconstituting with up to 60% isopropanol and 80% ethanol respectively
Table 3: Composition of Wash Buffer 1
Reagent Stock Concentration pH
Tris HCl Up to 1M 6-8
Sodium acetate =3M 4-6
Guanidine thiocyanate =1M NA
Sodium azide Upto 1% NA
Nuclease free water for volume make up 5-7
Final pH 4.5-5.5
Table 4: Composition of Wash Buffer 2
Reagent Stock Concentration pH
Tris HCl Up to 1M 6-8
Sodium acetate =3M 8-10
Sodium azide Upto 1%
Nuclease free water for volume make up 5-7
Final pH 7.0-8.0
About 700 µl of Wash buffer 1 was added to the sample tube, mixed well with the contents, followed by magnetic separation. The supernatant was discarded.
Then up to 900 µl of Wash Buffer 2 was added to the sample tube, followed by mixing and magnetic separation. The supernatant was again discarded, and the sample was dried.
The washed and dried DNA bound paramagnetic particles were eluted using an elution composition prepared using DEPC treated autoclaved water.
For performing the elution step, up to 100 µl of elution composition (pH-5-6) was added to the sample tube and the resuspended paramagnetic particles were vortexed and incubated for up to 5 minutes at around 80°C.
Magnetic separation was performed again and the supernatant containing HPV DNA was transferred to a new tube for further analysis, or downstream applications or stored at -20°C or -80°C until further use.
Alternatively, the extraction of HPV DNA can be carried out using POC (Point of Care) Extraction in less than 16 minutes.
For the POC extraction, HPV DNA was extracted from up to 100µl of the clinical sample.
The lysis composition was prepared by mixing the reagents shown in Table 5.
Table 5: Lysis composition for 100 µl sample volume
Sr. No. Reagents Concentration
1 Tris HCl (Tris hydrochloric acid) (pH = 8) 5mM – 15mM
2 Magnesium chloride 5mM – 15mM
3 Sucrose 250mM – 350mM
4 Triton 100 = 1.0 %
First, the sample lysis was carried out by mixing up to 100µl of the clinical sample with up to 100µl of lysis composition (Table 5) and up to 50µl of proteinase K (up to 20mg/mL concentration) in the sample tube and incubating at around 56°C for up to 10 minutes.
Then, proteinase K inactivation was carried out by incubating the sample tube at around 98°C for up to 5 minutes to completely extract HPV DNA.
The extracted HPV DNA was transferred to a new tube for further analysis, or downstream applications or stored at -20°C or -80°C until further use.
The compositions described in the present example (Tables 1-5) can be partially or wholly in liquid form.
Alternatively, the compositions described in the present example (Tables 1-5) can be partially or wholly lyophilised.
Further, the working of the present example can be partially or wholly automated.
Example 2
Simultaneous detection of at least one of the 15 high-risk HPV subtypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68), while discriminating HPV16 and HPV18 from other high-risk HPV subtypes in a single reaction tube using real-time PCR
For this example, the extracted HPV DNA from Example 1 was used as the sample.
The screening was monitored using High Risk Positive Control Mix containing synthetic templates against HPV 16, HPV 18 and HPV 31 and IC and No Template Control (NTC)/nuclease free water or negative control.
Table 8 describes the components of PCR for detecting high-risk HPV subtypes.
PCR Mix was prepared using Taq polymerase enzyme, and buffer components. Detection compositions (A-K) were prepared using primers and dual-labelled probes specific to HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68.
Then, a cocktail mixture was prepared by mixing the detection composition and PCR Mix in a single reaction tube, further comprising primer and dual-labelled probe sequences specific to IC. (See Table 6)
Table 6: Components of the detection composition for detecting high-risk HPV subtypes
Detection composition SEQ ID NO Composition
A SEQ ID NO: 1, 5, and 17 Primer and dual-labelled probe sequences for HPV16, HPV35, HPV31, HPV33, HPV58, and HPV52
B SEQ ID NO: 2, 6, and 18 Primer and dual-labelled probe sequences for HPV18, HPV45, and HPV51
C SEQ ID NO: 3, 7, and 19 Primer and dual-labelled probe sequences for HPV68, HPV39, and HPV59
D SEQ ID NO: 4, 8, and 20 Primer and dual-labelled probe sequences for HPV18
E SEQ ID NO: 9, 10, and 21 Primer and dual-labelled probe sequences for HPV18
F SEQ ID NO: 13, 14, and 22 Primer and dual-labelled probe sequences for HPV16
G SEQ ID NO: 15, 16, and 23 Primer and dual-labelled probe sequences for HPV16
H SEQ ID NO: 26, 27, and 28 Primer and dual-labelled probe sequences for HPV52 and HPV53
I SEQ ID NO: 29, 30, and 31 Primer and dual-labelled probe sequences for HPV16, HPV31, and HPV35
J SEQ ID NO: 32, 33, and 34 Primer and dual-labelled probe sequences for HPV51, and HPV56
K SEQ ID NO: 35, 37, and 37 Primer and dual-labelled probe sequences for HPV33, and HPV58
Primer and dual-labelled probe sequences for IC
The components of the cocktail mixture are described in Table 7.
Table 7: Components of the cocktail mixture
Components Volume/Rx
PCR Mix Up to 1X
Detection composition (one or more selected from Tables 6, 12, 14 and 16) Up to 1X (up to 15 pmol primer and up to 6 pmol probes cocktail)
Nuclease free water To compensate
Template Up to 50%
Total volume Up to 50µL
The compositions described in the present example are partially or wholly in liquid form.
Alternatively, one or more of the PCR Mix, the Detection composition or the cocktail mixture are lyophilised and rehydrated using suitable reconstitution buffer prior to use.
Most commercially available kits for real-time PCR based detection of pathogens involve liquid reagents that either require a cold supply chain of sub-zero temperatures for transport and storage. Lack of proper storage conditions causes increased susceptibility to loss of efficacy in the activity of the compositions. To address this and facilitate feasible testing in varied conditions, the present disclosure provides compositions in a lyophilised state, as a whole or in part.
PCR plate was set up for real-time PCR system using the extracted HPV DNA, positive controls, NTC, and the reaction mixture such that the total volume of the composition was up to 50µl per well.
Table 8: Components of PCR for detecting high-risk HPV subtypes
Contents Description
PCR Mix Taq polymerase, and buffer components
(Components may be optionally lyophilized by addition of excipient,)
Detection composition See Table 6
Nuclease free water RNase/DNase free water
Positive control Mix: Synthetic template for HPV 16, 18, 31 and IC
Internal Control (IC) Endogenous housekeeping gene
Reconstitution buffer (optional) Buffer components for reconstitution of lyophilized PCR mix
The PCR cycling conditions for the performed experiment are set forth in Table 9.
Table 9: PCR Run method
Parameter Stage Temperature Time
RT incubation Hold 95°C Up to 3 minutes
Enzyme inactivation Hold 95°C Up to 10 secs
PCR amplification Cycle
(=45 cycles) 46°C-60°C Up to 30 sec*
(*data collection)
After performing the PCR run, fluorescence data was collected during 60°C extension step.
The PCR plate was analysed using data analysing software and the result was saved.
The results were generated as per Table 10.
Table 10: Test validation and interpretation
Other High-risk HPV subtypes (Dye 1*) HPV18
(Dye 2*) HPV16
(Dye 3*) IC
(Dye 4*) Interpretation
Positive Positive Negative Positive HPV18 detected
Positive Negative Positive Positive HPV16 detected
Positive Negative Negative Positive High risk HPV subtype other than HPV16 and HPV18 detected
Negative Negative Negative Negative Inhibition of PCR reaction, repeat the test.

*Dye 1, 2, 3, and 4 refer to four distinct dyes selected from fluorescent reporter groups FAM™, HEX™, ROX™, JOE™, CY3®, VIC®, TET™, Texas Red®, NED™, Alexa®, TAMRA™, CY5.5® and CY5®.
Example 3
Simultaneous detection of at least one of 7 high-risk HPV subtypes (HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58), while discriminating HPV16 and HPV18 from the other 7 high-risk HPV subtypes in a single reaction tube using real-time PCR
For this example, the extracted HPV DNA from Example 1 was used as the sample.
The screening was monitored using High Risk Positive Control Mix containing synthetic templates against HPV 16, HPV 18 and HPV 31 and IC and No Template Control (NTC)/nuclease free water or negative control.
Table 11 describes the components of PCR for detecting the 7 high-risk HPV subtypes.
PCR Mix was prepared using Taq polymerase enzyme, and buffer components.
Table 11: Components of PCR for detecting 7 high-risk HPV subtypes
Contents Description
PCR Mix Taq polymerase, and buffer components
(Components may be optionally lyophilized by addition of excipient,)
Detection composition See Table 12
Nuclease free water RNase/DNase free water
Positive control Mix: Synthetic template for HPV 16, 18, 31 and IC
Internal Control (IC) Endogenous housekeeping gene
Reconstitution buffer (optional) Buffer components for reconstitution of lyophilized PCR mix
Detection compositions (D or E, F or G, and O-R) were prepared using primers and dual-labelled probes specific to HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58.
Then, the Detection compositions were transferred in a single reaction tube, further comprising primer and dual-labelled probe sequences specific to IC. (See Table 12)
Table 12: Components of the detection composition for detecting 7 high-risk HPV subtypes
Detection composition SEQ ID NO Composition
D SEQ ID NO: 4, 8, and 20 Primer and dual-labelled probe sequences for HPV18
E SEQ ID NO: 9, 10, and 21 Primer and dual-labelled probe sequences for HPV18
F SEQ ID NO: 13, 14, and 22 Primer and dual-labelled probe sequences for HPV16
G SEQ ID NO: 15, 16, and 23 Primer and dual-labelled probe sequences for HPV16
O SEQ ID NO: 47, 48, and 49 Primer and dual-labelled probe sequences for HPV52
P SEQ ID NO: 50, 51, and 52 Primer and dual-labelled probe sequences for HPV58, and HPV33
Q SEQ ID NO: 53, 54, and 55 Primer and dual-labelled probe sequences for HPV31
R SEQ ID NO: 56, 57, and 58 Primer and dual-labelled probe sequences for HPV45
Primer and dual-labelled probe sequences for IC
A cocktail mixture was prepared by mixing the PCR Mix the detection composition and in the reaction tube.
The components of the cocktail mixture are described in Table 7.
The PCR cycling conditions for the performed experiment are set forth in Table 9.
After performing the PCR run, fluorescence data was collected during 60°C extension step.
The PCR plate was analysed using data analysing software and the result was saved.
The results were generated as per Table 10.
Example 4
Simultaneous detection of 9 HPV subtypes (HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV6, and HPV11) in a single reaction tube using real-time PCR
For this example, the extracted HPV DNA from Example 1 was used as the sample.
The screening was monitored using High Risk Positive Control Mix containing synthetic templates against HPV 16, HPV 18 and HPV 31 and IC and No Template Control (NTC)/nuclease free water or negative control.
Table 13 describes the components of PCR for detecting the 9 HPV subtypes.
PCR Mix was prepared using Taq polymerase enzyme, and buffer components.
Table 13: Components of PCR for detecting 9 HPV subtypes
Contents Description
PCR Mix Taq polymerase, and buffer components
(Components may be optionally lyophilized by addition of excipient,)
Detection composition See Table 14
Nuclease free water RNase/DNase free water
Positive control Mix: Synthetic template for HPV 16, 18, 31 and IC
Internal Control (IC) Endogenous Housekeeping gene
Reconstitution buffer (optional) Buffer components for reconstitution of lyophilized PCR mix
Detection compositions (D or E, F or G, L, M, and O-R) were prepared using primers and dual-labelled probes specific to HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV6, and HPV11.
Then, the Detection compositions were transferred in a single reaction tube, further comprising primer and dual-labelled probe sequences specific to IC. (See Table 15)
Table 14: Components of the detection composition for detecting 9 HPV subtypes
Detection composition SEQ ID NO Composition
D SEQ ID NO: 4, 8, and 20 Primer and dual-labelled probe sequences for HPV18
E SEQ ID NO: 9, 10, and 21 Primer and dual-labelled probe sequences for HPV18
F SEQ ID NO: 13, 14, and 22 Primer and dual-labelled probe sequences for HPV16
G SEQ ID NO: 15, 16, and 23 Primer and dual-labelled probe sequences for HPV16
L SEQ ID NO: 38, 39, and 40 Primer and dual-labelled probe sequences for HPV6
M SEQ ID NO: 41, 42, and 43 Primer and dual-labelled probe sequences for HPV11
O SEQ ID NO: 47, 48, and 49 Primer and dual-labelled probe sequences for HPV52
P SEQ ID NO: 50, 51, and 52 Primer and dual-labelled probe sequences for HPV58, and HPV33
Q SEQ ID NO: 53, 54, and 55 Primer and dual-labelled probe sequences for HPV31
R SEQ ID NO: 56, 57, and 58 Primer and dual-labelled probe sequences for HPV45
Primer and dual-labelled probe sequences for IC
A cocktail mixture was prepared by mixing the detection composition and the PCR Mix in the reaction tube.
The components of the cocktail mixture are described in Table 7.
The PCR cycling conditions for the performed experiment are set forth in Table 9.
After performing the PCR run, fluorescence data was collected during 60°C extension step.
The PCR plate was analysed using data analysing software and the result was saved.
The results were generated as per Table 15.
Table 15: Test validation and interpretation
Other HPV subtypes (Dye 1*) HPV18
(Dye 2*) HPV16
(Dye 3*) IC
(Dye 4*) Interpretation
Positive Positive Negative Positive HPV18 detected
Positive Negative Positive Positive HPV16 detected
Positive Negative Negative Positive HPV subtypes from either HPV31, HPV33, HPV45, HPV52, HPV58, or HPV6, HPV 11 detected
Negative Negative Negative Negative Inhibition of PCR reaction, repeat the test.
After this experimentation, the tracking system directed the user to quadrivalent or nine valent vaccination if the test result was negative.
Correspondingly, the tracking system directed the user to get treated if the test result was positive.
Example 5
Simultaneous detection and discrimination of at least one of the 3 low-risk HPV subtypes (HPV6, HPV11, and HPV42) in a single reaction tube using real-time PCR
For this example, the extracted HPV DNA from Example 1 was used as the sample.
The screening was monitored using HPV low risk Positive control Mix and No Template Control (NTC)/nuclease free water or negative control.
Table 17 describes the components of PCR for detecting low-risk HPV subtypes.
PCR Mix was prepared using Taq polymerase enzyme, and buffer components. Detection compositions (L-N) were prepared using primers and dual-labelled probes specific to HPV6, HPV11, and HPV42.
Then, the Detection compositions were transferred in a single reaction tube, further comprising primer and dual-labelled probe sequences specific for IC. (See Table 18)
Table 16: Components of the detection composition for detecting low-risk HPV subtypes
Detection composition SEQ ID NO Composition
L SEQ ID NO: 38, 39, and 40 Primer and dual-labelled probe sequences for HPV6
M SEQ ID NO: 41, 42, and 43 Primer and dual-labelled probe sequences for HPV11
N SEQ ID NO: 44, 45, and 46 Primer and dual-labelled probe sequences for HPV42
Primer and dual-labelled probe sequences for IC
A cocktail mixture was prepared by mixing the detection composition and the PCR Mix in the reaction tube.
The components of the cocktail mixture are described in Table 7.
Table 17: Components of PCR for detecting low-risk HPV subtypes
Contents Description
PCR Mix Taq polymerase, and buffer components
(Components may be optionally lyophilized using excipient,)
Detection composition See Table 16
Nuclease free water RNase/DNase free water
Positive control Mix: Synthetic template for HPV 6, 11,42 and IC
Internal Control (IC) Endogenous housekeeping gene
Reconstitution buffer (optional) Buffer components for reconstitution of lyophilized PCR mix
The PCR cycling conditions for the performed experiment are set forth in Table 9.
After performing the PCR run, fluorescence data was collected during 60°C extension step.
The PCR plate was analysed using data analysing software and the result was saved.
The results were generated as per Table 18.
Table 18: Test validation and interpretation
HPV6
(Dye 1*) HPV11
(Dye 2*) HPV42
(Dye 3*) IC
(Dye 4*) Interpretation
Negative Positive Negative Positive HPV11 detected
Negative Negative Positive Positive HPV42 detected
Positive Negative Negative Positive HPV6 detected
Negative Negative Negative Negative Inhibition of PCR reaction, repeat the test.
*Dye 1, 2, 3, and 4 refer to four distinct dyes selected from fluorescent reporter groups FAM™, HEX™, ROX™, JOE™, CY3®, VIC®, TET™, Texas Red®, NED™, Alexa®, TAMRA™, CY5.5® and CY5®.
Example 7
A kit to simultaneously detect 15 high-risk (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, and HPV68) and 3 low-risk (HPV6, HPV11, and HPV42) HPV subtypes in up to 2 reaction tubes using real-time PCR.
This example demonstrates a kit in accordance with the instant disclosure.
In this example, HPV16 and HPV18 are further discriminated from other high-risk HPV subtypes.
Additionally, the low-risk HPV subtypes are further discriminated from each other.
For this example, the extracted HPV DNA from Example 1 was used as the sample.
The screening was monitored using HPV High Risk Positive control Mix , HPV Low risk Positive control Mix and No Template Control (NTC)/nuclease free water or negative control.
Table 19 describes the components of PCR for detecting high-risk and low-risk HPV subtypes.
PCR Mix was prepared using Taq polymerase enzyme, excipient, and buffer components.
Detection compositions (A-N) were prepared using primers and dual-labelled probes specific to HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV6, HPV11, and HPV42.
Then, the Detection compositions (A-K) were transferred in one reaction tube, further comprising primer and dual-labelled probe sequences specific for IC. (See Table 6);
and the Detection compositions (L-N) were transferred in another reaction tube, further comprising primer and dual-labelled probe sequences specific for IC. (See Table 16)
A cocktail mixture was prepared by mixing the PCR Mix in the reaction tubes.
The components of the cocktail mixtures are described in Table 7.
Table 19: Components of PCR for detecting high-risk and low-risk HPV subtypes (18 subtypes)
Contents Description
PCR Mix Taq polymerase, and buffer components
(Components may be optionally lyophilized along with excipient)
Detection composition- HPV High Risk See Table 6 and Table 18
Detection composition-HPV Low Risk
Nuclease free water /No template control RNase/DNase free water
HPV High Risk Positive control Mix: Synthetic template comprising HPV High Risk 16, 18, 31 and Internal Control
HPV Low Risk Positive control Mix: Synthetic template comprising HPV Low Risk 6,11 and 42
Internal Control (IC) RNase P
Reconstitution buffer (optional) Buffer components for reconstitution of lyophilized PCR mix
The present kit may further comprise compositions/buffers described in Example 1 for performing manual, automated, or semi-automated extraction of HPV DNA from ? 500µL urine or cervical sample.
The compositions of the kit, as a whole or in part may be in liquid form.
Alternatively, the compositions of the kit, as a whole or in part may be lyophilized. Additionally, the lyophilised compositions may be optionally placed in a tube or a container. Furthermore, a reconstitution buffer may be further utilised for rehydrating the lyophilised compositions.
Most commercially available kits for real-time PCR based detection of pathogens involve liquid reagents that either require a cold supply chain of sub-zero temperatures for transport and storage. Lack of proper storage conditions causes increased susceptibility to loss of efficacy in the activity of the compositions. To address this and facilitate feasible testing in varied conditions, the present disclosure provides compositions in a lyophilised state, as a whole or in part.
PCR plate was set up for real-time PCR system using the extracted HPV DNA, positive controls, NTC, and the reaction mixture such that the total volume of the composition was up to 50µl per well.
The PCR cycling conditions for the performed experiment are set forth in Table 8.
After performing the PCR run, fluorescence data was collected during 60°C extension step.
The PCR plate was analysed using data analysing software and the result was saved.
The results were generated as per Table 10 and Table 18.
Performance Characteristics
Limit of Detection (LOD or Analytical Sensitivity)
Limit of Detection (See Table 20) was tested against the 2-fold serial dilutions of NIBSC standard to make total 7 dilutions and tested each point in 20 replicates. The LOD was considered at 95% confidence interval.
Table 20: Limit of Detection
Sr. No. HPV Types NIBSC Control ID IU
HPV High Risk
1 HPV 16 06/202 ? 500 IU
2 HPV 35, HPV 68, HPV 39, HPV 59, HPV 51, HPV 53, HPV 56, HPV 66 Internal calibrators <2000 copies for HPV 53, 66, 59 and <1000 copies
3 HPV 31 14/258 ?1000 IU
4 HPV 33 14/260 ?1000 IU
5 HPV 58 14/264 ?2000 IU
6 HPV 52 14/262 ?2000 IU
7 HPV 18 06/206 ? 500 IU
8 HPV 45 14/104 ?500 IU
HPV Low Risk
9 HPV 6 14/256 ?1000 IU for HPV 6 and 11
?1000 copies for HPV 42
10 HPV 11 19/224
11 HPV 42 Internal calibrators
Linear Range
Linear range was calculated by 10-fold serial dilutions of NIBSC and Mylab internal calibrators. Each HPV high risk genotype covered over 5*10^8 IU/ml to 50 IU/ml
HPV High Risk Linearity (Tentative LoD): 5* 10^8 IU/ml to 50 IU/ml
Tentative limit of detection of all the targets were determined by performing ten-fold serial dilutions of respective NIBSC standards. The dilutions that were used in the determination of tentative LoD were 10^6 IU/ml to 50 IU/ml from NIBSC against Mylab internal calibrators covering at least 5* 10^8 IU/ml to 50 IU/ml.
HPV High Risk and Low Risk LoD
The analytical sensitivity (limit of detection: LOD) of the Pathodetect™ HPV High & Low Risk Detection Kit is defined as the concentration of HPV High Risk types DNA molecules that can be detected with a positivity rate of =95%. The analytical sensitivity was determined using a twofold dilution series with NIBSC standard. Experiments were carried out in triplicates and as three independent runs on RT qPCR instrument. The analytical detection limit was found to be as follows in Table 21.
Table 21: Analytical sensitivity for HPV high risk and HPV low risk
Sr. No. HPV Types NIBSC Control ID IU
HPV High Risk
1 HPV 16 06/202 ? 500 IU
2 HPV 35, HPV 68, HPV 39, HPV 59, HPV 51, HPV 53, HPV 56, HPV 66 Internal calibrators <2000 copies for HPV 53, 66, 59 and <1000 copies
3 HPV 31 14/258 ?1000 IU
4 HPV 33 14/260 ?1000 IU
5 HPV 58 14/264 ?2000 IU
6 HPV 52 14/262 ?2000 IU
7 HPV 18 06/206 ? 500 IU
8 HPV 45 14/104 ?500 IU
HPV Low Risk
9 HPV 6 14/256 ?1000 IU for HPV 6 and 11
?1000 copies for HPV 42
10 HPV 11 19/224
11 HPV 42 Internal calibrators
Analytical specificity and cross reactivity
• The specificity of HPV subtypes are first and foremost ensured by the selection of the primers and probes, as well as the selection of stringent reaction conditions. The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis.
• To estimate the specificity of the present kit, in silico analysis using the Basic Local Alignment Search Tool (BLAST) managed by the National Centre for Biotechnology Information (NCBI) was used to assess the designed primer probe for their inclusivity of all the required subtypes of HPV.
• A potential cross-reactivity of the HPV types was tested using the control group listed in the Table 22. None of the tested pathogens has been reactive. No cross-reactivity appeared with mixed infection.
Table 22: Cross-reactivity testing for HPV types
High priority pathogens, human gDNA and normal flora In silico primer BLAST analysis PCR Reaction
Chikungunya virus (taxid:37124) ? ?
Dengue virus (taxid:12637) ? ?
Plasmodium (taxid:5820) ? ?
Homo sapiens (taxid:9606) ? ?
Human coronavirus 229E (taxid:11137) ? ?
SARS (taxid:694009) ? ?
SARS-CoV-2 (taxid:2697049) ? ?
Chlamydia pneumoniae (taxid:83558) ? ?
Haemophilus influenzae (taxid:727) ? ?
Legionella pneumophila (taxid:446) ? ?
Mycobacterium tuberculosis (taxid:1773) ? ?
Streptococcus sp. (taxid:1301) ? ?
Candida sp. (taxid:1535326) ? ?
E. coli (taxid:562) ? ?
Pseudomonas aeruginosa (taxid:287) ? ?
Staphylococcus sp. (taxid:1279) ? ?
HSV 1 and 2 ? ?
Neisseria gonorrhoeae ? ?
“?”: cross reactivity checked in-silico as well as wet lab testing
Precision
(Intra-Assay Variability (Repeatability), Inter-Assay Variability (Reproducibility), and Inter-Lot Variability, and Inter Operator Variability.
Experiments were performed to establish the precision performance of PathoDetect™ HPV High & Low Risk Detection kit for repeatability and reproducibility on real time PCR system.
Repeatability and Reproducibility data were obtained by the analysis of known controls using PathoDetect™ HPV High & Low Risk Detection kit. Test was performed in three replicates each in five independent runs, between three different operators and using three different lots. The results are within permissible limit of =20% Coefficient of Variation.
Performance on clinical samples
The diagnostic evaluation was performed by testing 25 HPV known positive and 50 known negative clinical samples. Results were 100% concordant with the reference results of Hybrid Capture technology and Nucleic acid amplification technology.
Further, the present kit provides an advantage of automation. As known to a skilled person, automated systems have been increasingly utilized to minimize the risk of human errors such as failure to follow the exact protocol, measuring mishaps, laboratorial accidents, contamination of materials due to over-handling, and miscalculations of data. Risk of human error is the combination of the likelihood of occurrence of error and the severity of error, and could be disastrous, especially in a routine analytical process such as extraction of nucleic acids that may form the basis of health diagnosis and prognosis discussed hereinabove. In addition to the advantages enlisted herein, an automated system highlights numerous advantages over semi-automated and manual systems such as higher production rates and increased productivity, efficient use of raw materials, better product quality, assurance of high reproducibility, improved safety, and established protocol. It also encourages versatility and reliability to procure better results.
The components of the present method and kit are either in liquid form, or in lyophilized form, as a whole or in parts.
A tracking system helps in communicating the screening results. As described in the above paragraphs, said system comprises: Module/program for collecting user details; Module/program for automatically inputting user’s HPV screening result; Module/program for linking the user details and the user’s HPV screening result to the specific user; and Module/program for communicating HPV screening result to said user.
The HPV screening result is at least one of positive result or negative result.
If the HPV screening result is a positive result, directs the user “to treatment”.
If the HPV screening result is a negative result, directs the user “to vaccinate”.
The system further tracks the treatment of the user if the user is positive.
It also further tracks the vaccination of the user if the user is negative.
Currently, three HPV vaccines 9-valent HPV vaccine (Gardasil 9, 9vHPV), quadrivalent HPV vaccine (Gardasil, 4vHPV), and bivalent HPV vaccine (Cervarix, 2vHPV)—have been licensed by the U.S. Food and Drug Administration (FDA). To be eligible to vaccinate, HPV screening forms a pre-requisite.
Due to its feasibility, the subject matter disclosed in the present disclosure helps in encouraging vaccination within the community.
The embodiments, examples and alternatives of the preceding paragraphs or the description and drawings, including any of their various aspects or respective individual features, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible.
The preferred embodiments of the present invention are described in detail above. It should be understood that ordinary technologies in the field can make many modifications and changes according to the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should fall within the protection scope determined by the claims.

, Claims:WE CLAIM:
1. An in-vitro method for screening one or more HPV subtypes from a user’s test sample; characterized in that, said method comprises:
collecting the test sample using a self-sampling device;
transporting the collected test sample using a transport medium;
carrying out simultaneous detection of one or more HPV subtypes from the collected test sample using real time PCR; and
using a tracking system for indicating/communicating HPV screening results to the users.
2. The method as claimed in claim 1, wherein the simultaneous detection of one or more HPV subtypes from the collected test sample is carried out by: extracting HPV DNA from the collected test sample using extraction compositions; and
contacting the extracted HPV DNA with one or more detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in a non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes using real time PCR.
3. The method as claimed in claim 2, wherein the detection compositions enable simultaneous detection of one or more HPV subtypes using up to two reaction tubes.
4. The method as claimed in claim 2, wherein one or more of the detection compositions comprises primer and probe sequences selected from SEQ ID NO: 1 to SEQ ID NO: 58.
5. The method as claimed in claim 1, wherein the collected test sample volume is selected ?500 µL.
6. The method as claimed in claim 2, wherein the extraction of HPV DNA is carried out using at least one of paramagnetic particles-based extraction or POC (Point of care) extraction.
7. The method in claim 2, wherein the detection compositions are lyophilized, as a whole or in part.
8. An in-vitro kit to screen one or more HPV subtypes from a user’s test sample; characterized in that, said kit comprises:
self-sampling device for collecting the test sample;
transport medium for transporting the collected test sample;
real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample; and
tracking system for communicating HPV screening results to the user.
9. The kit as claimed in claim 8, wherein the collected test sample volume is selected ?500 µL.
10. The kit as claimed in claim 8, wherein real time PCR-based simultaneous detection of one or more HPV subtypes from the collected test sample is carried out using:
Extraction compositions for extracting HPV DNA from the collected test sample; and
one or more Detection compositions comprising primer and probe sequences that hybridize with target sequences in HPV DNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection of one or more HPV subtypes.
11. The kit as claimed in claim 8, wherein the Detection compositions enable simultaneous detection of one or more HPV subtypes using up to two reaction tubes.
12. The kit as claimed in claim 8, wherein, the HPV subtypes are at least one of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV6, HPV11, and HPV42.
13. The kit as claimed in claim 10, wherein one or more Detection compositions enable simultaneous discrimination of HPV16 and HPV18 in one reaction tube.
14. The kit as claimed in claim 10, wherein one or more Detection compositions enable simultaneous detection and discrimination of HPV6, HPV11, and HPV42 in one reaction tube.
15. The kit as claimed in claim 10, wherein one or more Detection compositions comprise:
Detection composition A comprising primer and probe sequences for HPV16, HPV35, HPV31, HPV33, HPV58, and HPV52;
Detection composition B comprising primer and probe sequences for HPV 18, HPV 45, and HPV51;
Detection composition C comprising primer and probe sequences for HPV 68, HPV39, and HPV59;
Detection composition D and E comprising primer and probe sequences for HPV18;
Detection composition F and G comprising primer and probe sequences for HPV16;
Detection composition H comprising primer and probe sequences for HPV52, and HPV53;
Detection composition I comprising primer and probe sequences for HPV 16, HPV 31, and HPV 35;
Detection composition J comprising primer and probe sequences for HPV 51, and HPV 56;
Detection composition K comprising primer and probe sequences for HPV 33, and HPV 58;
Detection composition L comprising primer and probe sequences for HPV 6;
Detection composition M comprising primer and probe sequences for HPV 11;
Detection composition N comprising primer and probe sequences for HPV 42;
Detection composition O comprising primer and probe sequences for HPV 52;
Detection composition P comprising primer and probe sequences for HPV 58 and 33;
Detection composition Q comprising primer and probe sequences for HPV 31;
Detection composition R comprising primer and probe sequences for HPV 45;
wherein, said primer and probe sequences are specific for one or more HPV subtypes;
such that, said primer and probe sequences hybridize with target sequences in HPV DNA in a non-overlapping and non-cross-reactive manner to simultaneously detect one or more HPV subtypes using real time PCR.
16. The tracking system as claimed in claim 1 and 8, wherein said system comprises:
Module/program for collecting user details;
Module/program for automatically inputting user’s HPV screening result;
Module/program for linking the user details and the user’s HPV screening result to the specific user; and
Module/program for communicating HPV screening result to said user.
17. The tracking system as claimed in claim 16, wherein said HPV screening result is at least one of positive result or negative result.
18. The tracking system as claimed in claim 17, wherein said system directs the user “to treatment” if the HPV screening result is a positive result.
19. The tracking system as claimed in claim 17, wherein said system directs the user “to vaccinate” if the HPV screening result is a negative result.
Dated this 29th Day of September 2022
Priyank Gupta
Agent for the Applicant
IN/PA-1454