CN108018376B

CN108018376B - Probe and method for hepatitis C virus typing and drug-resistant site detection

Info

Publication number: CN108018376B
Application number: CN201610929662.6A
Authority: CN
Inventors: 钟宏彬; 朱小龙; 姚秋林; 汪智蛟; 陈俊清; 刘娜
Original assignee: Shenzhen Huada Yinyuan Pharmaceutical Technology Co Ltd
Current assignee: Shenzhen Huada Yinyuan Pharmaceutical Technology Co Ltd
Priority date: 2016-10-31
Filing date: 2016-10-31
Publication date: 2020-11-03
Anticipated expiration: 2036-10-31
Also published as: CN108018376A

Abstract

The invention discloses a probe and a method for hepatitis C virus typing and drug-resistant site detection. The nucleotide sequence of the probe is shown as SEQ ID NO: 1-17. The probe provided by the invention can be used for hepatitis C virus typing and drug-resistant site detection, can be used for typing and drug-resistant site detection of multiple (total number > 46) HCV virus subtypes, can effectively screen appropriate drugs, avoids using immunized drugs, and achieves personalized treatment.

Description

Probe and method for hepatitis C virus typing and drug-resistant site detection

Technical Field

The invention relates to the technical field of hepatitis C virus detection, in particular to a probe and a method for hepatitis C virus typing and drug-resistant site detection.

Background

Hepatitis C Virus (HCV) is a single positive-stranded RNA virus with a genome of approximately 9.6kb in length. The HCV genome consists of two non-coding regions (UTRs) at both ends and an Open Reading Frame (ORF) in the middle. The ORF region is further divided into a C region, an E1 region, an E2 region, an NS1 region, an NS2 region, an NS4A region, an NS4B region, an NS5A region and an NS5B region in sequence. Because the polymerase on which HCV replicates lacks a proofreading function and is itself prone to mutation for environmental adaptation and evasion of immune surveillance by the host, a number of mutant strains, i.e., genotypes, are gradually formed during long-term evolution. HCV genotypes are divided into 6 genotypes (1-6), and each genotype is divided into various subtypes such as a/b/c, and at present, more than 80 subtypes are existed.

The hepatitis C caused by different genotypes of HCV, the judgment of the disease condition degree, the use of antiviral drug treatment and the like have obvious differences. HCV type 1B is currently thought to be associated with severe hepatitis and progressive disease, accounting for 80% of the total. It is also common in type 1b among patients in decompensated stage of cirrhosis, primary liver cancer and recurrence after liver transplantation. Amoroso et al (1998) found that 92% of patients with acute HCV type 1b infection develop chronic hepatitis C, while the probability of transformation of other genotypes is 35% -50%. Studies in zingiberone et al (2015) found that infection with HCV of different genotypes was associated with viral concentration of HCV in blood, with patients with genotype 1 having higher HCV concentrations than patients with non-type 1; patients with different genotypes of HCV need different antiviral drugs, for example, patients with type III HCV infection have a significantly higher complete response rate to IFN-alpha 2b than those with type II HCV infection (P <0.005), while those with type III HCV infection have a significantly lower non-response rate to IFN-alpha 2b than those with type II HCV infection (P < 0.005). The combination of Telaprevir with pegylated interferon (PEG-IFN)/ribavirin is limited to patients infected with HCV genotype 1; the european association for hepatopathy (EASL) published the latest "hepatitis c treatment guidelines" online 4 months 2014, giving corresponding treatment regimens for various genotypes of HCV, and indicating that HCV genotypes and genotype 1 subtype (1a/1b) should be evaluated to determine the choice of treatment regimen before initiating treatment.

The site mutation caused by the characteristics of high natural mismatch rate and low fidelity of RNA polymerase when HCV virus is replicated forms virus resistance after the medicine is used for a period of time, causes virus rebound, and limits the clinical application of DAA. The HIV Research association Forum (Forum for collectivity HIV Research) reported clinically relevant HCV mutation sites and drug resistance analysis in 2012. In contrast, the agency and drug evaluation and Research Center (Center for drug evaluation and Research) published a more detailed and more drug-resistant site report related to HCV DAA drug therapy in 2015, which lists mutation sites related to the regions of HCV NS3, NS5A, and NS5B, site mutations generated after various DAA drugs were used, and wild-type replicon fold analysis (fold-change), and drug-resistant sites, HCV genotypes, and drug-resistance analysis (fold-resistance) corresponding to various DAA drugs.

HCV genotyping methods mainly include molecular methods (including sequencing, specific primer PCR, specific probe hybridization (LIPA), gene chip, Restriction Fragment Length Polymorphism (RFLP), etc.), serological methods (including recombinant immunoblot assay (RIBA) and Enzyme Immunoassay (EIA)). Among them, the sequencing method amplifies representative gene fragments such as 5' NTR region, C region, NS5B region by PCR and then performs nucleotide sequence determination, which is the "gold standard" for HCV genotyping. These methods are effective in distinguishing major genotypes, but only direct sequencing of nucleotides is effective in distinguishing subtypes.

The existing HCV genotyping technology mainly aims at detecting and analyzing a specific certain gene region in a genome, and part of methods are unstable and have low accuracy, and can only be used for genotyping to the genotype, while the subtype typing capability is very limited. The existing typing method also cannot detect drug-resistant sites basically.

Disclosure of Invention

The invention provides a probe and a method for typing of hepatitis C virus and detecting drug-resistant sites, which can be used for typing of various HCV virus subtypes and detecting drug-resistant sites, can effectively screen appropriate drugs, avoids using immunized drugs and achieves personalized treatment.

According to a first aspect of the present invention, the present invention provides a probe for hepatitis c virus typing and drug-resistant site detection, the nucleotide sequence of the probe is as shown in SEQ ID NO: 1-17.

According to a second aspect of the present invention, the present invention provides a kit for hepatitis c virus typing and drug-resistant site detection, the kit comprising a probe, the nucleotide sequence of the probe is as shown in SEQ ID NO: 1-17.

Further, the kit further comprises: a reagent component for extracting hepatitis C virus RNA.

Further, the kit further comprises: a reagent composition for reverse transcription of hepatitis c virus RNA.

Further, the kit further comprises: and (3) a component for constructing a library of cDNA obtained by reverse transcription of hepatitis C virus RNA.

Further, the above components for constructing a cDNA library include: a component for end repair, a component for adding A bases to the 3' end, a component for linker ligation, and a component for PCR amplification.

Further, the kit further comprises: components for library capture.

According to a third aspect of the present invention, the present invention provides a gene chip for hepatitis c virus typing and drug-resistant site detection, the gene chip comprising a solid support and a probe, the nucleotide sequence of the probe is as shown in SEQ ID NO: 1-17.

According to a fourth aspect of the present invention, there is provided a method for typing hepatitis C virus, the method comprising the steps of: extraction and treatment of nucleic acid: extracting virus RNA from whole blood or separated plasma sample by using RNA virus extraction reagent; then carrying out reverse transcription to synthesize RNA into double-stranded cDNA; construction of the library: constructing a library of the double-stranded cDNA obtained above; library capture: using a nucleotide sequence as set forth in SEQ ID NO: 1-17 to perform hybrid capture on the constructed library; sequencing: performing high-throughput sequencing on the captured library; information analysis: and removing the human source sequence from the obtained original off-line data, and then comparing the obtained original off-line data with the existing HCV reference sequence to obtain the final sample HCV whole gene sequence and the hepatitis C virus typing.

According to a fifth aspect of the present invention, there is provided a method for hepatitis c virus resistance site detection, the method comprising the steps of: extraction and treatment of nucleic acid: extracting virus RNA from whole blood or separated plasma sample by using RNA virus extraction reagent; then carrying out reverse transcription to synthesize RNA into double-stranded cDNA; construction of the library: constructing a library of the double-stranded cDNA obtained above; library capture: using a nucleotide sequence as set forth in SEQ ID NO: 1-17 to perform hybrid capture on the constructed library; sequencing: performing high-throughput sequencing on the captured library; information analysis: and removing the human source sequence from the obtained original off-line data, and then comparing the obtained original off-line data with the existing HCV reference sequence to obtain the final sample HCV whole gene sequence, and corresponding mutation information and drug resistance sites.

The probe provided by the invention can be used for hepatitis C virus typing and drug-resistant site detection, can be used for typing and drug-resistant site detection of multiple (total number > 46) HCV virus subtypes, can effectively screen appropriate drugs, avoids using immunized drugs, and achieves personalized treatment.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments.

The invention carries out probe design based on the genome regions of 46 HCV gene subtypes, can provide more accurate and stable detection results through capture and next-generation sequencing technologies, and can accurately classify most HCV subtypes; meanwhile, the invention can be used for detecting drug-resistant sites and provides more accurate guidance for drug administration.

Firstly, designing and synthesizing a probe:

downloading 46 HCV gene subtypes from NCBI (http:// www.ncbi.nlm.nih.gov /) as reference sequences for probe design; removing repeated sequences among genomes, and only keeping one copy; by comprehensively considering the information of probe length, Tm value, repetitive sequence ratio, GC content, specificity, target region coverage depth and the like, each probe length is designed to be 90bp, and the allowed maximum GC content is 60%. After the probe is designed, an oligonucleotide (oligo) synthesizer is used for synthesis, and a matched using reagent is configured.

Probe sequences designed based on genomes of two HCV subtypes 2a and 1b common in China and related to drug-resistant sites are core sequences, and are specifically shown in Table 1.

TABLE 1

Secondly, the probe uses an experimental technology:

1. extraction and processing of nucleic acids

The sample is whole blood or separated plasma, plasma virus is extracted by adopting an RNA virus extraction kit, then reverse transcription is carried out, double-stranded DNA is synthesized, and RT-PCR (reverse transcription-PCR amplification) is carried out for subsequent experiments.

2. Construction of the library

And constructing the Hiseq library by adopting a trace nucleic acid library construction process on the obtained cDNA.

3. Chip capture

The constructed library was hybrid captured using HCV virus capture probes (table 1).

4. Sequencing

For the captured library, a Hiseq sequencing platform was used to complete high throughput sequencing using the sequencing length of PE 90.

5. Information analysis strategy

Removing the human source sequence from the obtained original offline data, and then comparing the existing HCV reference sequences to obtain the final sample HCV whole gene sequence and corresponding mutation information.

The technical solutions and technical effects of the present invention are described in detail by the following specific embodiments, and it should be understood that the embodiments are only exemplary and do not limit the scope of the present invention.

Examples

In this example, blood samples of 40 HCV patients were used for detection, and the overall procedure included RNA extraction, reverse transcription of RNA into cDNA, construction of a second generation library, sequencing on a computer, and information analysis.

RNA extraction (using QIAampultrans Virus Kit):

1. 1mL of plasma was taken from each sample and placed in a 2mL centrifuge tube, 0.8mL Buffer AC and 5.6. mu.g carrier RNA solution (carrier RNA solution) were added, mixed by inversion and incubated for 10 minutes;

2. centrifuging after finishing incubation at the rotating speed of 1200 Xg for 3 minutes, and removing supernatant;

3. adding 300. mu.L buffer AR (earlier incubation at 60 ℃) and 20. mu.L proteinase K, shaking and mixing until the magnetic beads are completely resuspended, placing the mixture on a 40 ℃ incubator for incubation for 10 minutes, and slightly centrifuging;

4. adding 300. mu.L buffer AB, mixing and centrifuging;

5. transferring 700 μ L of the mixture to a purification column (QIAamp spin column), centrifuging at 4000 Xg for 1min, and removing the filtered liquid;

6. add 500. mu.L buffer AW1, centrifuge for 1min at 6000 Xg, remove filtrate, and transfer the column to a new 2mL collection tube, add 500. mu.L buffer AW2, centrifuge for 3 min at 20000 Xg;

7. transferring the purified column to a new 1.5mL collection tube, adding 30. mu.L buffer AVE to dissolve RNA, and centrifuging at 6000 Xg for 1 min; then 30. mu.L of buffer AVE was added, and the mixture was centrifuged at 6000 Xg for 1 min;

8. the purification column was removed to obtain a purified product after filtration.

Second, RNA reverse transcription (reagent from INVITROGEN)

1. mu.L of 5 Xfirst strand buffer (one-strand synthesis buffer) was added to 10. mu.L of RNA in each sample for disruption, and the mixture was immediately placed on ice at 94 ℃ for 10min on a PCR instrument;

2. to the RNA of the previous step was added 0.5. mu. L N6 primer (0.1. mu.g/. mu.L). The PCR instrument was kept on ice at 65 ℃ for 5 min.

3. The reaction mixture was prepared according to the following ratio: multiple samples were formulated with about 10% margin.

4. The above mixture (Mix) was added to RNA, mixed and reacted on a PCR instrument according to the following procedure:

5. the synthesized single-stranded cDNA (16 μ L total) was placed on ice to formulate the following reagent Mix: multiple samples were formulated with about 10% margin.

6. The mixture (Mix) was added to the single strand cDNA and mixed well before being placed on a constant temperature mixer (Thermomixer) at 16 ℃ for 2h (300rpm intermittent shaking for 15s, resting for 2 min). After completion, the cDNA product was purified using Ampure XP Beads, dissolved in 43. mu.L EB solution.

Third, library construction

1. End repair (reagent from ENZYMATICS)

The reaction mixture was prepared according to the following ratio:

the reaction was carried out in a thermostatic mixer (Thermomixer) at 20 ℃ for 30 min. After completion of the reaction, the end-repair product was purified with 90. mu.L of Ampure XPBeads (1.8X) and dissolved in 21. mu.L of EB solution.

Addition of ' A ' base to the 3 ' end of cDNA (reagent from ENZYMATICS Co.)

The reaction mixture was prepared according to the following ratio:

the reaction was carried out in a thermostatic mixer (Thermomixer) at 37 ℃ for 30 min. This step did not require purification.

Ligation of DNA adapter and purification of ligation product (reagent from Huada Gene Co., Ltd.)

The reaction mixture was prepared according to the following ratio:

the reaction was carried out in a thermostatic mixer (Thermomixer) at 20 ℃ for 20 min. The ligation was then purified with 47.7. mu.L Ampure XPBeads (1.8X) and finally the sample was dissolved in 25. mu.L EB solution.

Note that: the volume of the solution at this step was adjusted according to the state of the sample. If the initial amount of sample is low or the quality is poor, this step is dissolved in 15.5. mu.L of EB solution and the PCR reaction is performed in its entirety.

PCR (reagents from INVITROGEN Co.)

Dissolving primer (primer)1.0 and label sequence primer (Index primer) in pure water to make its concentration reach 100. mu. mol/L, placing at-20 deg.C, and storing as stock solution; appropriate primer1.0 and Index primer were diluted to 10. mu. mol/L working solution as required and frozen at-20 ℃. If the DNA in the above step is dissolved in 25. mu.L of EB solution, 15.1. mu.L of the DNA is taken out for PCR reaction, and the rest is stored in a refrigerator at-20 ℃.

PCR system and reaction conditions:

the amplification system was as follows:

note that: in addition to DNA and tag sequence primers (10. mu.M), the remaining reagents can be formulated as a reaction mixture (Mix), with multiple samples formulated at about 10% margin. DNA and tag sequence primers (10. mu.M) were added separately.

Reaction procedure: 94 ℃ for 2 min; 15 cycles of 94 ℃ for 15s, 62 ℃ for 30s and 72 ℃ for 30 s; 10min at 72 ℃; infinity at 16 ℃.

After the PCR reaction, the mixture was purified using magnetic beads and dissolved in 15. mu.L of EB solution.

Fourth, library capture (reagent from Huada gene)

1. Probes for the synthesis of HCV subtype reference sequences (table 1);

2. 500ng of each library was placed in a new 1.5mL centrifuge tube, evaporated to dryness by vacuum centrifugation, and re-solubilized with 3.4. mu.L of water.

3. Block # 12.5. mu. L, Block # 22.5. mu.L, common P1Block 1.5. mu.L and Index Block N (200. mu.M) 1.5. mu.L were added, transferred to a PCR tube, denatured at 95 ℃ for 5 minutes, and incubated at 65 ℃ for 5 minutes.

4. A hybridization buffer mixture was also prepared, as shown in Table 2:

TABLE 2

5. 13 mu L of the hybridization buffer mixture is respectively added into the system in the step 3, 5 mu L of HCV probe, 0.5 mu L of RNaseBlock and 1.5 mu L of water are added, and the mixture is mixed evenly. Hybridization was carried out for 24 hours.

6. Preparing Dynabeads magnetic beads:

a) dynabeads M-280Streptavidin magnetic beads were vigorously shaken with a vortex mixer until well mixed.

b) 50 μ L of Dynabeads M-280Streptavidin magnetic beads were taken for each hybridization reaction in a 1.5mL labeled centrifuge tube.

c) Add 200. mu.L Binding Buffer.

d) And (5) violently oscillating and mixing by using a vortex mixer.

e) Placing the centrifuge tube on a magnetic frame, and removing supernatant after the liquid becomes clear.

f) Repeat "step c) to step e)" 2 times.

g) Add 200. mu.L Binding Buffer to resuspend the beads.

7. The mixed system after 24 hours of hybridization was transferred to Dynabeads and incubated at room temperature for 30 minutes.

8. Washing the magnetic beads:

a) taking the sample off the blending device, and performing instantaneous centrifugation to ensure that no liquid remains on the tube cover;

b) transferring the centrifuge tube on Dynal magnetic frame, standing for 1-2min until the liquid in the tube is clear, and removing supernatant;

c) keeping the centrifuge tube on a magnetic frame, adding 500 mu L of SureSelect Wash Buffer #1 heavy suspension magnetic beads, and oscillating on a vortex mixer for 10s to mix the sample uniformly;

d) incubating the sample at room temperature for 15 min;

e) after centrifugation, placing the sample tube on a Dynal magnetic frame, standing for 1-2min until the sample tube is clear, and removing supernatant;

f) keeping the centrifuge tube on a magnetic frame, adding 500 mu L of SureSelect Wash Buffer #2 heavy suspension magnetic beads at 65 ℃, and oscillating on a vortex mixer for 10s to mix the sample uniformly;

g) incubating the sample in a dry bath at 65 ℃ for 10 min;

h) inverting the centrifuge tube to mix the sample, and performing instantaneous centrifugation;

i) placing the centrifuge tube on Dynal magnetic frame, standing for 1-2min to clarify, and removing supernatant;

j) repeat "step f) to step i)" 2 times; removing the Wash Buffer #2 as clean as possible;

k) add 33.5. mu.L of nucleic-Free Water.

Post-PCR reaction system (reagents from Agilent Corp.):

post PCR Mix was prepared according to Table 3 below, 16.5. mu.L Mix was added to each tube, mixed well and centrifuged briefly before being placed in the PCR instrument.

TABLE 3

Reagent	Volume (μ L)
		DNA with magnetic beads	33.5
5X Herculase II Reaction Buffer (Reaction Buffer)	10
		dNTP(100mM)	0.5
HS-EXON-FC-1.1(10μM)	2.5
		HS-EXON-FC-1.2(10μM)	2.5
Herculase II Fusion DNA Polymerase (Fusion DNA Polymerase)	1
		Total volume	50

Post-PCR reaction conditions: 2min at 98 ℃; 20s at 98 ℃, 30s at 60 ℃ and 30s at 72 ℃ for 16 cycles; 5min at 72 ℃; keeping the temperature at 4 ℃.

10. After completion of PCR, purification was performed using magnetic beads, and finally, the solution was dissolved in 30. mu.L of Elution Buffer (Elution Buffer) to complete capture.

Fifth, computer sequencing

Sequencing the library on a computer by using Hiseq 4000 and finally obtaining the data quantity of each sample of which the data quantity is more than 1G by adopting a PE91 sequencing strategy.

Sixthly, data analysis

1) And (4) processing the second-generation sequencing data to remove the influence caused by sequencing errors. The method comprises the following steps: removing the sequence comprising the sequencing linker; removing low quality sequences; a sequence comprising more than five percent of indeterminate bases; the repetitive sequences introduced during the PCR were removed. The output result format is FASTQ, and the quality system is Phred + 64.

2) The host sequence is removed. Since the host of HCV virus is human, and the sequencing result includes a part of human DNA, it is necessary to remove the human-derived sequence by alignment. The comparison software SOAP2(http:// SOAP. genetics. org. cn/# down2), the reference genome hg19(http:// hgdown load. cse. ucsc. edu/goldenPath/hg19/bigZips /), was used, allowing 13 mismatches due to the large difference between the HCV viral genome and the human genome; the results of the simulation data evaluation showed that the proportion of the genomes of the different genotypes of HCV compared to the human genome under the same parameters was less than 0.1%.

3) And (4) HCV genotype identification. Identification is carried out by comparing genome sequences of HCV different genotypes/subtypes (genotype/subtype) and calculating genome coverage; generally, more than 90% coverage is required, more than 100 layers of sequencing depth are required, and the genome type with the optimal coverage depth is selected as the genome type of the sample; the types of listings in the database include 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6 a. Under the same alignment parameters, simulation data evaluation is used, and the results show that the correct genome types are all 99 +% coverage, the proportion of other types is below 30%, and the coverage rate of different assembly results of the same type is above 90%. The alignment software used was BWA (http:// bio-bw. sourceforce. net /); the software that calculates the depth of coverage is soap, coverage (http:// soap, genomics. org. cn/# down 2).

4) And (4) genome assembly. The method is used for the subsequent detection of the mutation sites, and can also be used for the genotype identification of mixed sample types. And automatically selecting qualified parameters through analysis to assemble the genome, wherein the size of the genome in a normal assembly result is 8 k-10 k, and whether HCV viruses with different genotypes are mixed or not needs to be judged for a sample with an assembly result larger than 10 k. The software SOAPdenovo2(https:// github. com/aquaskyline/SOAPdenovo2) was used for genome assembly.

5) And detecting a variation Site (SNP). Wild type genomes H77 and Con1, respectively, are currently selected for reference mainly against common HCV infection genotypes 1a, 1 b. In order to ensure the accuracy and the detectable rate of the found mutation sites, two groups of software, namely mulmer + SOAPpileup and BWA + GATK, are selected for analysis, and results are merged and provided with a credibility label. The sources of the reference genome and related software are as follows:

H77(http://www.ncbi.nlm.nih.gov/nuccore/22129792/)；

Con1(http://www.ncbi.nlm.nih.gov/nuccore/AJ238799)；

mummer(http://mummer.sourceforge.net/)；

GATK(https://software.broadinstitute.org/gatk/)。

6) and (4) gene annotation. At present, HCV virus inhibitors mainly aim at three genes of NS3/NS5A/NS 5B. And annotating the corresponding gene according to the result of the variation detection, and marking the corresponding amino acid variation information on the gene. Non-synonymous mutations in three genes associated with viral inhibitors are of major concern.

7) And (5) detecting drug-resistant sites. The drug resistance site information is derived from published literature data, and mainly comprises two types of HCV 1a and 1 b. And analyzing and annotating the annotated variation detection result and the drug-resistant site database to obtain the drug-resistant site detection result of each sample, including the genotype, the existence or nonexistence of the drug-resistant site, the specific variation condition of the drug-resistant site, the drug-resistant information and the like of each sample.

Seventhly, information analysis results

The results of the typing of the 40 samples are shown in Table 4, and the analysis content includes Rawdata (raw data), cleardata (quality qualified data), Effect data (valid data matched to HCV sequences), HCVgenotype (HCV typing) and coverage (proportion of the reference sequence covered by data).

TABLE 4

After the drug-resistant site detection, only 2 samples are found to have the drug-resistant sites, and the results are shown in table 5.

TABLE 5

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A probe set for hepatitis C virus typing and drug-resistant site detection, comprising a nucleotide sequence set as shown in SEQ ID NO: 1-17.

2. A kit for hepatitis C virus typing and drug-resistant site detection is characterized by comprising a probe set, wherein the probe set comprises a nucleotide sequence shown in SEQ ID NO: 1-17.

3. The kit of claim 2, further comprising: a reagent component for extracting hepatitis C virus RNA.

4. The kit of claim 2, further comprising: a reagent composition for reverse transcription of hepatitis c virus RNA.

5. The kit of claim 2, further comprising: and (3) a component for constructing a library of cDNA obtained by reverse transcription of hepatitis C virus RNA.

6. The kit of claim 5, wherein the components for performing library construction of cDNA comprise: a component for end repair, a component for adding A bases to the 3' end, a component for linker ligation, and a component for PCR amplification.

7. The kit of claim 5 or 6, further comprising: components for library capture.

8. A gene chip for hepatitis C virus typing and drug-resistant site detection is characterized by comprising a solid carrier and a probe set, wherein the probe set comprises a nucleotide sequence shown as SEQ ID NO: 1-17.