Preparation and application of guiding gene chip for HCV infection individual treatment

A hepatitis C virus, gene chip technology, applied in biochemical equipment and methods, microorganism-based methods, chemical libraries, etc., can solve the problems of mixed virus infection, insufficient sensitivity, difficult to promote, etc., and achieve high specificity. Effect

Inactive Publication Date: 2015-02-18
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological methods are fast, simple, low-cost, and widely used clinically, but it is difficult to distinguish when there is mixed virus infection or virus mutation
[0005] 2) Direct sequencing method: direct sequencing using PCR products is the gold standard for typing, but the sensitivity is insufficient and limited by sequencing equipment, it is not easy to promote at the grassroots level
However, this method requires a special fluorescent quantitative PCR instrument and supporting reagents
[0007] 4) Nested PCR method: use two pairs of PCR primers to amplify the fragment, the first pair of PCR primers amplifies

Method used

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  • Preparation and application of guiding gene chip for HCV infection individual treatment
  • Preparation and application of guiding gene chip for HCV infection individual treatment
  • Preparation and application of guiding gene chip for HCV infection individual treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Development of guidance gene chip for individualized treatment of hepatitis C virus infection

[0046] 1. Primer probe design and screening

[0047] First download the HCV gene sequence and the human IL28B gene sequence from the NCBI gene database. After the sequence is downloaded, use the AlignX program in the Vector NTI Advance 10 (invitrogen) software package to perform a global alignment of the gene sequences of each pathogen according to the default parameter settings. Design specific oligonucleotide probes and universal primers at conservative positions in the gene sequence according to the alignment results. The results of agarose electrophoresis of the amplified products of rs12979860 gene fragment are attached figure 2 . After screening, a total of 8 upstream and downstream primers were determined, and the reversed-phase primers were labeled with bio at the 5'end as the primers used in the chip; 16 specific detection probes were determined, and the 3'en...

Embodiment 2

[0057] Example 2: Determination of the criteria for determining the positive determination of the guide gene chip for individualized treatment of hepatitis C virus infection

[0058] Cutoff value is the criterion for judging whether the gene chip signal value is positive. Each typing probe selects non-HCV virus (ie negative strain) and blank control for gene chip hybridization. Through repeated experiments and data statistics, the average background statistics of negative strains and blank control + 2SD are used as each probe The Cutoff value of the probe. The discrimination ability of each mutation detection probe above 2.5 times is used as the criterion for judging whether the site has mutation.

Embodiment 3

[0059] Example 3: Specific evaluation of guidance gene chip for individualized treatment of hepatitis C virus infection

[0060] Specificity is the most important evaluation indicator of the diagnostic method. The gene chip of the present invention uses optimized systems and conditions to detect HIV positive, HBV positive, syphilis positive, adenovirus positive serum and normal blood donor serum. Figure 4 It can be seen that HIV-positive, HBV-positive, syphilis-positive, adenovirus-positive serum and normal blood donor serum are all HCV-negative, and the internal standard probe has a normal signal, indicating that the system is working normally and has good specificity. The present invention detects 5 subtypes of HCV. image 3 It can be seen that all five subtypes can be clearly distinguished, indicating that the present invention has good specificity.

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Abstract

The invention relates to a guiding gene chip for HCV infection individual treatment. The preparation method of the guiding gene chip comprises the following steps: preparing a universal primer; preparing an HCV hypotype nucleic acid parting probe and a human IL-28B rs12979860 investigation of polymorphism probe; preparing an oligonucleotide chip; building a polyfunctional RT-PCR system; building a hybrid system. The gene chip prepared by the method can discriminate five hypotypes of 1b, 2a, 3a, 3b and 6a of HCV at the same time, can detect polymorphism of CC, TT and CT three genes of human rs12979860, has the advantages that the gene chip is quick, accurate, and high in flux and specificity, and can provide guiding for HCV clinical diagnosis and individual treatment.

Description

Technical field [0001] The invention relates to the preparation and application of a guide gene chip for individualized treatment of hepatitis C virus infection, and belongs to the technical field of gene chip detection. Background technique [0002] Hepatitis C virus (hepatitis c virus, HCV) infection is a serious hazard. 170 million patients worldwide are infected with HCV, and there are 3 million new cases each year. 50-80% of people infected with HCV can develop a chronic state, of which 20-30% develop cirrhosis or liver cancer. There are differences in the geographical distribution of different genotypes or subtypes of chronic hepatitis C infection, the effect of antiviral therapy, and the severity of the disease. The genetic polymorphism of human IL-28B is closely related to the viral response rate of antiviral therapy. It is an important predictor of the success of treatment for hepatitis C patients. Individualized treatment of HCV infection can significantly increase th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C40B50/18C12R1/93
CPCC12Q1/707C12Q1/6883C12Q2600/106C12Q2600/156
Inventor 王升启刘琪琦王娈娈陈苏红张敏丽孙晓彦
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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