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Amplification primer, sequencing primer, kit and method for detection of hepatitis C virus genotyping

A technology for hepatitis C virus and genotyping, which is applied in the field of amplification primers, sequencing primers, and kits for detecting hepatitis C virus genotyping, and can solve the problems of inconvenient operation, high price, low sensitivity and specificity and other problems, to achieve the effect of easy detection, high sensitivity and strong specificity

Active Publication Date: 2018-02-02
英潍捷基(上海)贸易有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of reverse hybridization is high, about 5%, but it is expensive and not easy to operate, especially in developing countries, it cannot be fully promoted; PCR-RFLP and real-time PCR can only detect known, single-site Mutations
Other methods have low sensitivity and specificity, can only distinguish several main genotypes, and have insufficient ability to subtype

Method used

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  • Amplification primer, sequencing primer, kit and method for detection of hepatitis C virus genotyping
  • Amplification primer, sequencing primer, kit and method for detection of hepatitis C virus genotyping
  • Amplification primer, sequencing primer, kit and method for detection of hepatitis C virus genotyping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 detects the primers of hepatitis C virus genotyping

[0036] Primers for detection of hepatitis C virus genotyping based on Sanger sequencing technology, including amplification primers and sequencing primers.

[0037]Design PCR primers and corresponding sequencing primers for the core / E1 region of HCV genomic RNA, use primer design software, such as Primer Premier 5, to select a specific sequence from the core / E1 region sequence of HCV genomic RNA, and then base , to design specific primers. The primer sequences are SEQ ID No: 1-2, respectively.

[0038] name

serial number

Sequence 5'-3'

CE-F

SEQ ID NO.1

CGCAATGTGCGTAACGTCAACGATAC

CE-R

SEQ ID NO.2

GTAGGCGACGACTTCTTCATCA

[0039] The design of the sequencing primer is similar to that of the specific primer used to amplify the viral RNA in the sample, but only one sequencing primer is required, and the primer sequence is SEQ ID No:3.

[0040] n...

Embodiment 2

[0041] Embodiment 2 detects the kit of hepatitis C virus genotyping

[0042] The test kit of the detection hepatitis C virus genotyping of the present embodiment comprises the following table components:`

[0043]

[0044] 1. PCR detection reagents, including amplification primers (with base sequences shown in SEQ ID No: 1 and SEQ ID No: 2), Tris hydrochloride Buffer, hot start Taq enzyme (hot start Taq enzyme adopts chemical modification, through Heat shock at 95°C for 15 minutes, Taq enzyme can play a role, hot start Taq enzyme can reduce the formation of primer dimers and increase reaction specificity), reverse transcriptase.

[0045] 2. PCR sequencing reagents, including sequencing primers (with the base sequence shown in SEQ ID No: 3), sequencing dye (BigDye), sequencing reaction solution (BigDye Sequencing Buffer), high-purity deionized formamide (Hi-DiFormamide )

[0046] 3. Quality control products, including negative quality control products and HCV typing positi...

Embodiment 3

[0048] Embodiment 3 detects the method for hepatitis C virus genotyping

[0049] see figure 1 , is the operation flowchart of the method for detecting hepatitis C virus genotyping of the present invention; The method for detecting hepatitis C virus genotyping of the present embodiment comprises the following steps:

[0050] 1. Take out the HCV RT-PCR reaction solution and HCV RT-PCR enzyme system from the kit, thaw at room temperature, shake and mix well, and centrifuge at 8,000rpm for a few seconds before use.

[0051] Take N (N = number of samples to be tested + HCV typing positive quality control + negative quality control) reaction tubes,

[0052] The HCV single RT-PCR amplification system is prepared in the following table

[0053] components

HCV RT-PCR reaction solution

HCV RT-PCR Reaction Solution Enzyme System

total capacity

Dosage

27μl

3μl

30μl

[0054] Mix the components thoroughly, and then centrifuge for a short time...

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Abstract

The invention discloses an amplification primer, a sequencing primer, a kit and a method for detection of hepatitis C virus genotyping. The amplification primer has the base sequences shown as SEQ IDNo:1 and SEQ ID No:2, and the sequencing primer has the base sequence shown as SEQ ID No:3. The method for detection of hepatitis C virus genotyping provided by the invention adopts the originally created primers for amplification and sequencing, and can be used for detection of common genotypes 1b, 2a, 3a, 3b and 6a of human hepatitis C virus.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and more specifically, the invention relates to an amplification primer, a sequencing primer, a kit and a method for detecting hepatitis C virus genotyping. Background technique [0002] Hepatitis C Virus (HCV) is a blood-borne RNA virus that causes chronic liver disease. Its nucleic acid is single-stranded, linear, and positive-strand RNA. The HCV genome is highly heterogeneous due to the lack of proofreading function of the polymerase upon which HCV RNA replication depends. The current international HCV genotype nomenclature system is the Simmonds system, which uses nucleic acid sequencing and phylogenetic tree analysis results to divide HCV into six main genotypes (indicated by 1-6) and a series of subtypes (indicated by a,b) , c etc.). Patients infected with different HCV genotypes have different clinical manifestations, severity of liver disease, and progression of chronic dis...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6869C12R1/93
CPCC12Q1/6869C12Q1/707C12Q2600/112C12Q2531/113C12Q2521/107C12Q2535/101
Inventor 李葆华蒋析文刘悦甘小洪马道亮
Owner 英潍捷基(上海)贸易有限公司
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