CN117363793A - Primer probe composition for detecting hepatitis C virus and integrated microfluidic chip kit - Google Patents
Primer probe composition for detecting hepatitis C virus and integrated microfluidic chip kit Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a primer probe composition for detecting hepatitis C virus and an integrated microfluidic chip kit. The primer probe composition provided by the invention has the advantages of good sensitivity and accuracy, high amplification efficiency, simplicity and convenience in operation, rapidness and time saving when used for detecting the hepatitis C virus. The invention integrates nucleic acid extraction, purification, amplification and detection by utilizing an integrated chip microfluidic mode, solves the problem of rapid pathogen detection, realizes a POCT detection scheme of 'sample in and result out', directly adds the sample into equipment after sampling, and purifies and amplifies the sample to obtain the result within 60 minutes.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a primer probe composition for detecting hepatitis C virus and an integrated microfluidic chip kit.
Background
Hepatitis C virus (Hepatitis C virus, HCV) belongs to the genus hepatitis C virus of the family Flaviviridae, and has a genome of single-stranded positive strand RNA of about 9.6kb in length, as an enveloped, spherical virus particle having a diameter of 30-60 nm. According to the characteristics of HCV genome, it can be divided into 6 genotypes and more than 90 different subtypes, and genotypes 1b and 2a are common in China, mainly type 1 b.
HCV is mainly transmitted by blood sources, 30-90% of hepatitis after transfusion is hepatitis C, and 1/3 of hepatitis C in hepatitis after transfusion in China. It becomes necessary to identify HCV viruses in blood samples quickly and easily. Currently, there are two main types of clinically common methods for detecting HCV: serological methods for determining HCV antibodies or PCR methods for detecting HCV ribonucleic acid in serum or plasma. The serology method has lower sensitivity, is easy to generate false negative results, has longer detection period, and is unfavorable for early detection and early treatment of viruses.
The real-time fluorescent quantitative PCR technology is the most widely applied molecular detection method at present, and has the characteristics of high sensitivity, strong specificity, high degree of automation and the like. However, many devices are bulky and the samples typically require pretreatment, and are relatively demanding in terms of experimental conditions and are not suitable for field detection.
Based on the above problems, the applicant has developed for several years, and in the previous application CN202110057507.0, a novel on-site rapid nucleic acid detection device CarryOn P1000Q is disclosed, which is a "hand-held full-automatic closed nucleic acid qPCR analysis system", and can complete on-site automatic nucleic acid detection of related pathogens within 60 minutes, so as to realize rapid detection of "sample in and result out". The application is further developed on the basis of the technology, and can be better matched with the reagent invention of the equipment, namely the integrated microfluidic chip kit for detecting the hepatitis C virus.
The existing detection kit and method have long operation time and complicated operation steps, so that the kit for detecting the hepatitis C virus, which has the advantages of high sensitivity, strong specificity, wide coverage, simple and convenient operation, time saving and cost saving, and the use method thereof are needed to be provided.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a primer probe composition for detecting hepatitis C virus and an integrated microfluidic chip kit. The primer probe composition can detect the hepatitis C virus, has better sensitivity and accuracy, high amplification efficiency, simple and convenient operation, and is rapid and time-saving.
In order to achieve the above object, the present invention provides the following technical solutions:
in one aspect, the invention provides a primer probe composition for detecting hepatitis C virus, which comprises a primer probe composition for detecting hepatitis C virus and a primer probe composition for detecting internal reference.
Specifically, the primer probe composition for detecting the hepatitis C virus comprises an HCV primer probe composition.
Specifically, the primer probe composition for detecting the internal reference is an IPC primer probe composition.
Further specifically, the method comprises the steps of,
(1) The HCV primer probe composition comprises:
F：SEQ ID NO:1：5'-ACTACTGTCTTCACGCAGAAAGC-3'；
R：SEQ ID NO:2：5'-TTCCGCAGACCACTATGGC-3'；
P：SEQ ID NO:3：5'-TCGTGCAGCCTCCAGGACCCC-3'；
(2) The IPC primer probe composition comprises:
F：SEQ ID NO:4：5'-AGTTGCAGTGTAACCGTCATGTA-3'；
R：SEQ ID NO:5：5'-TCGACGAGACTCTGCTGTTAA-3'；
P：SEQ ID NO:6：5'-CAGTAATCTGCGTCGCACGTGTGCA-3'。
specifically, the primer probe composition for detecting the hepatitis C virus is used for detecting 5' -non coding region of a hepatitis C virus genome.
Specifically, the 5 'end of the probe is marked with a fluorescence report group, and the 3' end of the probe is marked with a quenching group; the fluorescent reporter group is one or more of CY3, CY5, CY5.5, FAM, HEX, VIC, JOE or ROX, and the quenching group is a fluorescence quenching group.
More specifically, the fluorescent reporter group at the 5 'end of the probe for detecting the hepatitis C virus is FAM, and the quenching group at the 3' end is BHQ1; the fluorescence reporter group at the 5 'end of the probe for detecting the internal reference IPC is CY5, and the quenching group at the 3' end is BHQ3.
In another aspect, the invention provides application of the primer probe composition in preparation of a hepatitis C virus product.
In particular, the products include, but are not limited to, stand-alone reagents, chips or kits.
In yet another aspect, the present invention provides a product for detecting hepatitis C virus, said product comprising the primer probe composition described above.
In particular, the products include, but are not limited to, stand-alone reagents, chips or kits.
Specifically, the product also comprises a purifying reagent, an air-dried RT-qPCR reaction reagent and a freeze-dried IPC process quality control product.
The purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads.
Further specifically, the lysate comprises 6M guanidine hydrochloride, 0.4M sodium acetate (pH 4.7) and 2% Triton X-100, and the eluate comprises 10mM Tris-HCl (pH 8.5).
Further specifically, the sequence of the quality control product in the freeze-drying IPC process is SEQ ID NO 7.
Further specifically, the lyophilized IPC process quality control is RNA pseudovirus.
Further specifically, the quality control product for the freeze-drying IPC process further comprises mannitol, trehalose, bovine serum albumin, an antifoaming agent, 2-hydroxypropyl-beta-cyclodextrin, tris-HCl (pH 8.0), naCl and Tween20.
Further specifically, the freeze-drying IPC process quality control product is packaged in an integrated micro-fluidic chip in the form of freeze-drying balls.
In still another aspect, the invention also provides an integrated microfluidic chip kit for detecting hepatitis C virus, which comprises the primer probe composition.
Specifically, the product also comprises a purifying reagent, an air-dried RT-qPCR reaction reagent and a freeze-dried IPC process quality control product.
The purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads.
Further specifically, the lysate comprises 6M guanidine hydrochloride, 0.4M sodium acetate (pH 4.7) and 2% Triton X-100, and the eluate comprises 10mM Tris-HCl (pH 8.5).
Further specifically, the sequence of the quality control product in the freeze-drying IPC process is SEQ ID NO 7.
Further specifically, the lyophilized IPC process quality control is RNA pseudovirus.
Further specifically, the quality control product for the freeze-drying IPC process further comprises mannitol, trehalose, bovine serum albumin, an antifoaming agent, 2-hydroxypropyl-beta-cyclodextrin, tris-HCl (pH 8.0), naCl and Tween20.
Further specifically, the freeze-drying IPC process quality control product is packaged in an integrated micro-fluidic chip in the form of freeze-drying balls.
Specifically, the kit completes the extraction, purification, amplification and detection of the nucleic acid of the sample on an integrated chip.
In particular, the microfluidic chip kit can be used for Carryon P1000Q rapid nucleic acid detection equipment.
In yet another aspect, the invention provides the use of the primer probe composition, product or kit described above for detecting hepatitis C virus.
In yet another aspect, the present invention provides a method for detecting hepatitis c virus, the method being a non-disease diagnosis and treatment method, the method comprising detecting hepatitis c virus in a sample to be detected using the primer probe composition, product or kit described above.
Specifically, the method comprises the following steps:
(1) Extracting RNA of a sample by using the purifying reagent and purifying the RNA;
(2) Amplifying the sample RNA extracted and purified in step (1) using the primer probe composition described above;
(3) And analyzing the amplification result.
Further specifically, the steps are performed on an integrated microfluidic chip by using CarryOn P1000Q rapid nucleic acid detection equipment.
Compared with the prior art, the kit provided by the invention has the following beneficial effects:
(1) The primer probe composition provided by the invention can detect the hepatitis C virus, has better sensitivity and accuracy, high amplification efficiency, and is simple and convenient to operate, rapid and time-saving.
(2) The problem of pathogen rapid detection is solved by utilizing an integrated chip microfluidic mode, a POCT detection scheme of sample inlet-outlet is realized, equipment is directly added after sampling, and the results are obtained within 60 minutes after extraction, purification, amplification and detection of nucleic acid are integrated.
Drawings
FIG. 1 is a graph showing the detection result of the sensitivity of the kit.
FIG. 2 is a graph of the results of the reproducibility of the kit.
FIG. 3 is a graph showing the specificity of the control sample in HBV virus DNA chamber, B in HIV-1 virus RNA chamber, C in human genome, and D in coligenome.
Fig. 4 is a front and back physical diagram of the integrated microfluidic chip.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
The examples are not to be construed as a specific technique or condition, and are carried out according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sam Brooks et al, J. Mi. Cloning Experimental guidelines, third edition, scientific Press, et al, huang Peitang et al) or according to the specifications of the product.
1. Reagent and instrument
(1) The information on the reagents used in this application is detailed in Table 1 below.
Table 1 reagents and manufacturer
(2) The instrument information used in this application is detailed in table 2 below.
Table 2 instrument
| Instrument for measuring and controlling the intensity of light | Manufacturer' s | Model number |
| One ten thousandth balance | METTLER-TOLEDO, Inc. | AL104 |
| Real-time fluorescent quantitative PCR instrument | Thermo Fisher Scientific | ABI 7500 |
| Small-sized desk type vortex mixing instrument | HANGZHOU MIU INSTRUMENTS Co.,Ltd. | MIX-28+ |
| Palm type centrifugal machine | Linbel instruments manufacturing Co.Ltd in Jiangsu sea | LX-100 |
| Blowing drying box | SHANGHAI YIHENG INSTR Co.,Ltd. | DHG-9030A |
| Vacuum freeze drier | Sihuan Keyi (Tianjin) Technology Co.,Ltd. | LGJ-20 |
The whole equipment used in the application, namely a PCR reaction device is shown in patent 202110057507.0, and a chip device for detecting nucleic acid is shown in 202110055537.8.
Example 1 primer probe composition for detecting hepatitis C Virus
The primer probe composition comprises a primer probe composition for detecting the hepatitis C virus and a primer probe composition for detecting an internal reference.
The primer probe composition for detecting the hepatitis C virus is an HCV primer probe composition.
The primer probe composition for detecting the internal reference is an IPC primer probe composition.
(1) The HCV primer probe composition comprises:
F：SEQ ID NO:1：5'-ACTACTGTCTTCACGCAGAAAGC-3'；
R：SEQ ID NO:2：5'-TTCCGCAGACCACTATGGC-3'；
P：SEQ ID NO:3：5'-TCGTGCAGCCTCCAGGACCCC-3'；
(2) The IPC primer probe composition comprises:
F：SEQ ID NO:4：5'-AGTTGCAGTGTAACCGTCATGTA-3'；
R：SEQ ID NO:5：5'-TCGACGAGACTCTGCTGTTAA-3'；
P：SEQ ID NO:6：5'-CAGTAATCTGCGTCGCACGTGTGCA-3'。
the primer probe composition of the hepatitis C virus is used for detecting 5' -non coding region of a hepatitis C virus genome.
Example 2 Integrated microfluidic chip kit for detecting hepatitis C Virus
(1) The primer probe composition of example 1 was included, wherein the final concentration of HCV primer was 0.2. Mu.M, probe was 0.15. Mu.M, the final concentrations of IPC primer were 0.15. Mu.M, and probe concentrations were 0.1. Mu.M.
(2) The method also comprises a purifying reagent, an air-drying RT-qPCR reaction reagent and a freeze-drying IPC process quality control product.
1) The purification reagent comprises 400 mu L of lysate and 850 mu L of eluent, and 15 mu L of magnetic beads, wherein the lysate comprises 6M guanidine hydrochloride, 0.4M sodium acetate (pH 4.7) and 2% Triton X-100, the eluent comprises 10mM Tris-HCl (pH 8.5), the purification reagent is added into a sample layer in a chip, and the magnetic bead drying method is disclosed in patent 202110222434.6.
2) The air-dried RT-qPCR reaction reagent system is shown in Table 3 below.
TABLE 3 air-drying RT-qPCR reaction reagent system
| Composition of the components | |
| 4×Air-Dryable 1-Step RT-qPCR Mix | 12.5μL |
| Primer probe mixture | 2.5μL |
And adding the reagent in the table 3 into a reaction bin of the integrated microfluidic chip, placing the chip into a blast drying box, and performing Air drying operation according to an Air-dry 1-Step RT-qPCR Mix instruction book.
3) The quality control of the freeze-drying IPC process comprises the preparation of IPC pseudoviruses and a freeze-drying reagent system.
A pseudovirus of the IPC gene (SEQ ID NO: 7) was constructed and obtained according to the preparation method of the pseudovirus described in example 1 of the patent CN202110669475. X.
The lyophilized IPC reagent system is shown in table 4 below.
Table 4 freeze-dried IPC reagent system
| Composition of the components | Final concentration |
| IPC pseudovirus | 1×10 6 copies/mL |
| Mannitol | 10％ |
| Trehalose | 10％ |
| 2-hydroxypropyl-beta-cyclodextrin HP-beta-CD | 0.1％ |
| Bovine serum albumin BSA | 0.5mg/mL |
| Defoaming agent SE-15 | 0.009％ |
| Tris-HCl(pH 8.0) | 10mM |
| NaCl | 50mM |
| Tween20 | 0.05％ |
The above-mentioned reagents of Table 4 were lyophilized into pellets by dropping every 5. Mu.L into liquid nitrogen, and then lyophilized in a penicillin bottle according to the lyophilization procedure of the lyophilization machine of Table 5.
Table 5 lyophilization procedure
And (3) putting the freeze-dried balls on a chip of the air-dried reaction reagent, and assembling the integrated detection kit.
Example 3 method of Using hepatitis C Virus detection kit
The detection of hepatitis C virus described herein was performed using CarryOn P1000Q, the specific detection procedure is shown in Table 6 below.
TABLE 6
| Step (a) | Operation of | Time | Accumulated time | Environment (environment) |
| 1. Starting up the device | Manual work | 5 seconds | 00：05 | Quarantine site |
| 2. Preparation of nasopharyngeal swab samples | Manual work | For 1 minute | 01：05 | Quarantine site |
| 3. Taking appropriate amount of nasopharyngeal swab sample, and adding into chip | Manual work | 15 seconds | 01：20 | Quarantine site |
| 4. Clicking the "detect" button of the home page | Manual work | 5 seconds | 01：25 | Quarantine site |
| 5. Scanning chip two-dimensional code identification chip and inserting equipment | Manual work | 15 seconds | 01：40 | Quarantine site |
| 6. One-click operation device | Manual work | 5 seconds | 01：45 | Quarantine site |
| 7. Sample processing | Automatic machine | For 1 minute | 02：45 | On-chip |
| 8. Nucleic acid extraction purification | Automatic machine | 14 minutes | 16：45 | On-chip |
| 9. Reverse transcription (reagent-containing redissolution) | Automatic machine | For 10 minutes | 26：45 | On-chip |
| 10. Real-time fluorescence PCR (45 cycles) | Automatic machine | 31 minutes | 57：45 | On-chip |
| 11. Interpretation, output and reporting of the result | Automatic machine | 0 seconds | 57：45 | On-chip |
| 12. Taking out the chip and discarding the waste to a biosafety waste bin | Manual work | 10 seconds | 57：55 | Quarantine site |
| 13. Resetting the device, sterilizing the interior, waiting for inspection or shutting down | Automatic machine | 20 seconds | 58：15 | Inside the device |
The real-time quantitative PCR reaction procedure was: 55 ℃ for 10min;95 ℃ for 1min; (95 ℃ C. 10s,60 ℃ C. 20 s), 45cycles.
Experimental example 1 sensitivity detection
Hepatitis C virus ribonucleic acid serum liquid standard (BDS-BW-013) was purchased from Bruce Biotech Inc. Standard substance concentration of 1.62X10 5 IU/mL, 200. Mu.L of sample loading, 2X 10 of sample input 3 IU、5×10 2 The extracted nucleic acid samples were examined according to the systems and procedures of examples 1 to 3 described above, with IU, 125IU and 25IU, and the examination results are shown in Table 7 below, and the examination curves are shown in FIG. 1.
TABLE 7
As shown in Table 7, the primer composition and the kit thereof have better sensitivity, and the sensitivity can reach 125IU/mL.
Experimental example 2 accuracy test
Using the above-mentioned RNA standard substance for hepatitis C virus, the sample was diluted to 25IU (200. Mu.L in sample size), and the extracted nucleic acid samples were subjected to repeated six times of detection in accordance with the system and procedure of examples 1 to 3, and the detection results are shown in Table 8 below.
TABLE 8
| Sample of | HCV | IPC |
| First time | 37.43 | 27.49 |
| Second time | 37.76 | 27.51 |
| Third time | 37.20 | 28.15 |
| Fourth time | 37.53 | 27.38 |
| Fifth time | 37.25 | 27.97 |
| Sixth time | 37.49 | 28.46 |
| Maximum relative deviation | 0.85％ | 2.28％ |
| Coefficient of Variation (CV) | 0.54％ | 1.56％ |
As shown in Table 8, the primer composition and the kit thereof have good repeatability, the input amount of 25IU can be stably detected, and the amplification curve of the equipment result is shown in FIG. 2.
Experimental example 3 specificity detection
The specificity test of the kit is carried out by using human whole genome, colibacillus whole genome and hepatitis B virus DNA liquid serum standard substance and HIV-1RNA liquid indoor quality control substance. Wherein the sample volumes of the human genome and the E.coli genome were 200. Mu.L, and the input amount was 1. Mu.g. The hepatitis B virus DNA liquid serum standard substance (BDS-BW-009) and the HIV-1RNA liquid indoor quality control substance (BDS-IQC-151) are purchased from Guangzhou Bangding biosciences, inc., and 200 mu L of the hepatitis B virus DNA liquid serum standard substance and the HIV-1RNA liquid indoor quality control substance are directly taken and added into a sample bin of equipment for detection.
The above-extracted samples were examined according to the system and procedure of examples 1 to 3 described above, and the examination results are shown in Table 9 below.
TABLE 9
| Sample of | HCV | IPC | Target detection results |
| HBV DNA standard substance | — | 27.88 | Negative of |
| Indoor quality control product of HIV-1RNA | — | 27.65 | Negative of |
| Human genome | — | 28.74 | Negative of |
| Coli genome | — | 28.78 | Negative of |
As shown in FIG. 3, the results of the specific detection of HCV are negative in all samples, and the primer composition and the kit have better specificity.
The foregoing is a description of embodiments of the invention, which are specific and detailed, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Sequence listing
<110> Beijing midwifery Instrument science and technology Co., ltd
<120> primer probe composition for detecting hepatitis C virus and integrated microfluidic chip kit
<130> 20220617
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
ttccgcagac cactatggc 19
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<213> Artificial sequence (artificial sequence)
<400> 3
tcgtgcagcc tccaggaccc c 21
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<213> Artificial sequence (artificial sequence)
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agttgcagtg taaccgtcat gta 23
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<213> Artificial sequence (artificial sequence)
<400> 5
tcgacgagac tctgctgtta a 21
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<213> Artificial sequence (artificial sequence)
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cagtaatctg cgtcgcacgt gtgca 25
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<213> Artificial sequence (artificial sequence)
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agttgcagtg taaccgtcat gtaccagtaa tctgcgtcgc acgtgtgcac ctagtctaat 60
cacttatgac tcagataact taacagcaga gtctcgtcga 100
Claims (10)
1. A primer probe composition for detecting hepatitis c virus, characterized in that: the primer probe composition comprises a primer probe composition for detecting the hepatitis C virus and a primer probe composition for detecting an internal reference;
the primer probe composition for detecting the hepatitis C virus is an HCV primer probe composition;
the primer probe composition for detecting the internal reference is an IPC primer probe composition;
the HCV primer probe composition comprises:
F：SEQ ID NO:1：5'-ACTACTGTCTTCACGCAGAAAGC-3'；
R：SEQ ID NO:2：5'-TTCCGCAGACCACTATGGC-3'；
P：SEQ ID NO:3：5'-TCGTGCAGCCTCCAGGACCCC-3'；
the IPC primer probe composition comprises:
F：SEQ ID NO:4：5'-AGTTGCAGTGTAACCGTCATGTA-3'；
R：SEQ ID NO:5：5'-TCGACGAGACTCTGCTGTTAA-3'；
P：SEQ ID NO:6：5'-CAGTAATCTGCGTCGCACGTGTGCA-3'。
2. use of the primer probe composition of claim 1 in the preparation of a product for detecting hepatitis c virus.
3. The use according to claim 2, characterized in that: the product comprises independent reagents, chips or kits.
4. A product for detecting hepatitis c virus, characterized in that: the product comprising the primer probe composition of claim 1.
5. The product according to claim 4, wherein: the product also comprises a purifying reagent, an air-dried RT-qPCR reaction reagent and a freeze-dried IPC process quality control product.
6. The product according to claim 5, wherein: the purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads.
7. The product according to claim 5, wherein: the sequence of the quality control product in the freeze-drying IPC process is SEQ ID NO 7;
the quality control product in the freeze-drying IPC process is RNA pseudovirus;
the freeze-drying system of the freeze-drying IPC process quality control product comprises mannitol, trehalose, bovine serum albumin, a defoaming agent, 2-hydroxypropyl-beta-cyclodextrin, tris-HCl (pH 8.0), naCl and Tween20.
8. The product according to claim 5, wherein: the freeze-drying IPC process quality control product is packaged in an integrated micro-fluidic chip in a freeze-drying ball mode.
9. An integrated microfluidic chip kit for detecting hepatitis c virus, which is characterized in that:
the kit comprises the primer probe composition of claim 1;
the kit also comprises a purification reagent, an air-dried RT-qPCR reaction reagent and a freeze-dried IPC process quality control product;
the purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads;
the sequence of the quality control product in the freeze-drying IPC process is SEQ ID NO 7;
the quality control product in the freeze-drying IPC process is RNA pseudovirus;
the freeze-drying system of the freeze-drying IPC process quality control product comprises mannitol, trehalose, bovine serum albumin, a defoaming agent, 2-hydroxypropyl-beta-cyclodextrin, tris-HCl (pH 8.0), naCl and Tween20.
The freeze-drying IPC process quality control product is packaged in an integrated micro-fluidic chip in a freeze-drying ball mode.
The kit completes the extraction, purification, amplification and detection of the nucleic acid of the sample on an integrated chip.
10. A method for detecting hepatitis c virus, said method being a non-disease diagnostic and therapeutic method, characterized in that: the method comprises detecting hepatitis c virus in a sample to be detected using the primer probe composition of claim 1, the product of any one of claims 4-8, or the kit of claim 9.
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