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Detection method of hepatitis B virus drug-resistant mutation

A technology for hepatitis B virus and drug-resistant mutations, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., to achieve the effects of improving utilization, high detection specificity, and increased detection throughput

Inactive Publication Date: 2017-02-01
XIAMEN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In summary, although there are many methods for detecting HBV drug-resistant mutations at home and abroad, they all have certain limitations. Therefore, it is necessary to develop a fast, simple, sensitive, specific, stable and reliable high-throughput HBV drug-resistant mutation detection method. Detection method

Method used

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  • Detection method of hepatitis B virus drug-resistant mutation
  • Detection method of hepatitis B virus drug-resistant mutation
  • Detection method of hepatitis B virus drug-resistant mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The detection results of wild-type HBV (WT) and three drug-resistant mutant HBV (204V, 204I and 204S) in the ROX channel are as follows: Figure 4 shown.

[0044] The detection process of this embodiment is as follows:

[0045] (1) Prepare the following PCR amplification systems respectively: 75mmol / L Tris-HCl pH 8.5, 20mmol / L (NH 4 ) 2 SO 4 ,0.1% Tween20, 2.5mmol / L MgCl 2 , 200 μmol / L dNTPs, 1U Taq polymerase, 0.04 μmol / L upstream PCR amplification primer, 0.4 μmol / L downstream PCR amplification primer, 0.2 μmol / L fluorescent probe, wild-type HBV (WT) and three drug-resistant mutations Type HBV (204V, 204I and 204S) DNA template each 5μl. The base sequence of the upstream PCR amplification primer is 5'-AGACTCGTGGTGGACTTCTCTCA-3'; the base sequence of the downstream PCR amplification primer is 5'-TTGACATACTTTTCCAATCAAT-3'. The base sequence of the fluorescent probe is 5'-TGGCTTTCAGTTATGTGGATGATTGGT-3', the 5' end is marked with ROX fluorescent group, and the 3' en...

Embodiment 2

[0049] The detection results of wild-type HBV (WT) and two kinds of drug-resistant mutant HBV (173L and 173G) in the FAM channel are as follows: Figure 5 shown.

[0050] The detection process of this embodiment is as follows:

[0051] (1) Prepare the following PCR amplification systems respectively: 75mmol / L Tris-HCl pH 8.5, 20mmol / L (NH 4 ) 2 SO 4 , 0.1% Tween20, 2.5mmol / L MgCl2, 200μmol / L dNTPs, 1U Taq polymerase, 0.04μmol / L upstream PCR amplification primer, 0.4μmol / L downstream PCR amplification primer, fluorescent probe 0.2μmol / L, wild type 5 μl each of HBV (WT) and two drug-resistant mutant HBV (173L and 173G) DNA templates. The base sequence of the upstream PCR amplification primer is 5'-AGACTCGTGGTGGACTTCTCTCA-3'; the base sequence of the downstream PCR amplification primer is 5'-TTGACATACTTTTCCAATCAAT-3'. The base sequence of the fluorescent probe is 5'-CTATGGGAGTGGGCCTCAGT-3', the 5' end is marked with a FAM fluorescent group, and the 3' end is marked with a BH...

Embodiment 3

[0055] The present invention is to wild-type HBV (WT) detection sensitivity investigation result such as Figure 6 Shown; Drug-resistant mutant HBV (204V) detection sensitivity investigation result is as follows Figure 7 shown.

[0056] The detection process of this embodiment is as follows:

[0057] (1) Prepare the following PCR amplification systems respectively: 75mmol / L Tris-HCl pH 8.5, 20mmol / L (NH 4 ) 2 SO 4 ,0.1% Tween20, 2.5mmol / L MgCl 2 , 200 μmol / L dNTPs, 1U Taq polymerase, 0.04 μmol / L upstream PCR amplification primer, 0.4 μmol / L downstream PCR amplification primer, 0.2 μmol / L fluorescent probe, the concentration is 1×10 6 ,1×10 5 ,1×10 4 ,1×10 3 ,1×10 2 ,1×10 1 5 μl each of wild-type HBV (WT) and drug-resistant mutant HBV (204V) DNA templates / μL.

[0058] The base sequence of the upstream PCR amplification primer is 5'-AGACTCGTGGTGGACTTCTCTCA-3';

[0059] The base sequence of the downstream PCR amplification primer is 5'-TTGACATACTTTTCCAATCAAT-3'.

[...

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Abstract

The invention discloses a detection method of hepatitis B virus drug-resistant mutation, and relates to hepatitis B viruses. The detection method comprises the following steps: 1) designing a PCR amplification primer in a relatively conservative area on an HBV (hepatitis B virus) polymerase gene sequence; 2) designing a fluorescent probe which can cover HBV drug-resistant mutation sites; 3) identifying characteristic melting-point temperature values of the fluorescent probe and the PCR amplification product after hybridization by virtue of a melting curve analysis technology; and 4) judging whether the drug-resistant mutation occurs in the prepared HBV polymerase gene sequence, and identifying the type of the HBV drug-resistant mutation. The detection method is comprehensive in the coverage of drug-resistant mutation sites, high in detection efficiency and short in consumed detection time; the detection adopts homogeneous detection and closed-tube operation; the detection method is simple to operate and high in detection throughput; and the detection method is high in detection specificity and is capable of judging and reading results easily.

Description

technical field [0001] The invention relates to hepatitis B virus (HBV), in particular to a method for detecting the drug-resistant mutation of the hepatitis B virus. Background technique [0002] HBV is one of the main pathogens causing viral hepatitis, and the possibility of primary liver cancer in people with chronic HBV infection is more than 300 times that of normal people. There are two main categories of clinical drugs currently used in the treatment of HBV: interferons and nucleoside analogs. Interferon drugs mainly inhibit the virus by activating the expression of various antiviral proteins in cells. Interferon therapy has the advantage of low recurrence rate after successful treatment, but the longer onset time and more contraindications limit the clinical application of interferon. Nucleoside analogs mainly block viral replication by competitively binding to the dNTP binding site of HBV polymerase, thereby rapidly and effectively reducing the viral load in vivo....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6858C12Q1/706C12Q2600/156C12Q2563/107C12Q2527/107
Inventor 廖逸群李庆阁许晔宋娜杰占伟牟毅吴玉珍
Owner XIAMEN UNIV
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