Claims:WE CLAIM:
1. A method for simultaneously detecting and quantitating or discriminating one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample using real time RT-PCR, the said method comprising:
lysing the test sample using a lysis composition to obtain sample lysate;
obtaining RNA from the sample lysate; and
contacting the RNA with one or more detection compositions comprising oligonucleotide sequences;
wherein the oligonucleotide sequences comprise primers and probes that hybridize with target sequences in the HCV RNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection and quantitation or discrimination of one or more of HCV genotypes 1-6 and subtypes thereof.
2. The method as claimed in claim 1, wherein the method comprises additional steps of washing and/or eluting.
3. The detection compositions as claimed in claim 1, wherein the one or more detection compositions comprise primer sequences and probe sequences selected from SEQ ID NO: 1 to SEQ ID NO: 41;
wherein, the said sequences are specific for one or more of HCV genotypes 1-6 and subtypes thereof.
4. The method as claimed in claim 1, wherein the probes are labelled with different fluorescent reporter groups at their 5 'ends and a fluorescent quencher group at their 3' ends.
5. The method as claimed in claim 4, wherein the fluorescent reporter groups are 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.
6. The method as claimed in claim 1, wherein the method comprises quantitation of HCV; the said method further comprising one or more calibrators.
7. A kit for simultaneous detection and discrimination of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample, wherein the said kit comprises:
a lysis composition; and
one or more detection compositions comprising non-overlapping and non-cross-reactive primer sequences and probe sequences;
wherein the primer and probe sequences hybridize with heterogenous target sequences in HCV RNA to enable simultaneous detection and discrimination of one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
8. The detection compositions as claimed in claim 7, wherein the one or more detection compositions comprise;
detection composition A comprising primers and probes for HCV 1a and 3;
detection composition B comprising primers and probes for HCV 2 and 1b;
detection composition C comprising primers and probes for HCV 4;
detection composition D comprising primers and probes for HCV 6 and 5;
detection composition E comprising primers and probes for HCV 1a and 3b;
detection composition F comprising primers and probes for HCV 2, 1b, and 1; and
detection composition G comprising primers and probes for HCV 4 and 3a;
wherein, the said primers and probes hybridize in a non-overlapping and non-cross-reactive manner to simultaneously detect and discriminate one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
9. The detection compositions as claimed in claim 8, wherein the detection composition A comprises;
forward primer sequences comprising SEQ ID NO: 1 and 7;
reverse primer sequences comprising SEQ ID NO: 2 and 8; and
probe sequences comprising SEQ ID NO: 18 and 21;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 1a and HCV 3 using real time RT-PCR.
10. The detection compositions as claimed in claim 8, wherein the detection composition B comprises;
forward primer sequences comprising SEQ ID NO: 3, 5, and 15;
reverse primer sequences comprising SEQ ID NO: 4, 6, and 16; and
probe sequences comprising SEQ ID NO: 19 and 20;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 1b and HCV 2 using real time RT-PCR.
11. The detection compositions as claimed in claim 8, wherein the detection composition C comprises;
forward primer sequences comprising SEQ ID NO: 9;
reverse primer sequences comprising SEQ ID NO: 10; and
probe sequences comprising SEQ ID NO: 22;
wherein, the primers and probe hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 4 using real time RT-PCR.
12. The detection compositions as claimed in claim 8, wherein any of the detection compositions A-G further comprise primer and probe sequences for HCV common, wherein
forward primer sequences comprising SEQ ID NO: 26;
reverse primer sequences comprising SEQ ID NO: 27; and
probe sequences comprising SEQ ID NO: 25;
wherein, the primers and probe hybridize with homogenous target sequences in HCV RNA for detecting HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
13. The detection compositions as claimed in claim 8, wherein the detection composition D further comprises;
forward primer sequences comprising SEQ ID NO: 11 and 13;
reverse primer sequences comprising SEQ ID NO: 12 and 14; and
probe sequences comprising SEQ ID NO: 23 and 24;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 5 and HCV 6 using real time RT-PCR.
14. The detection composition as claimed in claim 8, wherein the detection composition E comprises;
forward primer sequences comprising SEQ ID NO: 1, 31, 37, and 40;
reverse primer sequences comprising SEQ ID NO: 2, 32, 38, and 41; and
probe sequences comprising SEQ ID NO: 18, 33, 39, and 17;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 1a and HCV 3b using real time RT-PCR.
15. The detection composition as claimed in claim 8, wherein the detection composition F comprises;
forward primer sequences comprising SEQ ID NO: 5, 3, 15, and 34;
reverse primer sequences comprising SEQ ID NO: 6, 4, 16, and 35; and
probe sequences comprising SEQ ID NO: 20, 19, and 36;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 2, HCV 1b, and HCV 1 using real time RT-PCR.
16. The detection composition as claimed in claim 8, wherein the detection composition G comprises;
forward primer sequences comprising SEQ ID NO: 9 and 28;
reverse primer sequences comprising SEQ ID NO: 10 and 29; and
probe sequences comprising SEQ ID NO: 22 and 30;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 4 and HCV 3a using real time RT-PCR.
17. A kit for the detection and quantitation of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample wherein, the said kit comprises:
a lysis composition;
one or more calibrators; and
a detection composition, wherein, the said detection composition comprises primer sequences and probe sequence claimed in claim 12 that hybridize with homologous target sequence in HCV RNA in non-cross reactive and non-overlapping manner to amplify, detect, quantitate one or more of HCV genotypes 1-6 and subtypes thereof, from the test sample.
18. The kit as claimed in claim 17, wherein the detection composition comprises primers and probes selected from SEQ ID NO: 26, 27, and 25 and wherein, the primers and probes are specific for HCV.
19. The kit as claimed in claim 17, wherein the one or more calibrators comprise in vitro RNA transcript specific for 5’ UTR of HCV RNA at a concentration ranging from 7X106 IU/mL to 7X103 IU/mL.
20. The method as claimed in claim 1, and kits as claimed in claims 7 and 17, wherein the test sample volume is up to 1mL of any sample likely to contain HCV RNA.
21. The method as claimed in claim 1, and kits as claimed in claims 7 and 17, wherein the lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, and isopropanol; and further comprises paramagnetic particles or silica support.
22. The method as claimed in claim 1, and kits as claimed in claims 7 and 17, wherein the method and kit compositions, as a whole or in part are lyophilized.
23. The method as claimed in claim 1, and kits as claimed in claims 7 and 17, wherein the use of the method and kit compositions, as a whole or in part is automated.
24. The method as claimed in claim 1, and kits as claimed in claims 7 and 17, wherein a synthetic internal control (IC) is exogenously added to the test sample prior to the lysis.
25. The method as claimed in claim 1, and kits as claimed in claims 7 and 17, 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 70 minutes.

Dated this 17th Day of February 2022

Priyank Gupta
Agent for the Applicant
IN/PA-1454
, 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 DETECTION AND QUANTITATION OR DISCRIMINATION OF HCV GENOTYPES AND KIT THEREOF

APPLICANT
MYLAB DISCOVERY SOLUTIONS PRIVATE LIMITED
An Indian entity
having address,
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 3,617 Byte ASCII (Text) file named "Seq Listing_HCV_filing," created on February 17, 2022.
TECHNICAL FIELD
The present invention relates to the field of Nucleic Acid Testing (NAT). Described subject matter herein, in general, relates to a method, composition and kit for the simultaneous detection and quantitation or discrimination of genotypes of Hepatitis C virus (HCV).
BACKGROUND
Chronic infection with Hepatitis C virus (HCV) is one of the major causes of liver cirrhosis and hepatocellular carcinoma. Globally, an estimated 58 million people are infected with HCV, adding about 1.5 million every year. According to WHO, in 2019, an estimated 2,90,000 people died of hepatitis C infection. In India, about 1% of the population (approximately 14 million people) are believed to be infected with HCV while about 2,50,000 people die of viral hepatitis or its sequelae every year.
HCV is an enveloped RNA virus belonging to family Flaviviridae. The HCV genome is a linear, single-strand, positive-sense, 9600-nucleotide RNA containing a single open reading frame (ORF) flanked by highly structured untranslated regions (UTRs), which are essential for RNA replication. The 5' UTR having 324 to 341 nucleotides encoding a polyprotein of 3,011 amino acids is the most conserved region among HCV strains, and the 3’-UTR is of about 27 bases (Beales et al., 2001). The high degree of conservation within the 5'UTR has made it an ideal region for detection of sequence-based genotypes of HCV.
While, extensive genetic heterogeneity at all levels (genotypes, subtypes, isolates/strains) is a distinctive characteristic of HCV, genotypes 1–6 still contain all the identified epidemiologically important HCV variants. Potentially endemic regions for genotypes 1, 2, 4 and 5 are mostly distributed around Africa, whereas type 3 prevails in the Northern Indian subcontinent, and type 6 within Southeast Asia.
In India, HCV 3 is predominant (~63.85%) followed by HCV 1 (25.72%). HCV 3 is more prevalent in the Northern, Eastern and Western parts of India and HCV 1 is more prevalent in the Southern India. HCV 4 has been found mostly in South Indian states of Andhra Pradesh and Tamil Nadu while HCV 6 has been found to be prevalent in North-Eastern parts of India.
The treatment of HCV has evolved from interferon (IFN)-based therapies with or without ribavirin (RBV) to pegylated-IFN (PEG-IFN) and RBV-based therapies, which are better tolerated by patients. HCV 1 is highly infectious compared to HCV 2 or 3, as seen from a comparatively much higher viral load for HCV 1 as compared to HCV 3 and 4 and responds to a-interferon (INF-a) therapy and ribavirin combination therapy with difficulty (Nolte, 2001). Several clinical trials of pegylated interferon/ribavirin therapy have revealed significant differences in response rates for various HCV genotypes. Individuals with HCV 2 and 3 are more likely to respond to therapy with alpha interferon or the combination of alpha interferon and ribavirin than individuals with HCV 1. Further, the treatment duration for chronic hepatitis C is 48 weeks for genotypes 1 and 4-24 weeks for genotypes 2,3,4,5 and 6, respectively. Thus, based on the genotypes and subtypes, there exists a huge disparity in the treatment methodology as well as duration of treatment. Knowing the viral genotype and subtype along with the viral load present in an infection helps the clinician to determine an optimal course of treatment.
Although FDA-approved serological tests have played a pivotal role in screening antigens within distinguished samples, kits employing ELISA (Enzyme linked Immuno Sorbant Assay) and RIBA (Recombinant Immuno Blot Assay), despite having adequate specificity, do not detect an active infection. Another limitation of the immunoassays is the immunological window, as the average incubation period for antibody onset is 7 to 8 weeks and may range between 2 to 26 weeks. Therefore, the most appropriate methods are those based on viral RNA detection (CDC, 2008; Consensus on Conduct of Viral Hepatitis B and C, 2005; WHO, 2000).
NAT is considered the ‘gold standard’ for detecting an active HCV replication and HCV RNA is detectable as early as 1 week after exposure via needle-stick or blood transfusion, at least 4-6 weeks prior to seroconversion as demonstrated in a number of transmission settings (Maheshwari A et al, Lancet. 2008; 372:321–32). Thus, NAT is highly recommended: (1) for confirmation of HCV RNA in cases where patients are HCV seropositive; (2) for detection and quantitation or discrimination of the genotypes to determine to appropriate course of treatment; (3) to confirm the presence of HCV viremia in patients who are seronegative but immuno-compromised such as HIV infected individuals; (4) in babies who are born to HCV positive mothers-as antibody testing in babies can give false positive results up to 18 months of age; (5) for determining the baseline value before starting the anti-viral therapy; and (6) monitoring course of disease during the treatment.
In India, HCV infection imposes a considerable burden of mortality, morbidity, and healthcare costs. Thus, there is a need in the art to develop kits, compositions and methods to distinguish and quantitate an increasingly large number of known HCV genotypes, including subtypes. Moreover, cost-effective protocols with better sensitivities at lower viral concentrations are always desirable.
The present invention discloses NAT-based methods, compositions and kits thereof for efficiently detecting and quantitating genotypes HCV genotypes 1-6 and subtypes thereof in a test sample with improved/comparable sensitivity.
SUMMARY
The present invention is directed to a method for simultaneously detecting and quantitating or discriminating one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample using real time RT-PCR, the said method comprising:
lysing the test sample using a lysis composition to obtain sample lysate; obtaining RNA from the sample lysate; and contacting the RNA with one or more detection compositions comprising oligonucleotide sequences; wherein the oligonucleotide sequences comprise primers and probes that hybridize with target sequences in the HCV RNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection and quantitation or discrimination of one or more of HCV genotypes 1-6 and subtypes thereof.
In an exemplary embodiment, the instant invention discloses a kit for simultaneous detection and discrimination of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample, wherein the said kit comprises: a lysis composition; and one or more detection compositions comprising non-overlapping and non-cross-reactive primer sequences and probe sequences; wherein the primer and probe sequences hybridize with heterogenous target sequences in HCV RNA to enable simultaneous detection and discrimination of one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
In another alternate embodiment, the present invention is directed to a kit for the detection and quantitation of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample wherein, the said kit comprises: a lysis composition; one or more calibrators; and a detection composition, wherein, the said detection composition comprises primer sequences and probe sequence for HCV common that hybridize with homologous target sequence in HCV RNA in non-cross reactive and non-overlapping manner to amplify, detect, quantitate one or more of HCV genotypes 1-6 and subtypes thereof, from the test sample.
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. 1 demonstrates PCR efficiency, R^2 and slope for quantitated HCV RNA linearity IU/mL wherein the X axis represents the expected value in IU/mL, and the Y axis represents observed value IU/mL.
Fig. 2 represents amplification plots for HCV calibrators (QS1-QS4)
Fig. 3 represents standard curve for HCV calibrators
DETAILED DESCRIPTION
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” 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.
Further, reference throughout the specification “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.
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 method for simultaneously detecting and quantitating or discriminating one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample using real time RT-PCR, the said method comprising: lysing the test sample using a lysis composition to obtain sample lysate; obtaining RNA from the sample lysate; and contacting the RNA with one or more detection compositions comprising oligonucleotide sequences; wherein the oligonucleotide sequences comprise primers and probes that hybridize with target sequences in the HCV RNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection and quantitation or discrimination of one or more of HCV genotypes 1-6 and subtypes thereof.

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 regions of HCV cDNA; wherein, the said target sequences may be amplified, detected, or otherwise analysed.

The instant disclosure further relates to a kit for simultaneous detection and discrimination of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample, wherein the said kit comprises: a lysis composition; and one or more detection compositions comprising non-overlapping and non-cross-reactive primer sequences and probe sequences; wherein the primer and probe sequences hybridize with heterogenous target sequences in HCV RNA to enable simultaneous detection and discrimination of one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.

The present disclosure also relates to a kit for the detection and quantitation of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample wherein, the said kit comprises: a lysis composition; one or more calibrators; and a detection composition, wherein, the said detection composition comprises primer sequences and probe sequence for HCV common that hybridize with homologous target sequence in HCV RNA in non-cross reactive and non-overlapping manner to amplify, detect, quantitate one or more of HCV genotypes 1-6 and subtypes thereof, from the test sample.

The first aspect of the present invention is related to a method for simultaneously detecting and quantitating or discriminating one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample using real time RT-PCR, the said method comprising: lysing the test sample using a lysis composition to obtain sample lysate; and obtaining RNA from the sample lysate.

In one embodiment, the “test sample” or “sample” is any sample suspected of containing HCV RNA such as, but is not limited to, K2EDTA blood, K2EDTA plasma, nasal swab, sputum, pharyngeal, dried blood, urine, and serum sample.

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 related embodiment the test sample volume is up to 1mL.
One embodiment comprises lysing the test sample using a lysis composition to obtain sample lysate. Depending on the type of the test sample, lysis composition components may be varied as is known to a person skilled in the art.

In an embodiment, the lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, and isopropanol.
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 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.
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.
Additionally, the lysis composition also comprises carrier RNA in one of the embodiments.
In another embodiment, the lysis composition may comprise alcohols such as ethanol or isopropanol; preferably 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 a related embodiment, the lysis composition further comprises paramagnetic particles or silica support.

Extraction of viral RNA 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.

As known to a skilled person, 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 in routine analytical processes like extraction of nucleic acids which 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 HCV RNA 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 comprise 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.
Alternatively, in an embodiment, lysed sample is washed with at least two washes using wash composition comprising buffer and alcohol. 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 is eluted using an elution composition to obtain HCV RNA that can be directly applied for further processing or analyses. In another 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.
In a preferred embodiment, extraction of HCV RNA is carried out using lysis, wash, and elution compositions in a single container; wherein the lysis composition performs sample lysis; the wash composition performs washing of the lysate; and the elution composition enables elution of HCV RNA.
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 like HCV.
The present disclosure further relates to contacting the RNA with one or more detection compositions comprising oligonucleotide sequences; wherein the oligonucleotide sequences comprise primers and probes that hybridize with target sequences in the HCV RNA in non-cross-reactive and non-overlapping manner to enable simultaneous detection and quantitation or discrimination of one or more of HCV genotypes 1-6 and subtypes thereof wherein, the said oligonucleotide sequences 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, primer or probe oligonucleotides, primer or probe sequences; 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” 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, one or more detection compositions comprise primer sequences and probe sequences selected from SEQ ID NO: 1 to SEQ ID NO: 41; wherein, the said sequences are specific for one or more of HCV genotypes 1-6 and subtypes thereof.
In a further embodiment, one or more detection compositions may comprise primer sequences and probe sequences specific for one or more of HCV genotypes 1-7 and subtypes thereof.
In a preferred embodiment, the primer and probe sequences hybridize with target sequences in HCV RNA 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 specific HCV RNA 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, 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 fluorescent reporter groups at their 5 ‘ends and a fluorescent quencher group at their 3’ ends.

Optionally, in some embodiments, the fluorescent reporter groups are selected from FAM™, HEX™, ROX™, JOE™, CY3®, VIC®, TET™, Texas Red®, NED™, Alexa®, TAMRA™, CY5.5® and CY5®. Further, 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 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 an embodiment IC may be added during the sample preparation procedure in lysis buffer 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, a synthetic internal control (IC) is exogenously added to the test sample prior to the lysis.
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 transcriptase 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 70 minutes.
In a further embodiment, the method comprises quantitation of HCV; the said method further comprising one or more calibrators.
In a preferred embodiment, the method may comprise quantitation of HCV genotypes 1-7 and subtypes thereof; the said method further comprising one or more calibrators.
The present invention further discloses a kit for simultaneous detection and discrimination of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample.
One embodiment of the present invention comprises a kit for simultaneous detection and discrimination of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample, wherein the said kit comprises: a lysis composition; and one or more detection compositions comprising non-overlapping and non-cross-reactive primer sequences and probe sequences; wherein the primer and probe sequences hybridize with heterogenous target sequences in HCV RNA to enable simultaneous detection and discrimination of one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
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 an embodiment, the test sample volume is up to 1mL of any sample likely to contain HCV RNA as described in earlier embodiments.
In another embodiment, sample lysis is carried out using a lysis composition for obtaining sample lysate.
In a related embodiment, the lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, and isopropanol; and further comprises paramagnetic particles or silica support as described in the previous embodiments.

In addition to sample lysis, in a preferred embodiment of the present invention, the kit optionally comprises additional steps of washing and/or eluting. The steps as well as their corresponding compositions have been detailed in other parts of the present disclosure.
The present disclosure further discloses detection compositions for carrying out simultaneous detection and discrimination of one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
In a related embodiment, the kit comprises one or more detection compositions comprising non-overlapping and non-cross-reactive primer sequences and probe sequences; wherein the primer and probe sequences hybridize with heterogenous target sequences in HCV RNA to enable simultaneous detection and discrimination of one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
In a preferred embodiment, the detection compositions comprise primer sequences and probe sequences selected from SEQ ID NO: 1 to SEQ ID NO: 41; wherein, the said sequences are specific for one or more of HCV genotypes 1-6 and subtypes thereof.
In an exemplary embodiment, detection composition A comprises primers and probes for HCV 1a and 3; detection composition B comprises primers and probes for HCV 2 and 1b; detection composition C comprises primers and probes for HCV 4; detection composition D comprises primers and probes for HCV 6 and 5; detection composition E comprises primers and probes for HCV 1a and 3b; detection composition F comprises primers and probes for HCV 2, 1b, and 1; and detection composition G comprises primers and probes for HCV 4 and 3a;
wherein, the said primers and probes hybridize in a non-overlapping and non-cross-reactive manner to simultaneously detect and discriminate one or more of HCV genotypes 1 -6 and subtypes thereof using real time RT-PCR.
The primers and probes in compositions A-G were grouped in a manner that avoided any cross reactivity between very closely associated genotypes, including template, primer, and probes heterobinding.
In an embodiment, the detection composition A comprises;
forward primer sequences comprising SEQ ID NO: 1 and 7;
reverse primer sequences comprising SEQ ID NO: 2 and 8; and
probe sequences comprising SEQ ID NO: 18 and 21;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 1a and HCV 3 using real time RT-PCR.
In another embodiment, the detection composition B comprises;
forward primer sequences comprising SEQ ID NO: 3, 5, and 15;
reverse primer sequences comprising SEQ ID NO: 4, 6, and 16; and
probe sequences comprising SEQ ID NO: 19 and 20;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 1b and HCV 2 using real time RT-PCR.
In an additional embodiment, the detection composition C comprises;
forward primer sequences comprising SEQ ID NO: 9;
reverse primer sequences comprising SEQ ID NO: 10; and
probe sequences comprising SEQ ID NO: 22;
wherein, the primers and probe hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 4 using real time RT-PCR.
In yet another embodiment, any of the detection compositions A-G further comprise primer and probe sequences for HCV common, wherein
forward primer sequences comprising SEQ ID NO: 26;
reverse primer sequences comprising SEQ ID NO: 27; and
probe sequences comprising SEQ ID NO: 25;
wherein, the primers and probe hybridize with homogenous target sequences in HCV RNA for detecting HCV 1-6 and subtypes thereof using real time RT-PCR.
In an embodiment, the detection composition D further comprises;
forward primer sequences comprising SEQ ID NO: 11 and 13;
reverse primer sequences comprising SEQ ID NO: 12 and 14; and
probe sequences comprising SEQ ID NO: 23 and 24;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 5 and HCV 6 using real time RT-PCR.
In an embodiment, the detection composition E comprises;
forward primer sequences selected from SEQ ID NO: 1, 31, 37, and 40;
reverse primer sequences selected from SEQ ID NO: 2, 32, 38, and 41; and
probe sequences selected from SEQ ID NO: 18, 33, 39, and 17;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 1a and HCV 3b using real time RT-PCR.
In another embodiment, the detection composition F comprises;
forward primer sequences comprising SEQ ID NO: 5, 3, 15, and 34;
reverse primer sequences comprising SEQ ID NO: 6, 4, 16, and 35; and
probe sequences comprising SEQ ID NO: 20, 19, and 36;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 2, HCV 1b, and HCV 1 using real time RT-PCR.
In a further embodiment, the detection composition G comprises;
forward primer sequences comprising SEQ ID NO: 9 and 28;
reverse primer sequences comprising SEQ ID NO: 10 and 29; and
probe sequences comprising SEQ ID NO: 22 and 30;
wherein, the primers and probes hybridize with heterogenous target sequences in HCV RNA for simultaneously detecting and discriminating HCV 4 and HCV 3a using real time RT-PCR.
Various modifications to the embodiments may be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments.
In an embodiment, the detection compositions as described in the previous embodiments may comprise primer and probe sequences specific for detecting and discriminating one or more of HCV genotypes 1-7 and subtypes thereof.
In further embodiment, a synthetic internal control (IC) is exogenously added to the test sample prior to the lysis as described in the previous embodiments.
In a preferred embodiment, 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 70 minutes.
The present disclosure further discloses a kit for the detection and quantitation of HCV RNA in a test sample.
One embodiment of the present disclosure comprises a kit for the detection and quantitation of one or more of hepatitis C virus (HCV) genotypes 1-6 and subtypes thereof from a test sample wherein, the said kit comprises:
a lysis composition;
one or more calibrators; and
a detection composition, wherein, the said detection composition comprises primer sequences and probe sequence for HCV common that hybridize with homologous target sequence in HCV RNA in a non-cross reactive and non-overlapping manner to amplify, detect, quantitate one or more of HCV genotypes 1-6 and subtypes thereof, from the test sample.
In another embodiment, the test sample volume is up to 1mL of any sample likely to contain HCV RNA as described in the earlier embodiments.
In a preferred embodiment, sample lysis is carried out using a lysis composition.
In a related embodiment, the lysis composition comprises lysis buffer, lysis enhancer buffer, carrier RNA, and isopropanol; and further comprise paramagnetic particles or silica support as described in earlier embodiments. Further, optionally additional steps of washing and/or eluting that have been described elsewhere in the specification may need to be performed in order to obtain HCV RNA that may be directly used for further processing or analyses.

In an embodiment, the detection composition comprise primers and probes selected from SEQ ID NO: 26, 27, and 25 and wherein, the primers and probes are specific for HCV to quantitate one or more of HCV genotypes 1-6 and subtypes thereof from the test sample.
In a further embodiment, the detection composition comprises primers and probes that are specific for HCV to quantitate one or more of HCV genotypes 1-7 and subtypes thereof from the test sample.

In an embodiment, a synthetic internal control (IC) is exogenously added to the test sample prior to the lysis as described in the earlier embodiments.
In an embodiment, the kit comprises one or more calibrators. The term “calibrator” or “calibrators” refers to a compound of known concentration used for performing quantitation.
In a related embodiment, the one or more calibrators comprise in vitro RNA transcript specific for 5’ UTR of HCV RNA at a concentration ranging from 7X106 IU/mL to 7X103 IU/mL.
In one embodiment, 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 70 minutes as described in the previous embodiments.

Alternatively, the method and kit compositions described in previous embodiments, 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 a preferred embodiment, lyophilized method and kit components exhibit increased shelf life, preferably more than 24 months.
In one embodiment, the use of method and kit compositions described in 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 viral 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 kits of the present disclosure may be coupled with any PCR machine having at least five channels.
In another aspect, the kit packets also carry 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 primers for HCV genotyping
SEQ ID NO Bases Sequence (5’ to 3’)
1 21 cacactccag tyaaytcctg g
2 21 cacctggaga gtaactgtgg a
3 21 cacactccag tyaaytcctg g
4 22 cacctggaga gtaactgtgg ag
5 21 ggtgagtaca ccggaattac c
6 16 ggggcacgcc caaatg
7 18 tctgcggaac cggtgagt
8 21 ctttcgcgacc caacactac t
9 20 agaccactat ggctctcccg
10 20 gtcttcacgc agaaagcgtc
11 21 agccatggcg ttagtatgag t
12 19 ttccgcagac cactatggc
13 21 ggccttgtgg tactgcctga t
14 14 gggcggcggt tggt
15 20 tgagtacacc ggaattgccg
16 16 ggggcacgcc caaatg
26 20 gccttgtggt actgcctgat
27 20 cacggtctac gagacctccc
28 21 ggtgagtaca ccggaatcgc t
29 18 gggcacgccc aaatttct
31 20 gatgaccggg tcctttcttg
32 17 ggcacgccca aatttcc
34 20 cgagcggcgt actgacaact
35 16 caagcggcgt gctgac
37 20 cgcaacytgg gtaaggtcat
38 22 cgacctcatg gggtacattc cg
40 19 aggctcggaa ggcaatcag
41 23 tggagccctt tgctgttrta cat
Table B: List of probes for HCV genotyping
SEQ ID NO Bases Sequence (5’ to 3’)
17 15 ctctcacrga gcggc
18 14 cagcttgaac aggc
19 14 caacttgaa aagc
20 17 agaaaggacc cagtctt
21 15 ccgcgagatc actag
22 18 tgtacaacac tcatacta
23 18 gtcgaacagc ctccagga
24 19 cacttccaaa accccaaag
25 17 agggtgcttg cgagtgc
30 14 aggacccggt cacc
33 15 acaacccgct caatg
36 14 gcctgtcgag cygc
39 19 gaagattccc tgttgcrta

Table C: Detection Compositions for HCV genotyping
Detection Compositions Target HCV genotype
A 1a, 3
B 2, 1b
C 4
D 5, 6
E 1a, 3b
F 1, 1b, 2
G 4, 3a
Examples:
The examples of the disclosure presented below are provided for illustrative purposes and are not intended to limit the scope of the invention. Many embodiments of the disclosure within the scope of the claims according to the examples will become apparent to those of ordinary skill in the art upon reading the foregoing description and the following examples.
Example 1: Extraction of HCV RNA using paramagnetic particles
In this example, the extraction of HCV RNA was carried out using 200µL (up to 500µL) of K2EDTA plasma sample from up to 5mL of whole blood (Table 1)
Table 1: Buffer volumes with 200µL and 500 µL sample input
Extraction components 200µL sample input (in µL) 500µL sample input (in µL)
Lysis Buffer and isopropanol combo 725 1000
Carrier RNA 25 25
Internal Control 25 25
Lysis Enhancer Buffer 50 50
Magnetic beads 50 50
Wash Buffer 1 (Reconstituted with Isopropanol) 700 700
Wash Buffer 2 (Reconstituted with ethanol) 1800**
(900 X2) 1800**
(900 X2)
Elution Buffer 100 100
The sample lysis was performed using lysis composition comprising paramagnetic particles as is known to a skilled person in the art to provide sample lysate containing RNA or extracted RNA for further analyses.
Alternatively, HCV RNA may also be extracted from the K2EDTA sample using an automated paramagnetic particle method.
The extract was transferred to a new tube for further analysis, or stored at -20°C or 80°C until further use.
Example 2: A kit for the detection and discrimination of HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5 and 6 by real time RT-PCR using detection compositions.
This example demonstrates a kit for amplifying, detecting, and discriminating one or more of HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5 and 6 from a test sample in accordance with the present disclosure.
For this example, the lysate from Example 1 was directly applied for the detection and discrimination of HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5 and 6 using real time RT-PCR.
The present kit comprises one or more detection compositions for screening the extracted HCV RNA for one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR.
The present kit may further comprise lysis, wash, and elution compositions for performing manual, automated, or semi-automated extraction of HCV RNA from a sample. Alternatively, the present kit maybe combined with any other lysis and extraction method for performing the extraction of HCV RNA from a sample.
In the present example, the screening was monitored using HCV positive controls A, B, C and D and nuclease free water was used as a no template control (NTC) or negative control. PCR Mix was prepared using RT enzyme, Taq polymerase and buffer components. Detection compositions A, B, C, and D were prepared using primers and probes specific to HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5 and 6 as detailed in Table C.
In this example, 200µL (up to 500 µL may be used) of K2EDTA plasma sample was used as the sample.
For performing real time RT-PCR, reaction mixture was prepared by mixing the reagents shown in Table 3.
To reaction tubes containing PCR mix, detection compositions A, B, C, D and extracted RNA was added. The extracted sample and the HCV positive controls A, B, C, D were also then added to the corresponding reaction tubes which were directly put in the PCR machine and the PCR cycle was performed as per Table 4.
PCR plate was set up on a real time RT-PCR system using the extracted RNA, positive controls and the reaction mix such that the total volume of the composition was 25µL per well. This step was followed by saving the data captured in the data analysing software and its analysis and interpretation.
Table 2: Components for RT-PCR
Reagent Volume per reaction (25µl volume) for working concentration
Up to 10X PCR Mix 1X
HCV Detection composition A/B/C/D 1X (up to 900uM primers and 200uM probes)
Nuclease free Water To compensate
Template addition Up to 50% of total reaction volume
Volume 25µl

Table 3: Composition of reaction mix
Contents Description
PCR Mix RT enzyme, Taq polymerase and buffer components
Detection compositions A, B, C, D Primers and probes specific to HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5, 6 and internal control (IC)
Nuclease free water RNase/DNase free water
Positive control A/B/C/D In vitro RNA transcript of genotype 1 (1a,1b), 2,3,4,5,6 and internal control
Internal Control (IC) Synthetic Internal control template
The PCR cycling conditions for the present experiment are set forth in Table 4.
Table 4: Run method
Parameter Stage Temperature Time
Reverse Transcription Hold 50°C Up to 15 min
RT inactivation Hold 95°C Up to 20 sec
PCR amplification Cycle
(=45 cycles) 95°C
60°C Up to 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 instrument and the results were saved.
Table 5: Procedure undertaken:
Reaction Target Reporter
A HCV 1a FAM
HCV 3 VIC/HEX
B HCV 1b FAM
HCV 2 VIC/HEX
C HCV 4 FAM
HCV common VIC/HEX
D HCV 5 FAM
HCV6 VIC/HEX
HCV IC Cy5
Table 6: Test validation and interpretation
Targets Mix A Mix B Mix C Mix D Results
Sample 1a 3 1b 2 4 HCV
common 6 5 IC
1 + - - - - + - - + Valid for Genotype 1a
2 - + - - - + - - + Valid for Genotype 3
3 - - + - - + - - + Valid for Genotype 1b
4 - - - + + - - + Valid for Genotype 2
5 - - - - + + - - + Valid for Genotype 4
6 - - - - + - + + Valid for Genotype 5
7 - - - - - + + - + Valid for Genotype 6
8 - - - - - + - - + Sample could not be
genotyped.
9 - - - - - - - - - PCR inhibition,
invalid the test and
repeat the experiment
Table 7: Kit components for RT-PCR Amplification
Contents Description
PCR Mix Reverse Transcriptase, Taq polymerase and buffer components
HCV Detection Composition A Primers and probe specific to subtype HCV 1a and 3
HCV Detection Composition B Primers and probe specific to subtype HCV 1b and 2
HCV Detection Composition C Primers and probe specific to genotype HCV 4 and HCV common
HCV Detection Composition D Primers and probe specific to genotype HCV 5, 6 and IC
HCV Positive control A Genotype 3 and 1a
HCV Positive control B Genotype 2 and 1b
HCV Positive control C Genotype 4
HCV Positive control D Genotype 6, 5 and Internal control.
Internal control Synthetic template for Internal control
Nuclease free water
Performance Characteristics:
Analytical sensitivity:
The Limit of Detection (LOD or analytical sensitivity) was determined for the present kit for HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5 and 6 using the WHO NIBSC standards or with clinical specimens specific for each genotype. 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 8a and 8b). The linear range was found to be about 500 to 8x107 IU/mL.
Optimum LOD:
For HCV common target, WHO International Standard 5th WHO International Standard for HCV NAT NIBSC code: 14/150 was used.
The optimum LOD was determined to be 40 IU/mL for 200µL sample volume (extracted using silica support) and 12 IU/mL for 500µL sample volume (extracted using paramagnetic particles) for HCV detection. Limits of detection (LOD) were estimated for each HCV genotype (1a, 1b, 2, 3, 4, 5 and 6).
For each genotype, a single HCV specimen was diluted in HCV negative human plasma to make panels containing the following HCV concentrations: 4000, 2000, 1000, 500, 250, 125, and 62.5 IU/mL. Each genotype dilutions were tested for total of 20 replicates within 5 days using real time PCR machine.
Table 8a: Comparative LOD for NAT tests (for HCV common): 200µL sample input
Quantity (IU/mL) Points tested Hits Hit rate
200 20 20 100%
100 20 20 100%
80 20 20 100%
60 20 20 100%
40 20 19 95%
20 20 18 90%
Table 8b: Comparative LOD for NAT tests (for different genotypes): 200µL sample input
HCV genotype (IU/mL) Replicates tested HCV genotype Hits
HCV 1a HCV 1b HCV2 HCV 3 HCV 4 HCV 5 HCV 6
4000 20 20 20 20 20 20 20 20
2000 20 20 20 20 20 20 20 20
1000 20 20 20 20 20 20 20 20
500 20 20
(100%) 19
95%) 19
(95%) 20
(100%) 19
(95%) 20
(100%) 19
(95%)
250 20 19(95%) 19 18 19(95%) 18 18 18
125 20 18 17 18 19 16 17 16
62.5 20 12 11 15 17 13 16 14
Based on the above results, LOD for HCV genotypes were determined as 500 IU/mL at 95% confidence interval.
Analytical specificity and genotype coverage
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 testing international reference standards. (NIBSC 3rd HCV RNA genotype panel for nucleic acid amplification (PN: 12/172)
A potential cross-reactivity of the present kit was tested using the control group comprising similar organisms such as Hepatitis B virus, HIV-1, HIV-2 Adenovirus 5, Human Herpes Virus, Herpes Simplex Virus Type 2, Chikungunya Virus Human Cytomegalovirus, Human T-Lymphotrophic Virus Type I, Influenza Virus A, Influenza virus B, Dengue 1 virus Varicella-Zoster Virus, , Herpes Simplex Virus Type 1, Staphylococcus epidermis, Staphylococcus haemolyticus, Escherichia coli, Streptococcus viridans, Staphylococcus aureus, Mycobacterium tuberculosis and Candida albicans. No cross reactivity was observed.
Following are the experimental data to showcase the individual detection (performance study, accuracy percentage and precision, comparative data with commercially available kits):
Precision:
Precision of the present kit was evaluated by testing a one moderately positive (7XLOD) and one low positive (3XLOD) HCV specimens representing HCV genotypes 1a, 1b, 2, 3, 4, 5 and 6. Three replicates of each specimen were tested during inter assay to check variability between operator (n-3), days (n-5) and instrument (n-3).
The precision was within permissible limit of =20% coefficient of variation.
Table 9: Specificity and the genotypes coverage
3rd HCV genotype NIBSC WHO Panel (PN 12/172)
Genotype HCV IC HCV1a HCV 1b HCV 2 HCV 3 HCV 4 HCV 5 HCV 6
HCV 1a + + + - - - - - -
HCV 1b + + - + - - - - -
HCV 2i + + - - + - - - -
HCV 3a + + - - - + - - -
HCV 4r + + - - - - + - -
HCV 5a + + - - - - - + -
HCV 6i + + - - - - - - +
Table 10: Precision for repeatability and reproducibility
HCV genotype Viral Load IU/mL Total Replicates Mean Ct Within Run Within Lot Within Instrument
SD %CV SD %CV SD %CV
HCV 1a Moderate 15 32.44 0.31 0.95 0.33 1.01 0.31 0.96
Low 15 33.97 0.30 0.88 0.22 0.64 0.36 1.07
HCV 1b Moderate 15 32.44 0.24 0.75 0.27 0.82 0.15 0.45
Low 15 33.41 0.22 0.65 0.23 0.69 0.21 0.63
HCV 2 Moderate 15 32.55 0.18 0.56 0.17 0.52 0.26 0.79
Low 15 34.27 0.17 0.50 0.18 0.54 0.12 0.34
HCV 3 Moderate 15 33.03 0.15 0.46 0.14 0.42 0.17 0.50
Low 15 34.54 0.15 0.42 0.13 0.36 0.20 0.58
HCV 4 Moderate 15 33.56 0.21 0.61 0.23 0.68 0.23 0.69
Low 15 34.62 0.14 0.39 0.12 0.36 0.14 0.41
HCV 5 Moderate 15 32.60 0.18 0.18 0.18 0.56 0.19 0.59
Low 15 34.70 0.57 0.51 0.17 0.50 0.13 0.37
HCV 6 Moderate 15 33.54 0.20 0.61 0.22 0.66 0.17 0.49
Low 15 34.57 0.22 0.63 0.22 0.64 0.21 0.60
HCV genotype kit was evaluated for specificity and genotype coverage using NIBSC WHO 3rd HCV genotype Panel (PN 12/172)
The physical attributes of the discrimination kit
a. HCV common target is incorporated in the test kit. HCV common target reflects to HCV conserved region in 5’UTR region which detects all genotypes. This primer probe sequence is used for HCV detection purpose. In turn, kit can be claimed for detection and genotyping discrimination both. Sometimes, if HCV viral load is less, genotypes would not be determined. But HCV common target gives positive result indicating HCV presence. It would avoid reporting as false negative by only genotyping.
b. All real time PCR reagents are provided either in liquid or freeze-dried format.
c. Reports are available in an hour time
d. Kit is compatible with commercially available real time PCR machines providing open system scope.
e. Kit is optionally provided with lysis compositions to carry out manual, semi-automated, or automated HCV RNA extraction.
f. Kit is compatible with other extraction systems.
g. 3a and 3b HCV genotype discrimination scope is available
Example 3: A kit for the detection and discrimination of HCV 1 (subtypes 1a, 1b), 2, 3 (subtypes 3a, 3b), and 4 by real time RT-PCR using detection compositions.
This example demonstrates a kit for amplifying, detecting, and discriminating one or more of HCV 1 (subtypes 1a, 1b), 2, 3 (subtypes 3a, 3b), and 4 in a test sample in accordance with the present disclosure.
For this example, the extract from Example 1 was directly applied for the detection and discrimination of HCV 1 (subtypes 1a, 1b), 2, 3 (subtypes 3a, 3b), and 4 using real time RT-PCR.
The present kit comprises one or more detection compositions for screening the extracted HCV RNA for one or more of HCV genotypes 1-6 and subtypes thereof using real time RT-PCR. The present kit may further comprise lysis, wash, and elution compositions for performing manual, automated, or semi-automated extraction of HCV RNA from a sample. Alternatively, it may be combined with any other extraction system for performing the extraction of HCV RNA from a sample.
In the present example, the screening was monitored using HCV positive controls E, F, and G and nuclease free water was used as a no template control (NTC) or negative control. PCR Mix was prepared using RT enzyme, Taq polymerase and buffer components. Detection compositions E, F, and G were prepared using primers and probes specific to HCV 1, HCV 1a, 1b, 2, 3, 3a, 3b, 4 and internal control as detailed in Table C.
In this example, 200µL (up to 500 µl may be used) of K2EDTA plasma sample was used as the sample.
For performing real time RT-PCR, reaction mixture was prepared by mixing the reagents shown in Table 12. First the detection compositions E, F, and G were added to a reaction tube containing the PCR mix. The extracted sample and the HCV positive controls E, F, and G 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 4.
PCR plate was set up on a real time RT-PCR system using the extracted RNA, positive controls and the reaction mix such that the total volume of the composition was 25µL per well. This step was followed by saving the data captured in the data analysing software and its analysis and interpretation.
Table 11: Components for RT-PCR
Reagent Volume per reaction (25µl volume)
Up to 10 PCR Mix 1X
HCV Detection composition E/F/G 1X (Up to 900 mM primers, 200 mM probes)
Nuclease free Water To compensate
Template addition Up to 50% total volume
Volume 25µl

Table 12: Composition of reaction mix
Contents Description
PCR Mix RT enzyme, Taq polymerase and buffer components
Detection compositions E, F, G Primers and probes specific to HCV 1 (subtypes 1a, 1b), 2, 3 (subtypes 3a, 3b), 4and internal control (IC)
Nuclease free water RNase/DNase free water
Positive control E/F/G In vitro RNA transcript of genotype 1 (1a,1b), 2,3 (3a, 3b),4, and internal control
Internal Control (IC) Synthetic Internal control template
The PCR cycling conditions for the present experiment are set forth in Table 4. After performing the PCR run, fluorescence data was collected during 60°C extension step. The PCR plate was analysed using data analysing instrument and the results were saved.
Table 13: Procedure undertaken:
Reaction Target Reporter
E HCV 1a FAM
HCV 3b NED/ROX
IC Cy5
F HCV 2 FAM
HCV 1b VIC/HEX
HCV 1 NED/ROX
G HCV 4 FAM
HCV 3a NED/ROX
HCV common VIC/HEX
Table 14: Test validation and interpretation
Targets Mix E Mix F Mix G Results
Sample 1a 3b IC 1b 1 2 3a 4 HCV
1 + - + - - - - - + Valid for Genotype 1a
2 - + + - - - - - + Valid for Genotype 3b
3 - - + + - - - - + Valid for Genotype 1b
4 - - + - + - - - + Valid for Genotype 1
5 - - + - - + - - + Valid for Genotype 2
6 - - + - - - + - + Valid for Genotype 3a
7 - - + - - - - + + Valid for Genotype 4
8 - - + - - - - - + Sample could not be genotyped.
9 - - - - - - - - - PCR inhibition, invalid the test and repeat the experiment
Table 15: Kit components for discrimination using RT-PCR
Contents Description
PCR Mix Reverse Transcriptase, Taq polymerase and buffer components
Detection Composition E Primers and probe specific to HCV1a and 3b
Detection Composition F Primers and probe specific to HCV 1, 1b, and 2
Detection Composition G Primers and probe specific to HCV 3a, 4; add HCV common
HCV Positive control E Genotype 1a, 3b; add IC
HCV Positive control F Genotype 1b, 1, and 2
HCV Positive control G Genotype 3a, 4; add HCV common
Internal control Synthetic template for Internal control
Nuclease free water RNase and DNase Free
Example 4: Detection and quantitation of HCV RNA using real time RT-PCR
The extract from Example 1 was used for the detection and quantitation of HCV RNA using real time RT-PCR.
For quantitation of unknown HCV sample, four calibrators were provided ranging from 7x106 IU/ML to 7X103 IU/ML.
Calibrators were prepared using in vitro transcribed RNA using dilution series and then calibrated against NIBSC code: 14/150. First, HCV 5’UTR specific PCR products containing an RNA polymerase promoter site was used as a template for in vitro transcription with the MEGAscript® Kit. Then, in vitro transcribed RNA obtained as a result was resuspended in TE buffer at a concentration of 0.5–1 µg/µL. The in vitro transcription was carried out using equal volumes of four ribonucleotide solutions together. For performing the same, 8 µL of the dNTPs mixture was directly added to a standard 20 µL reaction. Then, 10X buffer and T7 RNA polymerase enzyme mix were added to the reaction mixture.
The whole content was then incubated at 370C for up to 12 hours. Post incubation, RNA precipitation was carried out using ethanol method.
Ten-fold serial dilutions of transcribed RNA products were then tested with respective gene primer probe sets for specific detection and limit of detection.
For the present example, four calibrators were used as detailed in Table 17. These calibrators when tested in real time RT-PCR, generate a standard curve (the standard curve is generated by machine software using the quantity provided Vs Ct value detected for each quantity). For performing HCV quantitation, reaction Mix was prepared using PCR Mix and Detection Mix.
First, PCR Mix was prepared using RT enzyme, Taq polymerase and buffer components (see Table 2). Then, detection mix was prepared using primers and probes specific to HCV and internal control. (Table 16). Calibrators and/or HCV RNA extracted from clinical samples were added as template in the reaction mix.
Table 16: Components in the reaction mix used for HCV quantitation
Contents Description
PCR Mix RT enzyme, Taq polymerase and buffer component
Detection Mix Primers and probe specific to HCV and internal control
Table 17: Calibrators for HCV quantitation
HCV Quantitation Calibrators QS1-7*106 IU/mL
QS2 -7*105 IU/mL
QS3-7*104 IU/mL
QS4-7*103 IU/mL
PCR plate was set up for real time RT-PCR system using the extracted HCV RNA or calibrators and the reaction mix such that the total volume of the composition was 25µL per well. The PCR cycling conditions for the performed experiment are set forth in Table 4.
In 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.
For quantitative analysis: For quantitative experiment testing, real time PCR software displays the values for unknown specimen in the result column in IU/mL for HCV target. Negative control (NTC) should not show any value in the result column.
Samples showing no amplification for HCV target, should show amplification for Internal control. Internal Control must be valid and Ct values should be =37, only then the results should be considered.

Amplification Plot (Fig. 2) and Standard Curve analysis (Fig. 3) for HCV
Interpret the values for unknown samples, only if
(i) R2 is > 0.98
(ii) Slope of calibrators is between -3.0 to – 3.7
(iii) PCR efficiency is between 85%-115%
Example 5: Kit for detecting and quantitating one or more of HCV genotype 1-6 and subtypes thereof.
The present kit comprises HCV detection composition for detecting and quantitating one or more of HCV genotypes 1-6 and subtypes thereof from the test sample.
The present kit also comprises one or more calibrators for detecting and quantitating one or more of HCV genotypes 1-6 and subtypes thereof from the test sample.
The present kit may further comprise lysis, wash, and elution compositions for performing manual, automated, or semi-automated extraction of HCV RNA from a sample. Alternatively, the present kit may be combined with any other extraction system for performing the extraction of HCV RNA from a sample.
In this example, up to 200 µL or 500 µL or 1mL of K2EDTA plasma sample was prepared using up to 3mL of whole blood and HCV RNA was extracted as per Example 1.
The extract was then directly incorporated for HCV detection and quantitation using real time RT-PCR.
For performing detection and quantitation of HCV RNA, reaction mix was prepared. The extracted RNA sample and calibrators 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 4.
The calibrators used were prepared as per Example 4 as detailed in Table 17.
During 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 analytical sensitivity (Limit of quantitation: LOQ) of the present kit is defined as the concentration of HCV RNA molecules that can be detected with a positivity rate of = 95%. The analytical sensitivity in consideration with nucleic acid extraction was determined using a dilution series of the 5th WHO International Standard for HCV NAT NIBSC code: 14/150 spiked into HCV negative EDTA plasma. Experiments were carried out in replicates and as five independent runs for total of 20 replicates each dilution using real time PCR machine.
The analytical detection limit of the present kit is 40IU/mL when tested with 200µL initial sample input and extracted using silica support and 12 IU/mL for 500µL initial sample input and extracted using paramagnetic particles.
Linear Range
The linear range of the present kit was determined by analysing a logarithmic dilution series of the 5th WHO International Standard for HCV NAT NIBSC code: 14/150 and Mylab™ HCV calibrators. Each NIBSC dilution was extracted and tested in replicates along with the Mylab™ calibrators and analysed on real time PCR system.
The linear range of the present kit has been determined to cover concentrations from 40 IU/mL to 7X107 IU/mL when tested with 200µL sample volume extracted using silica support and 12 IU/mL to 1X108 IU/mL when tested with 500µL sample volume extracted using paramagnetic particles.
Conversion Factor
The conversion factor 1 IU/mL = 1.13 HCV copies/mL
Specificity
The specificity of the present kit is ensured by the selection of the primers and probes and 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 detectability of all relevant subtypes and genotypes has also been ensured by using NIBSC 3rd HCV RNA genotype panel for nucleic acid amplification (PN: 12/172). The specificity was validated with 60 different HCV negative plasma samples. These did not generate any signals with the HCV specific primers and probes.
A potential cross-reactivity of the present kit was tested using the control group list comprising Hepatitis B virus, HIV-1, HIV-2 Adenovirus 5, Human Herpes Virus, Herpes Simplex Virus Type 2, Chikungunya Virus Human Cytomegalovirus, Human T-Lymphotrophic Virus Type I, Influenza Virus A, Influenza virus B, Dengue 1 virus Varicella-Zoster Virus, , Herpes Simplex Virus Type 1, Staphylococcus epidermis, Staphylococcus haemolyticus, Escherichia coli, Streptococcus viridans, Staphylococcus aureus, Mycobacterium tuberculosis and Candida albicans. None of the tested pathogens has been reactive. No cross-reactivity appeared with mixed infections.
Precision: Intra assay precision- repeatability & inter assay precision- reproducibility
Experiments were performed to establish the precision performance of the present kit for repeatability and reproducibility on real time PCR system.
Repeatability and Reproducibility data were obtained by testing clinical samples of known viral load (moderate 7X LOD) and Low (3X LOD) using the present kit. Test was performed in replicates for intra assay, inter assay, inter operator and inter lot variation. The resulting data is given in Table 18.
Table 18: Repeatability and reproducibility
HCV Standard variation % Coefficient variation
Intra assay N-10 0.36 (log 2.7) 0.018 (log 4.7) 11.63% 0.40%
Inter assay N-5 0.14 (log 2.7) 0.17 (log 4.7) 4.75 3.83
Inter operator N-3 0.34 (log 2.6) 13.09
Inter lot N-3 0.046 (log 3.6) 1.31
Performance study on clinical samples
The diagnostic evaluation was performed by testing 60 HCV negative and 30 HCV positive plasma samples which have been previously analysed using CE/IVD approved real time PCR HCV assay. All of the negative samples were found negative, and the positive samples were in correlation with comparative kit. The present kit for detection and quantitation of HCV RNA was further compared with commercially available kits for its Linear range/LOD. The % LOD used for this analysis was 95%.
Table 19: Comparative LOD for 200µL sample volume and silica column-based isolation
Parameter Qiagen artus® HCV RG RT-PCR Kit Mylab™ HCV quantitative PCR assay
Sample volume (µL) 500 200
Extraction Technology Column based Column based
Sensitivity (IU/mL) 33.6 40
Linear range (IU/mL) 65 to 1 x 106 40 to 7x107
According to the comparative data in Table 19, the present kit exhibited better sensitivity and linear range than the commercially available kit.
Table 20: Comparative LOD for 500µL sample volume and paramagnetic particle-based isolation
Abbott HCV assay VERSANT® HCV RNA V1.0 Mylab™
Sample volume (µL) 500 500 500
Extraction Technology Magnetic particles Magnetic particles Magnetic particles
Sensitivity (IU/mL) 12 15 12
Linearity (IU/mL) 12 to 1x10^8 15 to 1X108 12 to 1X108
According to the comparative data in Table 20, the present kit exhibited comparative sensitivity and linearity with the commercially available kits.
Table 21: Comparative LOD for genotyping kits
The limit of detection of the Abbott Real Time HCV Genotype II assay was compared with the present kit.
Organism Abbott RealTime HCV genotype II Mylab™
HCV genotypes 500 IU/mL 500 IU/mL
In accordance with the comparative data presented in Table 21, it could be 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 HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5 and 6 as opposed to the simultaneous discrimination of HCV 1 (subtypes 1a, 1b), 2, 3, 4, 5 by Realtime HCV genotype II by Abbott Molecular showcasing advancement in an ability to detect multiple viral agents in a single test sample. Also, 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 current disclosure 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 Realtime HCV genotype II.
The current disclosure further offers indigenous and cost-effective detection and quantitation compositions that may be used even at low sample concentrations to achieve reproducible results with high accuracy. This disclosure may hence help in the identification and quantitation of the HCV even at their lowest concentrations, preventing any further false negative results.
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 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.