A kit for detecting drug-resistant mutations of hepatitis B virus

A hepatitis B virus and drug-resistant mutation technology, applied in the field of medical devices, to achieve good specificity, low false positives, and high degree of automation

Inactive Publication Date: 2011-12-21
李艳 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no literature reporting kits for the detection of drug-resistant mutations in HBV whole gene sequence analysis using gene sequencing technology

Method used

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  • A kit for detecting drug-resistant mutations of hepatitis B virus
  • A kit for detecting drug-resistant mutations of hepatitis B virus
  • A kit for detecting drug-resistant mutations of hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of the kit of the invention

[0033] The composition of the kit of the present invention is as follows:

[0034] (1) HBV DNA extraction reagent: 10mmol / L Tris-HCl (pH8.0), 5mmol / L EDTA, 0.5% SDS, 150μg / ml proteinase K

[0035] (2) Primer:

[0036] The sequence of the upstream primer of HBV DNA Pol gene is: 5’-CTGCTGGTGGCTCCAGTT-3’

[0037] The downstream primer sequence of HBV DNA Pol gene is: 5’-GAGTTCCGCAGTATGGATCG-3’

[0038] The above primer sequence was synthesized by Shanghai Life Technology Company.

[0039] (3) Negative control and positive control: deionized water is used as a negative control, and samples containing HBV DNA are used as a positive control.

[0040] (4) PCR reaction solution: 10×PCR Premix (mixed solution), 0.25 pmol / μL primer, 2.5-4.0 mM MgCl 2 , 2U Taq enzyme, 0.2~0.4 mM dNTPs, 0.3~0.6 mM dUTP, usually 1~2 μL template.

[0041] (5) The setting of PCR amplification program: on the ABI9700 instrument, it is usually 95℃ for 5 min, then 9...

Embodiment 2

[0046] Example 2 Using the kit prepared in Example 1 to detect HBV nucleoside analog resistance gene mutation

[0047] To detect HBV nucleoside analog resistance gene mutations in peripheral blood samples of 20 hepatitis B patients.

[0048] Detection process:

[0049] First, design specific primers based on the HBV RT region gene sequence provided by the HBV nucleic acid database. Obtain peripheral blood samples of clinical hepatitis B patients and quickly extract HBV DNA; configure PCR reaction solution for PCR amplification, then recover PCR amplification products, perform sequencing reactions, and then sequence reaction products after purification, and finally in the NCBI nucleic acid database Perform nucleic acid sequence comparison to find the gene variants of 202, 204, 173, 180, 181, 169, 236, 184, 202, 250 in the reverse transcriptase region.

[0050] Specific steps are as follows:

[0051] (1) Extraction of serum HBV DNA: Extract HBV DNA from peripheral blood of hepatitis B ...

Embodiment 3

[0059] Example 3 Evaluation of the detection capability of the kit

[0060] The detection kit (gene sequencing method) of Example 1 was used to detect HBV resistance in the serum of 30 patients with hepatitis B detected by the probe hybridization method. Experiments show that the sensitivity, specificity and sensitivity of this kit are better than those of probes. The hybrid method is more precise and fully meets the current practical requirements of clinical diagnosis and treatment:

[0061] Comparison of two methods for detecting HBV resistance mutation

[0062]

[0063] among them:

[0064] (1) Specificity: 100%;

[0065] (2) Sensitivity: 95%;

[0066] (3) Positive predictive value: the positive predictive value reaches 100%;

[0067] (4) Negative predictive value: the negative predictive value reaches 91%;

[0068] (5) Repeatability: The results of repeated experiments are consistent;

[0069] (6) Time-consuming: The testing time for a clinical specimen is about 12-14 hours, which is...

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Abstract

The invention relates to a test kit for detecting drug-resistant mutations of hepatitis B virus, comprising HBVDNAPol gene region amplification primers, HBVDNA extraction reagents, negative controls and positive controls, PCR reaction solution and PCR sequencing reagents, wherein the upstream primer sequence of the HBVDNAPol gene is As shown in SEQIDNo.1, the downstream primer sequence of HBVDNAPol gene is shown in SEQIDNo.2. The detection kit of the invention has high detection sensitivity, good specificity and high speed, and can be completed within 12 to 14 hours.

Description

technical field [0001] The invention relates to a kit for detecting drug-resistant mutations of hepatitis B virus, in particular to a kit for detecting drug-resistant mutations of hepatitis B virus by using gene sequencing technology, and belongs to the field of medical devices. Background technique [0002] Hepatitis B is one of the most serious health problems in the world. There are about 2 billion people infected with hepatitis B virus (HBV), of which 350 million are chronically infected. In China, there are about 120 million HBV infections. Hepatitis B infection is the main cause of liver fibrosis, liver failure and liver cancer. Drug resistance of the virus has been a major problem in the treatment of hepatitis B for a long time. HBV drug resistance will lead to serious clinical consequences, and HBV drug resistance needs to be closely monitored during clinical treatment. [0003] At present, the first-line anti-hepatitis B drugs (nucleoside analogues) approved by SF...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 李艳童永清顾剑郑红云
Owner 李艳
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