CN115261518A - Primer combination, kit and application for genotyping of hepatitis C virus - Google Patents
Primer combination, kit and application for genotyping of hepatitis C virus Download PDFInfo
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Abstract
The invention discloses a primer combination, a kit and application for genotyping hepatitis C virus, wherein the primer combination consists of a reaction solution A and a reaction solution B; the reaction solution A comprises hepatitis C virus type 1 primers and probes, hepatitis C virus type 2 primers and probes and hepatitis C virus type 6 primers and probes, and the reaction solution B comprises hepatitis C virus type 3 primers and probes, hepatitis C virus type 4 primers and probes and hepatitis C virus type 5 primers and probes; the primer combination is used for detecting 6 genotypes of hepatitis C virus. The invention has the advantages that the invention is based on the fluorescence PCR technology, utilizes different fluorescence channels to carry out genotyping on the hepatitis C virus, assists in the selection of antiviral drugs, is simple and quick, and is beneficial to clinical accurate treatment.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a primer combination and a kit for genotyping hepatitis C virus and application thereof.
Background
Hepatitis C Virus (HCV) belongs to the Family Flaviviridae (Family Flaviviridae) and is a positive-strand RNA virus with an envelope. HCV infection of human liver cells can cause diseases such as chronic hepatitis, hepatic fibrosis, liver cirrhosis, liver cancer and the like, and no vaccine is used for immunoprophylaxis of hepatitis C virus. According to the related literature, about 1400 million people with positive hepatitis C virus antibody exist in China, and the adult hepatitis C virus antibody positive rate is about 1.3%. Hepatitis C has high hiding degree and is easy to miss diagnosis, and the public has less cognition rate on hepatitis C and low treatment level, thus causing the spreading of hepatitis C.
The total length of the hepatitis C virus genome is about 9.6kb, only 1 piece of RNA of the hepatitis C virus is taken as genetic information and is divided into 1, 2, 3, 4, 5 and 6 genotypes, and each genotype is divided into subtypes from a, c, d, e, f, g to t and the like. The HCV genome has high mutation probability, and the conservation degree of different genome regions among subtypes is obviously different, wherein the 5' UTR region difference and the evolutionary rate are low, and the HCV genome has the highest conservation among different subtypes. The distribution of the various genotypes of HCV is significantly different, with types 1, 2, and 3 being widely distributed globally, type 4 being mainly distributed in North Africa and middle east countries, type 5 being mainly distributed in the southern Africa, and type 6 being prevalent mainly in the southeast Asia. HCV infection in northern China mainly takes 1b and 2a types as main types, mostly is transmitted by blood, such as blood transfusion, compensated blood donation and the like, while subtypes 3a, 3b and 6a in regions in southwest and south China are gradually increased, and subtypes 2b, 4a, 5a, 6b and the like are discovered successively.
HCV genotyping is of great significance in epidemiological studies and clinical treatment of hepatitis c, HCV genotypes have significant differences in response to drugs and treatment regimens, and most current studies suggest that HCV types 1 and 4 respond poorly to interferon therapy, while HCV types 2 and 3 respond well, whereas type 1a is susceptible to RAVs when treated with DAAs. Differences in IFNa treatment between HCV genotypes were observed in clinical studies, and with the same treatment schedule, patients infected with genotypes 2 and 3 had SVR rates for interferon 2-3 times that of genotype 1, and recurrence rates after 24 weeks of PEGIGNa treatment for genotype 1 infection were about 60%, significantly higher than other genotypes (33.3%). The SVR for type 4 infection is similar or lower than that for type 1 infection, the SVR for type 5 infection is close to that for type 2 and type 3, and the SVR for type 6 infection is between type 1, type 2 and type 3 when treated with IFNa + ribavirin. The gene-generic direct antiviral drug DAAs marks that the treatment of hepatitis C enters a new era, but DAAs drugs such as dalastavir, asunaprevir, darbuvir and the like are suitable for HCV type 1 infected people, while Elba Weida Norvir is suitable for patients with gene types 1 and 4, and other patients with gene types have poor treatment effect or are easy to have RAVs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a primer combination, a kit and an application for genotyping of hepatitis C virus, and the invention designs a composition of a primer and a probe aiming at the specificity of hepatitis C virus type 1-6, which can specifically identify HCV RNA in human serum or blood plasma, and realizes qualitative detection and genotyping of HCV RNA through reverse transcription-real-time fluorescence PCR reaction, thereby providing reference for epidemiological research and clinical treatment of hepatitis C virus.
In order to achieve the purpose, the invention adopts the following technical scheme:
a primer combination for genotyping hepatitis C virus, which consists of a reaction solution A and a reaction solution B; the reaction solution A comprises hepatitis C virus type 1 primers and probes, hepatitis C virus type 2 primers and probes and hepatitis C virus type 6 primers and probes, and the reaction solution B comprises hepatitis C virus type 3 primers and probes, hepatitis C virus type 4 primers and probes and hepatitis C virus type 5 primers and probes; the primer combination is used for detecting 6 genotypes of hepatitis C virus, wherein:
the hepatitis C virus type 1 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATCAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
the hepatitis C virus type 2 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATAAACCCACTCTATGCCCGGCC, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
the hepatitis C virus type 6 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGAG, the 5 'end of the probe is ROX, and the 3' end is BHQ2;
the hepatitis C virus type 3 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
the hepatitis C virus type 4 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATTAACCCGCTCAATGCCCGGAAAT, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
the hepatitis C virus 5 type primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATAAACCCGCTCAATGCCCGGAGAT, and the 5 'end of the probe is ROX and the 3' end is BHQ2.
The reaction solution a and the reaction solution B both have rnaspep primer sets as internal standards, and the rnaspep primer sets specifically include:
forward primer (F): AGATTTGGACCTGCGAGCG;
reverse primer (R): GAGCGGCTGTCTCCACAAGT;
fluorescent probe (P): TTCTGACCTGAAGGCTCTGCGCG, and the 5 'end of the probe is CY5 and the 3' end is BHQ3.
The invention provides a kit with a primer combination for genotyping hepatitis C virus, which is characterized by comprising a primer combination consisting of a reaction solution A and a reaction solution B.
The invention also provides application of the primer combination kit for genotyping of the hepatitis C virus, and the kit is used for detecting the hepatitis C virus.
The hepatitis c virus includes hepatitis c viruses of 6 genotypes.
The invention has the advantages that the invention is based on the fluorescence PCR technology, utilizes different fluorescence channels to carry out genotyping on the hepatitis C virus, assists in the selection of antiviral drugs, is simple and quick, and is beneficial to clinical accurate treatment.
Drawings
FIG. 1 is a specific detection chart of the kit of the present invention;
FIG. 2 is a graph of the effect of interfering substances on the detection of hepatitis C virus type 1 (including control curves);
FIG. 3 is a graph of the effect of interfering substances on the detection of type 2 hepatitis C virus (control curve included);
FIG. 4 is a graph of the effect of interfering substances on the detection of hepatitis C virus type 3 (including control curves);
FIG. 5 is a graph of the effect of interfering substances on the detection of hepatitis C virus type 4 (including control curves);
FIG. 6 is a graph of the effect of interfering substances on the detection of hepatitis C virus type 5 (including control curves);
FIG. 7 is a graph of the effect of interfering substances on the detection of hepatitis C virus type 6 (including control curves);
FIG. 8 shows the result of the detection of HCV type 1b national Standard substance gradient (200-2 gamma 107 IU/mL);
FIG. 9 shows a gradient of national standard substance (200-2 gamma 10) for HCV type 2a 7 IU/mL) detection result;
FIG. 10 shows a gradient of national standard substance for HCV type 6a (300-3 gamma 10) 7 IU/mL) detection result;
FIG. 11 shows a gradient of national standard substance (200-2 gamma 10) for HCV type 3a 7 IU/mL) detection result;
FIG. 12 shows a gradient of national standard substance for HCV type 4 (300-2 gamma 10) 7 IU/mL) detection result;
FIG. 13 shows a gradient of national standard substance for HCV type 5 (300-2 gamma 10) 7 IU/mL) detection result;
Detailed Description
The present invention will be further described below, and it should be noted that the following examples are provided to illustrate the detailed embodiments and specific procedures based on the technical solution, but the scope of the present invention is not limited to the examples.
The invention relates to a primer combination for genotyping hepatitis C virus, which consists of a reaction solution A and a reaction solution B; the reaction solution A comprises hepatitis C virus type 1 primers and probes, hepatitis C virus type 2 primers and probes and hepatitis C virus type 6 primers and probes, and the reaction solution B comprises hepatitis C virus type 3 primers and probes, hepatitis C virus type 4 primers and probes and hepatitis C virus type 5 primers and probes; the primer combination is used for detecting 6 genotypes of hepatitis C virus, wherein:
the hepatitis C virus type 1 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATCAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
the hepatitis C virus type 2 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATAAACCCACTCTATGCCCGGCC, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
the hepatitis C virus type 6 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGAG, and the 5 'end of the probe is ROX, the 3' end is BHQ2;
the hepatitis C virus type 3 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
the hepatitis C virus type 4 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATTAACCCGCTCAATGCCCGGAAAT, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
the hepatitis C virus 5 type primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA;
reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG;
fluorescent probe (P): TTGGATAAACCCGCTCAATGCCCGGAGAT, and the 5 'end of the probe is ROX and the 3' end is BHQ2.
The reaction solution a and the reaction solution B both have rnaspep primer sets as internal standards, and the rnaspep primer sets specifically include:
forward primer (F): AGATTTGGACCTGCGAGCG;
reverse primer (R): GAGCGGCTGTCTCCACAAGT;
fluorescent probe (P): TTCTGACCTGAAGGCTCTGCGCG, and the 5 'end of the probe is CY5 and the 3' end is BHQ3.
The invention provides a kit with a primer combination for genotyping hepatitis C virus, which is characterized by comprising a primer combination consisting of a reaction solution A and a reaction solution B.
The invention also provides application of the primer combination kit for genotyping of the hepatitis C virus, and the kit is used for detecting the hepatitis C virus.
The hepatitis c virus includes hepatitis c viruses of 6 genotypes.
Example 1 extraction of nucleic acids from plasma samples
1. Total nucleic acid samples, including RNA, were extracted.
After a clinical sample containing the target nucleic acid is subjected to cell lysis or virus particle lysis by a lysis solution and nucleic acid release, the high-purity nucleic acid is obtained by washing and elution processes by utilizing the principle that silicon magnetic beads are combined with the nucleic acid and the nucleic acid is released. The nucleic acid extraction steps are as follows:
1) 200 mu L of sample, 20 mu L of proteinase K and 600 mu L of sample treatment solution are added into a 1.5mL centrifuge tube, and after shaking and mixing for 5 seconds, the mixture is placed on a constant temperature mixer with the temperature of 60 ℃ and the rpm of 1600 and shaken and mixed for 3 minutes.
2) 20 mu L of the magnetic bead suspension and 200 mu L of isopropanol are added into a centrifuge tube, and the mixture is placed on a constant temperature mixer at room temperature and 1600rpm and is shaken and mixed for 3 minutes.
3) Placing the centrifuge tube on a magnetic frame, placing for 2 minutes, and removing liquid;
4) Adding 700 mu L of rinsing liquid I into a centrifuge tube, shaking and mixing uniformly for 1 minute on a constant-temperature mixer at room temperature and 1600rpm, placing on a magnetic frame, placing for 1 minute, and removing liquid;
5) Adding 700 mu L of rinsing liquid II into a centrifuge tube, shaking and mixing uniformly for 1 minute on a constant-temperature mixer at room temperature and 1600rpm, placing on a magnetic frame, placing for 1 minute, and removing liquid;
6) Air-drying the centrifuge tube for 2-5 minutes;
7) Adding 50-100 mu L of eluent into a centrifuge tube, placing the centrifuge tube on a constant-temperature mixing instrument with the temperature of 60 ℃ and the rpm of 1600, and uniformly mixing the eluent for 3 minutes in a shaking way;
8) And (3) placing the centrifugal tube on a magnetic frame, placing for 2 minutes, collecting the nucleic acid solution in a new centrifugal tube, and carrying out subsequent tests on the eluted nucleic acid.
Example 2 genotyping assay for hepatitis C Virus
And (3) checking the principle: the hepatitis C virus genotyping detection kit is a single-tube multiplex PCR technology, and the technology can detect and analyze 4 target genes at the same time in one PCR.
1) Reaction solution 1 contains primers and probes for HCV types 1, 2 and 6
Hepatitis c virus type 1:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATCAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
hepatitis c virus type 2:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATAAACCCACTCTATGCCCGGCC, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
hepatitis c virus type 6:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGAG, the probe has ROX at its 5 'end and BHQ3 at its 3' end;
rnaspep primer set:
forward primer (F): AGATTTGGACCTGCGAGCG
Reverse primer (R): GAGCGGCTGTCTCCACAAGT
Fluorescent probe (P): TTCTGACCTGAAGGCTCTGCGCG, the probe has CY5 at its 5 'end and BHQ3 at its 3' end;
2) Reaction solution 1 contains primers and probes for HCV types 3, 4, and 5
Hepatitis c virus type 3:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
hepatitis c virus type 4:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATTAACCCGCTCAATGCCCGGAAAT, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
hepatitis C Virus 5 type
Forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATAAACCCGCTCAATGCCCGGAGAT, the 5 'end of the probe is Cy5, and the 3' end is BHQ3;
rnaspep primer set:
forward primer (F): AGATTTGGACCTGCGAGCG
Reverse primer (R): GAGCGGCTGTCTCCACAAGT
Fluorescent probe (P): TTCTGACCTGAAGGCTCTGCGCG, the 5 'end of the probe is ROX, and the 3' end is BHQ2;
PCR amplification
(1) Preparation of mixed liquid
And melting and uniformly mixing the PCR reagents, and performing instantaneous centrifugation for later use. 18.5 mu L of reaction liquid and 1.5 mu L of enzyme mixed liquid are shaken and mixed evenly to form PCR mixed liquid, and after instantaneous centrifugation, the PCR mixed liquid is kept in the dark at 4 ℃ for standby.
(2) Template addition
The sample nucleic acid template, the negative control template, and the positive control template treated in example 1 were added to the reaction tube containing the 20. Mu.L of the mixture, 10. Mu.L each, and the tube was closed and centrifuged briefly at low speed.
(3) Amplification procedure
The method adopts a one-step reverse transcription amplification technology to amplify the sample nucleic acid, and comprises the following specific procedures:
note: detection of fluorescence selection: FAM channel, HEX channel and ROX channel respectively detect type 1 and type 3, type 2 and type 4, type 6 and type 5, and CY5 channel detection internal standard.
3. Interpretation of results
The invention adopts a Taqman fluorescent probe method, during the PCR amplification and extension process, the probe is cut off by the exonuclease activity of Taq enzyme, a 5 'end report group and a 3' end quenching group are separated to generate a fluorescent signal which can be detected by a fluorescent quantitative PCR instrument, and the fluorescent signal is automatically drawn in real time. And after the reaction is finished, judging the standard analysis result according to the quality control standard and the result.
(1) Quality control standard
The above requirements are met simultaneously in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.
(2) Analysis of results
And analyzing results under the conditions that the instrument is normal, and the negative control and the positive control meet the requirements.
When the Ct value of the CY5 channel of the internal standard in the experiment is more than 35 or no Ct value, the sample contains PCR reaction inhibitor, the extraction fails or the internal standard is missed in the experiment process, and resampling, re-extraction or re-detection is recommended. Positive results suggest that the patient's diagnosis should be considered in conjunction with a comprehensive analysis of the treatment, including symptoms, medical history, other laboratory examinations, etc.
Example 3 verification of the Performance of the hepatitis C Virus genotyping test kit
According to related technical data and national related documents at home and abroad and by combining the practical application condition of the kit, the minimum detection limit, the analysis specificity and the anti-interference index of the kit are evaluated.
1. Minimum detection limit
The ability of the kit to detect samples near the minimum detection limit was examined. The detection is carried out by using national reference substances and national standard substances, and the detection result is shown in the following table.
2. Analysis of specificity
The kit is used for genotyping of hepatitis C virus, experiments show that the kit has no cross reaction on common respiratory pathogens (haemophilus influenzae, staphylococcus aureus, rubella virus, measles virus, mumps virus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, bordetella pertussis, moraxella catarrhalis, streptococcus pyogenes, pseudomonas aeruginosa, streptococcus salivarius, streptococcus agalactiae and candida albicans) and blood pathogens (hepatitis A virus, hepatitis B virus and HIV), and the experimental result is shown in figure 1.
3. Effect of interfering substances on the kit of the invention
Therapeutic drugs such as common polyethylene glycol, interferon, antibiotics and the like are added into the samples in a certain proportion respectively, so that the detection of the kit is not influenced. 10g/L of mucin, 5% of human blood, 300mg/L of ribavirin, 37.5mg/L of oseltamivir and 500mg/L of mupirocin are respectively detected, the results show that the interference substances have no influence on the detection results, and the experimental results are shown in figures 2-7.
Example 4 sensitivity verification
The sensitivity was verified using hepatitis C virus national standards (1 b, 2a, 6a, 3a, 4, 5). The standard substance is subjected to gradient dilution according to the labeled concentration of the standard substance, and the standard substance is extracted and tested, so that the reagent can achieve detection of 200-300IU/mL, and the sensitivity is higher than the national requirement. The results of the experiments are shown in FIGS. 8-13.
Various corresponding changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.
Claims (5)
1. A primer combination for genotyping hepatitis C virus is characterized by consisting of a reaction liquid A and a reaction liquid B; the reaction solution A comprises hepatitis C virus type 1 primers and probes, hepatitis C virus type 2 primers and probes and hepatitis C virus type 6 primers and probes, and the reaction solution B comprises hepatitis C virus type 3 primers and probes, hepatitis C virus type 4 primers and probes and hepatitis C virus type 5 primers and probes; the primer combination is used for detecting 6 genotypes of hepatitis C virus, wherein:
the hepatitis C virus type 1 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATCAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
the hepatitis C virus type 2 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATAAACCCACTCTATGCCCGGCC, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
the hepatitis C virus type 6 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGAG, the probe has ROX at 5 'end and BHQ2 at 3' end;
the hepatitis C virus type 3 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): CATTGGATCAAACCCGCTCAATGCCTGGA, the 5 'end of the probe is FAM, and the 3' end is BHQ1;
the hepatitis C virus type 4 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATTAACCCGCTCAATGCCCGGAAAT, the 5 'end of the probe is HEX, and the 3' end is BHQ1;
the hepatitis C virus type 5 primer and the probe are as follows:
forward primer (F): GTCTGCGGAACCGGTGAGTACACCGGAA
Reverse primer (R): AAAGGCCTTGTGGTACTGCCTGATAG
Fluorescent probe (P): TTGGATAAACCCGCTCAATGCCCGGAGAT, and the 5 'end of the probe is ROX and the 3' end is BHQ2.
2. The primer combination for genotyping of hepatitis c virus according to claim 1, wherein the reaction solution a and the reaction solution B each have an RNaseP primer set as an internal standard, and the RNaseP primer set specifically comprises:
forward primer (F): AGATTTGGACCTGCGAGCG
Reverse primer (R): GAGCGGCTGTCTCCACAAGT
Fluorescent probe (P): TTCTGACCTGAAGGCTCTGCGCG, and the 5 'end of the probe is CY5 and the 3' end is BHQ3.
3. A kit comprising the primer set for genotyping hepatitis C virus according to claim 1 or 2, wherein the kit comprises a primer set comprising reaction solution A and reaction solution B.
4. Use of a kit of primer combinations for genotyping hepatitis c virus according to claim 3, for the detection of hepatitis c virus.
5. Use of a kit of primer combinations for genotyping hepatitis c virus according to claim 4, wherein the hepatitis c virus comprises hepatitis c virus of 6 genotypes.
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