Group of nucleotide sequences for detecting gene mutants of hepatitis B virus (HBV) and use thereof

A nucleotide sequence, nucleotide technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low sensitivity, high cost, low efficiency, etc., to improve detection efficiency, The effect of reducing inspection costs

Inactive Publication Date: 2011-08-10
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The purpose of the present invention is to provide a group of nucleotide sequences for detecting HBV gene mutations, and provide the application of the above-mentioned nucleotide sequences in the process of detecting HBV gene mutations, aiming to solve the sensitivity of existing HBV gene mutation site detection methods Low, low efficiency and high cost problems

Method used

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  • Group of nucleotide sequences for detecting gene mutants of hepatitis B virus (HBV) and use thereof
  • Group of nucleotide sequences for detecting gene mutants of hepatitis B virus (HBV) and use thereof
  • Group of nucleotide sequences for detecting gene mutants of hepatitis B virus (HBV) and use thereof

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1: Use the nucleotide sequence of the present invention to detect artificially synthesized nucleotide fragments containing HBV gene mutation sites on a mass spectrometry platform

[0038] In this embodiment, by using the nucleotide sequence of the present invention, one or all of rtI169T, rtV173L, rtL180M, rtA181V, rtA181T, rtT184G, rtA194T, rtS202I, rtM204V, rtM204I, rtN236T and rM250V in HBV can be detected simultaneously. Several mutations.

[0039] (1) Artificially synthesized 3 nucleotide fragments with a length of 420bp, among which fragment 1 includes 4 gene mutation sites of rtI169T, rtV173L, rtL180M and rtA181V, and fragment 2 includes rtA181T, rtT184G, rtA194T, rtS202I and rtM204V these five gene mutation sites, segment 3 includes these four gene mutation sites rtM204I, rtN236T and rM250V. These three nucleotide fragments will be used as detection templates to verify the nucleotide sequence combination we invented.

[0040] (2) Primer design

[0...

Embodiment 2

[0088] Embodiment 2: Use the nucleotide sequence of the present invention to detect actual clinical samples on a mass spectrometry platform

[0089] In this embodiment, by using the nucleotide sequence of the present invention, one or all of rtI169T, rtV173L, rtL180M, rtA181V, rtA181T, rtT184G, rtA194T, rtS202I, rtM204V, rtM204I, rtN236T and rM250V in HBV can be detected simultaneously. Several mutations.

[0090] (1) PCR amplification

[0091] The PCR reaction fragment length is 300bp, the reaction system is 5 μL, which contains 1 μL artificially synthesized DNA template, 3.5mM Mg 2+ , 10×buffer, 100nM amplification primer, 500μM dNTP, 0.5U HotstarTaq enzyme (except primers, the rest were purchased from Qiagen, Germany); reaction conditions: 95°C for 15min; 45 cycles (94°C for 20s, 56°C for 30s, 72°C for 1min); 72°C for 3min. After the PCR is completed, the copy number of the nucleotide sequence with the mutation site in the artificially synthesized DNA template is amplifi...

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Abstract

The invention relates to a group of nucleotide sequences for detecting the gene mutants of HBV, which comprise at least one of the nucleotide sequences from SEQ ID No.1 to SEQ ID No.15. The invention also provides the use of the sequences in detection of the gene mutants of HBV. When the nucleotide sequences are used, 10 common gene mutation loci of HBV can be detected. When used for detecting the gene mutants of the HBV, the nucleotide sequences have the advantages of high throughput, high sensitivity and high specificity, are low in detection cost and have a practical clinic promotion and application value.

Description

technical field [0001] The invention belongs to the field of virus gene detection, and in particular relates to a group of nucleotide sequences for detecting HBV gene mutations and applications thereof. Background technique [0002] Hepatitis B virus (HBV) belongs to the hepadnaviridae family, and its genome is about 3.2kb in length, which is a partially double-stranded circular DNA structure. The HBV genome contains four partially overlapping open reading frames, namely pre-S / S region, pre-C / C region, P region and X region. There may be differences in the gene sequences of different HBV strains in the same patient, but they are highly related genetically. Studies have shown that the virus in each patient is a virus group composed of virus strains with differences in gene sequences. The dominant group and the disadvantaged group are relative and are always in constant change. The replication speed of HBV is very fast. It is estimated that HBV can replicate 10 times per 24 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张宇清刘海龙应康李建和
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