Claims:WE CLAIM:
1. A multiplex assay method for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said method comprising:
extracting viral nucleic acids from the test sample; and
running real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids using multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
2. The method as claimed in claim 1, wherein the extraction of the viral nucleic acids is carried out using paramagnetic particles.
3. The method as claimed in claim 1, wherein the extraction of the viral nucleic acids is carried out using lysis, wash, and elution compositions in a single container, wherein
the lysis composition performs sample lysis and binding of the viral nucleic acids to the paramagnetic particles;
the wash composition performs washing of the viral nucleic acids bound paramagnetic particles; and
the elution composition enables elution of the viral nucleic acids bound to paramagnetic particles.
4. The method as claimed in claim 1, wherein the test sample volume is up to 1ml.
5. The method as claimed in claim 1, wherein the use of the compositions as a whole or in part is automated.
6. The method as claimed in claim 1, wherein the compositions as a whole or in part are lyophilized.
7. The method as claimed in claim 3, wherein the lysis composition comprises lysis buffer and lysis enhancer buffer.
8. The method as claimed in claim 1, wherein the oligonucleotide sequences comprise primer and probe oligonucleotide sequences selected from SEQ ID NO. 1 to SEQ ID NO. 32 specific for HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, and internal control (IC) or combinations thereof.
9. The method as claimed in claim 8, wherein the oligonucleotide sequences for HIV-1 M/N amplification, detection, and discrimination are selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, and SEQ ID NO. 13, wherein
forward primers for HIV-1 M/N are selected from SEQ ID Nos. 1,3,5, or 7;
reverse primers for HIV-1 M/N are selected from SEQ ID Nos. 2,4,6, or 8;
hydrolysis probes for HIV-1 M/N are selected from SEQ ID Nos. 11 or 13;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within 5’-LTR of HIV-1 M/N, wherein, the said target sequence exhibits homogeneity among HIV-1 M and HIV-1 N groups.
10. The method as claimed in claim 8, wherein the oligonucleotide sequences for HIV-1 O amplification, detection, and discrimination are selected from SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 12, and SEQ ID NO. 13, wherein
forward primers for HIV-1 O are selected from SEQ ID Nos. 3, 5, 7, or 9;
reverse primers for HIV-1 O are selected from SEQ ID Nos. 4,6,8, or 10;
hydrolysis probes for HIV-1 O are selected from SEQ ID Nos. 12 or 13;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequences present within 5’-LTR of HIV-1 O.
11. The method as claimed in claim 8, wherein the oligonucleotide sequences for HIV-2 amplification, detection, and discrimination are selected from SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, ID NO. 18, and SEQ ID NO. 19, wherein
forward primers for HIV-2 are selected from SEQ ID Nos. 14 or 16;
reverse primers for HIV-2 are selected from SEQ ID Nos. 15 or 17;
hydrolysis probes for HIV-2 are selected from SEQ ID Nos. 18 or 19;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within gag region of HIV-2.
12. The method as claimed in claim 8, wherein the oligonucleotide sequences for HBV amplification, detection, and discrimination are selected from SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 31, wherein
forward primers for HBV are selected from SEQ ID Nos. 26 or 28;
reverse primers for HBV are selected from SEQ ID Nos. 27 or 29;
hydrolysis probes for HBV are selected from SEQ ID Nos. 30 or 31;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within S region of HBV, wherein, the said target sequence exhibits homogeneity among HBV genotypes A-H.
13. The method as claimed in claim 8, wherein the oligonucleotide sequences for HCV amplification, detection, and discrimination are selected from SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, and SEQ ID NO. 25, wherein
forward primers for HCV are selected from SEQ ID Nos. 20 or 22;
reverse primers for HCV are selected from SEQ ID Nos. 21 or 23;
hydrolysis probes for HCV are selected from SEQ ID Nos. 24 or 25;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within 5’-UTR of HCV, wherein, the said target sequence exhibits homogeneity among HCV genotypes 1-6.
14. The method as claimed in claim 8, wherein the oligonucleotide sequences for IC amplification, detection, and discrimination are selected from SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 32, wherein
SEQ ID No. 16 is a forward primer for IC;
SEQ ID No. 17 is a reverse primer for IC;
SEQ ID No. 32 is a hydrolysis probe;
wherein, the said hydrolysis probe is labelled with detectable reporter groups and hybridizes with IC.
15. The method as claimed in claim 8, wherein the internal control (IC) is exogenously added to the test sample prior to the extraction of viral nucleic acids.
16. The method as claimed in claim 1, wherein the real time RT-PCR is carried out using PCR buffer components, thermostable Reverse Transcriptase enzyme (RT), and Taq Polymerase, and wherein the Hot Start Fast Taq polymerase and thermostable RT enzyme complete real time RT-PCR run within the time range of 65 to 70 minutes.
17. A multiplex assay kit for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said kit comprising;
Compositions for sample lysis, wash, and elution to extract viral nucleic acids from the test sample; and
Multiplex detection composition for real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids; said multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
18. The kit as claimed in claim 16, wherein the extraction of the viral nucleic acids is carried out using paramagnetic particles.
19. The kit as claimed in claim 16, wherein the test sample volume is up to 1ml.
20. The kit as claimed in claim 16, wherein the oligonucleotide sequences comprise primer and probe oligonucleotide sequences selected from SEQ ID NO. 1 to SEQ ID NO. 32 wherein, the primer and probe oligonucleotide sequences are specific for HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, and internal control (IC) or combinations thereof.
21. The kit as claimed in claim 16, wherein the multiplex detection composition comprises;
forward primer with SEQ ID NO. 1; reverse primer with SEQ ID NO. 2; probe with SEQ ID NO. 11; for HIV-1 M/N wherein probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 9; reverse primer with SEQ ID NO. 10; probe with SEQ ID NO. 12; for HIV-1 O wherein probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 14 for HIV-2; reverse primer with SEQ ID NO. 15; probe with SEQ ID NO. 19; for HIV-2 wherein, probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 26; reverse primer with SEQ ID NO. 27; probe with SEQ ID NO. 30; for HBV wherein, probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 20; reverse primer with SEQ ID NO. 21; probe with SEQ ID NO. 24; for HCV wherein, probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 16; reverse primer with SEQ ID NO. 17; probe with SEQ ID NO. 32; for IC wherein, probe is labelled with detectable fluorophore;
such that the said primers and probes hybridize with target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
22. The kit as claimed in claim 16, wherein an internal control (IC) is exogenously added to the test sample prior to the extraction of viral nucleic acids.
23. The kit as claimed in claim 16, wherein the real time RT-PCR is carried out using PCR buffer components, thermostable Reverse Transcriptase enzyme (RT), and Taq Polymerase, wherein the Hot Start Fast Taq polymerase and thermostable RT enzyme complete real time RT-PCR run within the time range of 65 to 70 minutes.
24. The kit as claimed in claim 16, wherein the use of compositions of the kit, as a whole or in part is automated.
25. The kit as claimed in claim 16, wherein the compositions of the kit, as a whole or in part are lyophilized.
Dated this 28th Day of January 2022

PRIYANK GUPTA
Agent for the Applicant
IN/PA-1454 , Description:CROSS-REFERENCE TO RELATED APPLICATIONS
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 4,968 Byte ASCII (Text) file named "36071WOlORD_ST25.txt," created on January 28, 2022.
TECHNICAL FIELD
The present disclosure relates to the field of viral diagnosis. Described subject matter herein, in general, relates to a method for screening a plurality of viruses in a sample. More specifically, it relates to a method and a composition as well as a kit thereof for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-O, HIV-2, HBV, and HCV.
BACKGROUND
Blood and its components are transfused globally, on an everyday basis to save innumerable lives and improve health. The need for blood transfusion may arrive at any time in both rural as well as urban areas. An adequate and reliable supply of blood forms a key component of an effective health system. However, with every unit of blood, there is a 1% chance of acquiring transfusion-associated infections (TTIs), that form a major concern in the society. Hepatitis B virus (HBV), Hepatitis C virus (HCV) and Human Immunodeficiency virus (HIV) are the most prevalent blood-borne viruses that are transmitted primarily through blood transfusions. The 2021 WHO report suggests that approximately 296 million people are infected with HBV and 58 million people are infected with HCV, with 1.5 million new infections being recorded each year. On the other hand, Human Immunodeficiency virus (HIV) continues to be a major global public health issue, as declared by WHO in 2021, having claimed approximately 36.3 million lives so far.
Over the period, these viruses have undergone rapid mutations to achieve phylogenetically different groups, subtypes/types, etc. that are generally associated with the geographic distribution, ethnicity, and possible clinical outcomes. In addition, due to an increase in the feasibility for people to undertake geographical transitions, these viruses are no longer geographically restricted and have developed abilities to not only mutate into different groups/types/subtypes, cause cross-mutations, but also trigger coinfections within communities, globally.
Safety against these major transfusion transmitted viruses continues to be a worldwide concern despite the development of highly sensitive and specific serological tests and other surrogate markers, because of the time gap between the time of acquiring infection and development of seropositivity (window period). Any serological viral test undertaken during the window period (WP) leads to a false negative result because the antibodies are normally generated post this period. Thus, delaying the detection and posing a significant risk to the society.
To address this, nucleic acid test (NAT) was developed in the 1990s (Nucleic acid amplification testing in Indian blood banks: A review with perspectives, Ghosh K et al., Indian J Pathol Microbiol 2017: 60:313-8). Wherein, the nucleic acid sequences present in the pathogens, instead of the antibodies are targeted allowing earlier detection of infection and decreasing the dissemination of TTIs within the community. NAT is a highly sensitive and advanced technique which can reduce the WP of HBV TO 10.34 days, HCV to 1.34 days, and HIV to 2.93 days. (Datta S, Khillan K, Ranjan V, Wattal C. Nucleic acid amplification test: Bridging the gap in blood safety & re-evaluation of blood screening for cryptic transfusion-transmitted infection among Indian donors. Indian J Med Res. 2019 Mar;149(3):389-395).
In India, a country with a population of around 1.39 billion, TTIs form a crucial challenge since at present, all the blood banks follow the National Aids Control Organization guidelines that still emphasize the use of fourth generation, advanced antigen-antibody combined enzyme-linked immunosorbent test (ELISA) to screen blood for HIV, HBV, and HCV. NAT donors’ screening, which is effectually capable of truncating the prevalence of TTIs, is not asserted to be mandatory in hospitals and blood banks as of now in India. A review published in 2017 stated that NAT screening is carried out only in 2% blood banks and covers only 7% of all collected blood units in India, despite the substantial seroprevalence of TTIs in India, which is considerably higher than developed countries. (Nucleic acid amplification testing in Indian blood banks: A review with perspectives, Ghosh K et al., Indian J Pathol Microbiol 2017: 60:313-8)
Further, despite the advantages and the promises supplied by NAT, the implementation of these tests in low-resource countries remains challenging. While the World Health Organization (WHO) recommends diagnostic devices to be affordable, sensitive, and specific, in resource-limited countries, NAT remains technically demanding, incurs high costs, and requires dedicated infrastructure facility, equipment, consumables, and technical expertise.
Due to the challenges described herein above, there lingers a constant requirement to devise an economically feasible multiplex NAT, that would not only offer statistically significant advantage over the conventionally practiced serological tests for screening blood but also help in simultaneously detecting all the TTIs (HIV-1, HIV-2, HBV, and HCV, along with their groups/subtypes) in a test sample of pre-operational patients and persons partaking in blood donations within health institutions, globally.
SUMMARY
The instant disclosure relates to a multiplex assay method for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said method comprising; extracting viral nucleic acids from the test sample; and running real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids using multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
The present disclosure is also directed to a multiplex assay kit for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said kit comprising; Compositions for sample lysis, wash, and elution to extract viral nucleic acids from the test sample; and Multiplex detection composition for real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids; said multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Reference throughout the specification to “various embodiments,” “some embodiments,” “one 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 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 method, composition, and kit are described, it is to be understood that this disclosure is not limited to the particular method, composition, and kit 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.
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 pooled samples.
Also, 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 a multiplex assay method for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said method comprising; extracting viral nucleic acids from the test sample; and running real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids using multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
The present disclosure further relates to real time RT-PCR based simultaneous amplification using multiplex detection composition for the amplification, detection, and discrimination of the extracted viral nucleic acids. The exemplary embodiment pertains to a multiplex detection composition comprising primer and probe oligonucleotide sequences selected from SEQ ID NO. 1 to SEQ ID NO. 32 wherein, the primer and probe oligonucleotide sequences are specific for HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, and internal control (IC) or combinations thereof.
The present disclosure further relates to a multiplex assay kit for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said kit comprising; Compositions for sample lysis, wash, and elution to extract viral nucleic acids from the test sample; and Multiplex detection composition for real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids; said multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
First aspect of the present disclosure provides a multiplex assay method for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said method comprising, extracting viral nucleic acids from the test sample. Extraction of nucleic acids is a pivotal step in molecular biology, being routinely used in multiple areas of the biological and medical sciences, as this process forms the starting point in any molecular diagnosis such as nucleic acid testing (NAT).
Extraction of viral nucleic acids may be carried out using silica 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; also upon applying of a magnetic field to the sides of the vessel containing a mixture of the sample and such paramagnetic particles, they aggregate without denaturing or damaging the sample or its components. Moreover, lately, extraction methods using paramagnetic particles have become a popular approach due to their high potential for automation.
It is known to a skilled person, that automated systems are being 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. Such risks could be disastrous, especially in routine analytical processes such as extraction of nucleic acids as these may form the basis of health diagnosis and prognosis discussed hereinabove. Additionally, automated extraction methods pose numerous advantages over manual methods such as increased productivity and sample turnover, efficient use of raw materials, standardized protocols assuring high result reproducibility and improved safety. Automations also add versatility and reliability to the processes. In a preferred embodiment, the extraction of the viral nucleic acids is carried out using paramagnetic particles due to its above advantages as well as its potential for automation and semi-automation. Thus, in a related embodiment, the use of components of the kit, in whole or in part is automated.
It is also known to a skilled person that nucleic extraction methods using paramagnetic particles conventionally apply suitable buffers for carrying out lysis, wash, and elution steps depending on the type of extraction process to be devised.
In one embodiment, the extraction of the viral nucleic acids is carried out using lysis, wash, and elution compositions in a single container; wherein,
the lysis composition performs sample lysis and binding of the viral nucleic acids to the paramagnetic particles;
the wash composition performs washing of the viral nucleic acids bound paramagnetic particles; and
the elution composition enables elution of the viral nucleic acids bound to paramagnetic particles.
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 viral 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.
In a related embodiment, the lysis composition comprises lysis buffer and lysis enhancer buffer.
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, Tween 20; preferably sodium dodecyl sulphate.
Additionally, lysis buffer may comprise chaotropic salt selected from guanidinium thiocyanate, guanidine hydrochloride, guanidine isothiocyanate, cyanoguanidine isosulphate. In a preferred embodiment, the lysis buffer 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 ethylene diamine tetraacetic 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.
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.
Further, 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 comprise paramagnetic particles selected from magnetic beads, paramagnetic nanoparticles, microparticles that may be uncoated or coated with polymers, silica, or hydroxyapatite with terminal functionality.
Additionally, the lysis composition also comprises carrier RNA in one of the embodiments.
In another embodiment, the lysis composition may comprise isopropanol.
Sample lysis is carried out at a temperature ranging from 20°C to 70°C, preferably from 50°C to 60°C for up to 25 minutes at a pH ranging from 7.0 to 14, preferably from pH 7.0 to 10.
In another embodiment, lysed sample and bound viral nucleic acids to paramagnetic particles are washed with at least two washes using wash composition comprising buffer and alcohol in the same container. One wash preferably comprises at least 60% of isopropanol and the other wash comprises at least 80% of ethanol. Furthermore, the wash composition may comprise sodium salt selected from sodium citrate, sodium acetate, sodium chloride; preferably sodium acetate.
In yet another embodiment, washed lysed sample and viral nucleic acids bound to paramagnetic particles are eluted in the same container using elution composition.
Elution composition may be selected from water, buffer, polymeric alcohol, detergent, enzyme and/or salt or combinations thereof. In a preferred embodiment, elution composition comprises 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.
In the present disclosure, the test sample may be any sample that is suspected of carrying target sequences such as, but not limited to, K2EDTA blood, K2EDTA plasma, nasal swab, sputum, pharyngeal, dried blood, urine, and serum sample.
In one embodiment, the test sample volume is up to 1ml. 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. As known to a skilled person, a large sample size has obvious 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.
Subsequent to the extraction of viral nucleic acids, target sequences are amplified using engineered oligonucleotide sequences specific to HIV-1 M/N, HIV-O, HIV-2, HBV, and HCV; wherein, the said oligonucleotides refer to a polymeric form of nucleotides of any length, either a ribonucleotide (RNA) or a deoxyribonucleotide (DNA). These may be primer (forward and reverse) or probe oligonucleotides; wherein, the primers refer to nucleic acid sequence capable of acting as a point of initiation of DNA or RNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, and the probes refer to a nucleic acid of a unique sequence capable of hybridizing to a correctly amplified fragment. The term “hybridization” used herein indicates refers the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing.
Second aspect of the disclosure relates to a multiplex assay method comprising running real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids using a multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample. For the purpose of the instant disclosure, it is clarified that in an RT-PCR, target sequence pertains to specific regions of viral nucleic acids, which is DNA for DNA viruses or cDNA for RNA viruses. For the purpose of the current disclosure, the said oligonucleotide primer and probe sequences are specifically engineered for the target sequences pertaining to conserved regions of HIV-1 M/N, HIV-1 O, HIV-2, HBV, and HCV; wherein, the said target sequences may be amplified, detected, or otherwise analysed.
In an embodiment, the oligonucleotide sequences comprise primer and probe oligonucleotide sequences selected from SEQ ID NO. 1 to SEQ ID NO. 32 specific for HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, and internal control (IC) or combinations thereof.
Furthermore, in some embodiments of the present disclosure, any one or combinations of oligonucleotides 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.
Preferably, the probe oligonucleotide sequences described herein may be, but are not limited to hydrolysis probes or dual hybridization probes. More preferably, the probe oligonucleotide sequences in the present disclosure are hydrolysis probes. In some embodiments of the present disclosure, the probes are labelled with different detectable fluorescent reporter groups at their 5 'ends and fluorescent quencher groups at their 3' ends.
Optionally, in some embodiments, the detectable fluorescent reporter groups are selected from a group consisting of FAM™, HEX™, ROX™, JOE™, CY3®, VIC®, TET™, Texas Red®, NED™, Alexa®, TAMRA™, CY5.5® and CY5®. Further, the fluorescence quencher group may be selected from a group consisting of 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 actual needs, and whatever fluorescence reporters and quenchers are selected, they are within the scope of the present disclosure.
In the present disclosure, the oligonucleotides primer and probe sequences specifically engineered for HIV, HBV, HCV, and subtypes thereof, hybridize with the target sequences without cross-reacting or overlapping with any other nucleic acids present in the test sample. The non-cross reactive and non-overlapping nature helps in specifically amplifying the viral 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.
In an embodiment, the oligonucleotide sequences for HIV-1 M/N amplification, detection, and discrimination are selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, and SEQ ID NO. 13, wherein
forward primers for HIV-1 M/N are selected from SEQ ID Nos. 1,3,5, or 7;
reverse primers for HIV-1 M/N are selected from SEQ ID Nos. 2,4,6, or 8;
hydrolysis probes for HIV-1 M/N are selected from SEQ ID Nos. 11 or 13;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within 5’-LTR of HIV-1 M/N, wherein, the said target sequence exhibits homogeneity among HIV-1 M and HIV-1 N groups.
In another embodiment, the oligonucleotide sequences for HIV-1 O amplification, detection, and discrimination are selected from SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 12, and SEQ ID NO. 13. wherein
forward primers for HIV-1 O are selected from SEQ ID Nos. 3, 5, 7, or 9;
reverse primers for HIV-1 O are selected from SEQ ID Nos. 4,6,8, or 10;
hydrolysis probes for HIV-1 O are selected from SEQ ID Nos. 12 or 13;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequences present within 5’-LTR of HIV-1 O.
In an additional embodiment, the oligonucleotide sequences for HIV-2 amplification, detection, and discrimination are selected from SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, ID NO. 18, and SEQ ID NO. 19, wherein
forward primers for HIV-2 are selected from SEQ ID Nos. 14 or 16;
reverse primers for HIV-2 are selected from SEQ ID Nos. 15 or 17;
hydrolysis probes for HIV-2 are selected from SEQ ID Nos. 18 or 19;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within gag region of HIV-2.
Yet another embodiment discloses the oligonucleotide sequences for HBV amplification, detection, and discrimination are selected from SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 31, wherein
forward primers for HBV are selected from SEQ ID Nos. 26 or 28;
reverse primers for HBV are selected from SEQ ID Nos. 27 or 29;
hydrolysis probes for HBV are selected from SEQ ID Nos. 30 or 31;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within S region of HBV, wherein, the said target sequence exhibits homogeneity among HBV genotypes A-H.
Further embodiment relates to the oligonucleotide sequences for HCV amplification, detection, and discrimination are selected from SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, and SEQ ID NO. 25, wherein
forward primers for HCV are selected from SEQ ID Nos. 20 or 22;
reverse primers for HCV are selected from SEQ ID Nos. 21 or 23;
hydrolysis probes for HCV are selected from SEQ ID Nos. 24 or 25;
wherein, the hydrolysis probes are labelled with detectable reporter groups and hybridize with target sequence present within 5’-UTR of HCV, wherein, the said target sequence exhibits homogeneity among HCV genotypes 1-6.
Furthermore, an internal control (IC) containing synthetic fragment may 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 sequence specific to the viral nucleic acids that may be selected from a list comprising ICs such as buffer suspended IC and armored IC. Preferably IC is a synthetic sequence that is suspended in a stabilization buffer.
In a related embodiment, the internal control (IC) is exogenously added to the test sample prior to the extraction of viral nucleic acids.
In a further embodiment, the oligonucleotide sequences for IC amplification, detection, and discrimination are selected from SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 32, wherein,
SEQ ID No. 16 is a forward primer for IC;
SEQ ID No. 17 is a reverse primer for IC;
SEQ ID No. 32 is a hydrolysis probe;
wherein, the said hydrolysis probe is labelled with detectable reporter groups and hybridizes with IC.
Third aspect of the disclosure relates to a multiplex assay method for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample; the said method comprising running real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids using multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV,HCV, in the test sample.
In one embodiment, the real time RT-PCR is carried out using PCR buffer components, thermostable Reverse Transcriptase enzyme (RT), and Taq Polymerase.
In a related embodiment, the real time RT-PCR is carried out using requisite PCR Mix comprising PCR enzymes comprising reverse transcriptases selected from at least one of M-MLV reverse transcriptase or AMV reverse transcriptase and DNA polymerases selected from Taq, Fast Taq, Hot Start Fast Taq, Tfl, Pfu, or Tth DNA polymerase; preferably Hot Start Fast Taq polymerase and a thermostable RT enzyme such that the Hot Start Fast Taq polymerase and thermostable RT enzyme complete real time RT-PCR run within 60 to 80 minutes; preferably within the time range of 65 to 70 minutes.
Alternatively, the compositions described hereinabove, as a whole or in part are lyophilized.
In one embodiment, the use of the compositions described hereinabove, as a whole or in part is automated.
The present disclosure also discloses a multiplex assay kit for simultaneously detecting and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample, the said kit comprising;
Compositions for sample lysis, wash, and elution to extract viral nucleic acids from the test sample; and
Multiplex detection composition for real time RT-PCR based simultaneous amplification of target sequences within the extracted viral nucleic acids; said multiplex detection composition comprising oligonucleotide sequences that hybridize with the target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, from the test sample.
In the present disclosure, the term “kit” refers to a consumable or cartridge comprising a set of articles, components, cartridges, or compositions in a packet required for a specific purpose.
In one embodiment, for ease of automation, the extraction of the viral nucleic acids is carried out using paramagnetic particles.
In a related embodiment, extraction of viral nucleic acids is carried out using lysis, wash, and elution compositions in a single container as described in earlier embodiments. For purpose of present disclosure, a container is any vessel capable of holding the components or compositions of the kit for carrying out the present disclosure.
In a preferred embodiment, the test sample is any sample suspected of carrying target sequences as described in earlier embodiments.
In a related embodiment, the test sample volume is up to 1ml.
The present disclosure further discloses lysis, wash, and elution compositions for carrying out the extraction of viral nucleic acids.
In a related embodiment, the lysis composition comprises lysis buffer such as Tris HCl and lysis enhancer buffer comprising lysis enzyme such as proteinase K stabilized with calcium chloride and glycerol, chaotropic salt such as guanidinium thiocyanate, and may further comprise SDS, DTT, sodium azide to which may be added paramagnetic particles, carrier RNA, and isopropanol to bring about sample lysis and binding of nucleic acids to paramagnetic particles as elaborated in earlier embodiments.
In a further embodiment, lysed sample and viral nucleic acids bound to paramagnetic particles is washed with at least two washes of a wash composition comprising buffer and alcohol, preferably one wash comprising at least 60% of isopropanol and the other wash comprising at least 80% of ethanol. Further, salts such as sodium acetate and chaotropic agents such as guanidinium thiocyanate may be preferably added to the wash composition.
Washed viral nucleic acids bound to paramagnetic particles are eluted with elution composition preferably comprising nuclease free water. The eluted viral nucleic acids are transferred to PCR tube for running real time RT-PCR, to which may be added multiplex detection mix and PCR mix.
In a related embodiment, the oligonucleotide sequences comprise primer and probe oligonucleotide sequences selected from SEQ ID NO. 1 to SEQ ID NO. 32 wherein, the primer and probe oligonucleotide sequences are specific for HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, and internal control (IC) or combinations thereof.
In a preferred embodiment, the kit comprises oligonucleotide sequences that work in non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, and HCV, in a test sample.
In an exemplary embodiment of the kit, the multiplex detection composition comprises;
forward primer with SEQ ID NO. 1; reverse primer with SEQ ID NO. 2; probe with SEQ ID NO. 11; for HIV-1 M/N wherein probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 9; reverse primer with SEQ ID NO. 10; probe with SEQ ID NO. 12; for HIV-1 O wherein probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 14 for HIV-2; reverse primer with SEQ ID NO. 15; probe with SEQ ID NO. 19; for HIV-2 wherein, probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 26; reverse primer with SEQ ID NO. 27; probe with SEQ ID NO. 30; for HBV wherein, probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 20; reverse primer with SEQ ID NO. 21; probe with SEQ ID NO. 24; for HCV wherein, probe is labelled with detectable fluorophore;
forward primer with SEQ ID NO. 16; reverse primer with SEQ ID NO. 17; probe with SEQ ID NO. 32; for IC wherein, probe is labelled with detectable fluorophore;
such that the said primers and probes hybridize with target sequences in a non-cross reactive and non-overlapping manner to simultaneously amplify, detect, and discriminate one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in the test sample.
In a related embodiment, an internal control (IC) is exogenously added to the test sample prior to the extraction of viral nucleic acids
In one embodiment, the real time RT-PCR is carried out using PCR buffer components, thermostable Reverse Transcriptase enzyme (RT), and Taq Polymerase.
In a preferred embodiment, real time RT-PCR is carried out using PCR Mix comprising, a thermostable Reverse Transcriptase (RT), and Taq polymerase, preferably Hot Start Fast Taq polymerase and a thermostable RT enzyme, such that the Hot Start Fast Taq polymerase and thermostable RT enzyme complete real time RT-PCR run within the time range of 65 to 70 minutes.
Alternatively, the compositions of the kit described hereinabove, as a whole or in part are lyophilized. 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 one embodiment, the use of compositions of the kit described hereinabove, 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 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 viral 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.
In an embodiment, the kit of the present disclosure may be coupled with any PCR machine having at least five 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.
Example 1
Extraction of viral nucleic acid using paramagnetic particles
In this example, viral nucleic acids were extracted from up to 1ml of K2EDTA plasma sample or serum sample obtained from up to 5ml of whole blood using conventional methods known to a skilled person in the art.
A negative extraction control was used as a test control.
The lysis buffer was prepared in a sample tube by mixing the reagents shown in Table 2. The lysis enhancer buffer 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 using K2EDTA plasma sample was carried out by mixing the sample with the lysis/binding composition (Table 1) in the sample tube and incubating at 56°C for up to 25 minutes with continuous shaking at up to 900 rpm.
Table 1: Lysis/binding composition for 1ml sample volume
Sr. No. Reagents Volume (µl) /Rx
1 Lysis Buffer (see Table 2) 700
2 Lysis enhancer buffer 70
3 Paramagnetic particles 70
4 Internal Control (IC) 2.5
5 Carrier RNA 2.5
6 Isopropanol 1360
Total Volume 2065
Table 2: 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
Following the incubation, magnetic separation of the lysis composition in the sample tube was performed and the supernatant was discarded.
The nucleic acid 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 60% isopropanol and 80% ethanol respectively

Table 3: Composition of Wash Buffer 1
Reagent Stock Concentration pH
Tris HCl 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 4.6-5.5
Table 4: Composition of Wash Buffer 2
Reagent Stock Concentration pH
Tris HCl 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 1200 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 1800 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 nucleic acid bound paramagnetic particles were eluted using an elution composition prepared using DEPC treated autoclaved water. Thus, up to 120 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 viral nucleic acids was transferred to a new tube for further analysis, or downstream applications or stored at -20°C or -80°C until further use.
Example 2
Detection and discrimination of HIV-1 M/N, HIV-1 O, HIV-2, HBV, and HCV real time RT-PCR using multiplex detection compositions
The extracted nucleic acids from Example 1 were used for the detection and discrimination of HIV-1 M/N, HIV-1 O, HIV-2, HBV, and HCV using real time RT-PCR.
The screening was monitored using three positive controls and a no template control (NTC) or negative control.
PCR Mix was prepared using RT enzyme, Taq polymerase and buffer components.
Multiplex detection composition was prepared using primers and probes specific to HIV-1 M/N, HIV-1 O, HIV-2, HCV, HBV, and internal control.
Reaction mixture was prepared by mixing the reagents shown in Table 5.
PCR plate was set up for real time RT-PCR system using the extracted nucleic acids, positive controls and the reaction mix such that the total volume of the composition was 50µl per well.
Table 5: Composition of reaction mix
Reagent Final concentration of reagents per reaction
Upto 10X PCR Mix 1X
Nuclease free water or reconstitution buffer to compensate
Multiplex detection composition 1X
Template addition Up to 50% of total reaction volume
Total volume Up to 50µl

Table 6: Components for RT-PCR
Contents Description
PCR Mix RT enzyme, Taq polymerase and buffer components
Multiplex detection composition Primers and probes specific to HIV-1 M/N, HIV-1 O, HIV-2, HCV, HBV, and internal control
Nuclease free water RNase/DNase free water
Multiplex positive control In vitro transcript of HIV-1 M, HCV and IC, Plasmid for HBV
Positive control 1: HIV-1 O In vitro transcript of HIV-1 O target
Positive control 2: HIV-2 In vitro transcript of HIV-2 target
Internal Control (IC) Synthetic Internal control template
The PCR cycling conditions for the performed experiment are set forth in Table 7.
Table 7: PCR Run method
Parameter Stage Temperature Time
Reverse Transcription Hold 50°C Up to 15 minutes
RT inactivation Hold 95°C Up to 20 sec
PCR amplification Cycle
(=50 cycles) 95°C
60°C 05 sec
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.
Table 8: Test validation and interpretation
Control HIV target HCV target
HBV target
(ROX) Internal control Interpretation
Multiplex Positive Control Positive Positive Positive Positive Valid
HIV-1O positive control Positive UND UND UND Valid
HIV-2 positive control Positive UND UND UND Valid
Extraction control UND UND UND Positive Valid
No template control (NTC) UND UND UND UND Valid
UND: Undetermined/ No PCR amplification
The result generated as per the interpretation Table 8 indicated that the sample was HIV-1 and HIV-2 positive.
Example 3
This example demonstrates a kit for amplifying, detecting, and discriminating one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV, in a test sample in accordance with the present disclosure.
The present kit contains lysis, wash, and elution compositions for performing extraction of viral nucleic acids from a sample.
The present kit further contains multiplex detection composition for screening the extracted viral nucleic acids for one or more of HIV-1 M/N, HIV-1 O, HIV-2, HBV, HCV using real time RT-PCR.
In this example, up to 500 µl or 1ml of K2EDTA plasma sample was prepared using up to 5ml of whole blood.
.Viral nucleic acids (DNA and/or RNA) were extracted from the K2EDTA plasma sample using an automated paramagnetic particle method comprising a thermoshaker and a magnetic assembly with a base plate, a heating block, a single reaction container, a magnetic strip, a nylon plate and temperature sensors. For this, pre-filled extraction cartridge and PCR cartridge, comprising lyophilized compositions were placed on an automated platform (such as Mylab™ Compact series in this example) along with other consumables on the deck in designated place. The cartridges may comprise some or all of the compositions in a lyophilized state. Lyophilization ensures feasible storage and transport of the materials as known to a skilled person. Then, appropriate commands were given to start the pre-uploaded protocol. An automated platform such as from Mylab™ carries out the entire process from viral nucleic acid extraction to real time RT-PCR. Upon nucleic acid extraction, PCR amplification and detection were carried out and an in-built software analysed the data to generate results.
Programmable commands fed within the system help in extracting the viral nucleic acids under automated shaker and temperature conditions, facilitating minimized risk of human errors such as laboratorial accidents, contamination of materials due to over-handling, and miscalculations of data, as known to a skilled person. 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 extracted sample was then incorporated for viral screening using real time RT-PCR
The kit comprises a multiplex detection composition that was added to a reaction tube containing the PCR buffer prepared in accordance with Example 2.
The extracted sample and the positive controls were then added to the corresponding reaction tubes which were directly put in the PCR machine and the PCR cycle was performed as per Table 7.
After performing the PCR run, fluorescence data was collected during 60°C extension step.
This step was followed by saving the data captured in the data analysing software and its analysis and interpretation.
Performance Characteristics:
Analytical sensitivity
The Limit of Detection (LOD or analytical sensitivity) was determined for the present kit for HIV-1 Group M RNA, HIV-1 Group O RNA, HIV-2 RNA, HCV RNA, and HBV DNA using the following WHO NIBSC standards. For the WHO standards five independent dilution series of each viral standard were prepared with pooled human plasma collected in EDTA anticoagulant. Each dilution series was tested in triplicate. PROBIT analysis was used to estimate the 95% LOD. (Table 9)
Table: 9
Analyte Standard Units Average 95% LOD*
HIV-1 M NIBSC code: 10/152 IU/ml 17.5
HIV-1 O Mylab™ indigenous standard Copies/ml 18.5
HIV-2 NIBSC Code: 08/150 IU/ml 8.17
HCV NIBSC code: 14/150 IU/ml 7
HBV NIBSC code: 10/264 IU/ml 2
HIV-1: WHO International Standard 3rd HIV-1 International Standard NIBSC code: 10/152
HIV-2: WHO International Standard HIV-2 RNA International Standard NIBSC Code: 08/150
HCV: WHO International Standard 5th WHO International Standard for HCV NAT NIBSC code: 14/150
HBV: WHO International Standard 3rd WHO International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques NIBSC code: 10/264
Experiments were performed to establish the precision performance of the present kit for repeatability and reproducibility. Repeatability and Reproducibility data were obtained by the analysis of NIBSC standards. Test was performed in replicates in independent runs, between two different operators and using 3 different lots. The resulting data is given in Table 10.
Table: 10
HIV-1 HIV-2 HCV HBV
% CV % CV % CV % CV
Log 3.3 Log 2 Log2 Log 1.4 Log 3.3 Log 2 Log 2 Log 1.3
Intra assay=10
Inter assay N=5 1.73 2.28 1.41 1.38 1.73 2.28 1.11 1.4
1.41 3.23 1.14 0.87 1.41 3.23 1.26 1.54
Inter operator N=2 1.25 3.46 1.56 1.51 1.61 2.56 0.86 1.41
Inter Lot N=3 1.73 2.28 1.1 1.06 3.73 4.93 0.97 0.73
Analytical specificity
The specificity of the present kit was 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.
The genotype/subtype inclusivity of all relevant genotypes was ensured by checking with international reference standards.
A potential cross-reactivity of the present kit was tested using the control group comprising organisms such as Adenovirus 5, Human Herpes Virus, Candida albicans, Human Cytomegalovirus, Human T-Lymphotrophic Virus Type I, Influenza Virus A, Staphylococcus epidermis, Varicella-Zoster Virus, Staphylococcus haemolyticus, Herpes Simplex Virus Type 1, Escherichia coli, Herpes Simplex Virus Type 2, Chikungunya Virus, Streptococcus viridans, Staphylococcus aureus, Chikungunya, Dengue, and Mycobacterium tuberculosis.
Kit Comparisons:
The present kit was compared for its analytical sensitivity with the two commercially available kits, Ultrio Elite Assay and Cobas® MPX V 2.0 Assay. The % LOD used for this analysis was 95%.
Table 11 gives the comparative LOD of the NAT of the present invention.
Table 11: Comparative LOD for NAT tests
Organism Ultrio Elite Grifols Cobas® MPX Roche Present Kit
HIV-1 18 IU/ml 46.2 IU/ml 17.5 IU/ml
HIV-2 10.4 IU/ml 7.9 IU/ml 8.17 IU/ml
HBV 4.3 IU/ml 2.3 IU/ml 2.0 IU/ml
HCV 3.0 IU/ml 6.8 IU/ml 7.0 IU/ml
In accordance with the comparative data presented in Table 11, it was concluded that the kit showed comparative results with the currently practised commercially available kits.
Furthermore, the present kit could undertake simultaneous detection and discrimination of HIV-1, HIV-2, HBV, and HCV, as opposed to the simultaneous discrimination of HIV, HBV, and HCV by Cobas® MPX Roche., showcasing advancement in an ability to detect multiple viral agents in a single test sample. Also, unlike the requirement to perform dual tests for viral detection and discrimination, as practised in Ultrio Elite Grifols, the present kit could perform the detection and discrimination of plurality of viral agents in a single test, accelerating the viral screening process.
In addition, the present kit provides flexibility in the sample volume sizes, with the sample volume recommended to perform the current test sizing up to 1 ml, unlike the specific volume size of 500µl and 1 ml as recommended by Ultrio Elite Grifols and Cobas® MPX Roche respectively.
Table 12 discloses the total turnaround time taken by the present kit in comparison with the total turnaround time taken by with Ultrio Elite Grifols and Cobas® MPX Roche. Whereas Table 13 discloses the PCR run time taken by the present kit in comparison with the PCR run time taken by with Ultrio Elite Grifols and Cobas® MPX Roche. Table 12 and Table 13, in conjugation, as well as independently conclude that the kit disclosed in the present disclosure provides faster total turnaround time as well as PCR runtime compared to Ultrio Elite Grifols and Cobas® MPX Roche.
Table 12: Comparative data for turnaround time
Kit Sample to result time (total turnaround time)
Cobas® MPX Roche 5.5 hours for first 24 results and 24 results are completed every 1.5 hours thereafter.
Ultrio Elite Grifols 4-5 hours
Present Kit 2.5 hours for first 32 samples and next batches every 1 hour thereafter
Table 13: PCR run time/ test turnaround time
Kit PCR run time/ turnaround time
Present Kit ~68 minutes
Ultrio Elite Grifols 2-2.5 hours
Cobas® MPX Roche 2-2.5 hours
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 present kit also provides components or compositions in lyophilised form. 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 may ensure feasible room temperature storage, desirable stability, and unchallenging transport of the materials, ensuring that the kit showcases wider applications.
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.