Kit for detecting drug-resistant mutation in YMDD motif region of hepatitis B virus

A hepatitis B virus and drug-resistant mutation technology, which is applied in the direction of recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., can solve the detection of drug-resistant mutations in the hepatitis B virus YMDD motif region that has not been queried, etc. problem, achieve the effect of shortening the detection time, reducing the possibility of pollution, and improving efficiency

Inactive Publication Date: 2019-01-18
WUHAN CMLABS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This patent uses the combination of RNase H2 and PCR technology to develop a new type of detection method for drug-resistant mutations in the YMDD motif region of hepa...

Method used

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  • Kit for detecting drug-resistant mutation in YMDD motif region of hepatitis B virus
  • Kit for detecting drug-resistant mutation in YMDD motif region of hepatitis B virus
  • Kit for detecting drug-resistant mutation in YMDD motif region of hepatitis B virus

Examples

Experimental program
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Effect test

Embodiment 1

[0033] The present embodiment has designed the primer probe directed at the drug-resistant mutation of hepatitis B virus YMDD motif region, designed the specific primers SEQ ID NO.1 and SEQ ID NO.2 directed at the rtM204I mutation site, and directed at the rtM204V mutation site. Specific primers SEQ ID NO.1 and SEQ ID NO.3, and the probe SEQ ID NO.6 corresponding to the rtM204I / V mutation site; specific primers SEQ ID NO.4 and SEQ ID NO for the rtL180M mutation site .5, and the probe SEQ ID NO.7 corresponding to the rtL180M mutation site. The specific sequence is as follows:

[0034] 5'-AGTCCGTTTCTCTTGGCTC-3, the sequence is shown in SEQ ID NO.1;

[0035] 5'-CCCCAAWACCACATCATCaAAATA-x, the sequence is shown in SEQ ID NO.2;

[0036] 5'-CCAAWACCACATCATCCATCCAcATAAC-x, the sequence is shown in SEQ ID NO.3;

[0037] 5'-GGCCTCAGTCCGTTTCTCaTGG-x, the sequence is shown in SEQ ID NO.4;

[0038] 5'-AGCGGCATAAAGGGACTC-3', the sequence is shown in SEQ ID NO.5;

[0039] FAM-GGGAAAGCC...

Embodiment 2

[0043] This example prepares a kit for drug-resistant mutations in the YMDD motif region of hepatitis B virus.

[0044] The specific design of the kit is as follows:

[0045] (1) DNA extraction solution, including concentrated solution and lysis buffer;

[0046] (2) PCR reaction master mix 1: the main components are PCR buffer, 4 kinds of dNTPs, probe SEQ ID NO.6, and primers mixed with SEQ ID NO.1 and SEQ ID NO.2; among them, the probe SEQ ID NO.6 The concentration of ID NO.6 is 0.25 μM, and the concentration of primers SEQ ID NO.1 and SEQ ID NO.2 is 0.25 μM.

[0047] (3) PCR reaction master mix 2: the main components are PCR buffer, 4 kinds of dNTPs, probe SEQ ID NO.6, and primers mixed with SEQ ID NO.1 and SEQ ID NO.3; among them, the probe SEQ ID NO.6 The concentration of ID NO.6 is 0.25 μM, and the concentration of primers SEQ ID NO.1 and SEQ ID NO.3 is 0.25 μM.

[0048] (5) PCR reaction master mix 3: the main components are PCR buffer, 4 kinds of dNTPs, probe SEQ ID N...

Embodiment 3

[0054] In this example, the kit for detecting drug-resistant mutations in the YMDD motif region of hepatitis B virus prepared in Example 2 is used to detect samples, and the specific detection steps are as follows.

[0055] (1) Extracting the HBV DNA template: the HBV DNA template in the serum sample of hepatitis B virus was extracted with DNA extraction solution.

[0056] The specific process includes: adding 100 μL of concentrated solution to hepatitis B virus serum, centrifuging at 12000 rpm for 10 minutes; discarding the supernatant; adding 25 μL of lysis buffer and mixing, lysing at 100 °C for 10 minutes; finally centrifuging at 12000 rpm for 10 minutes, and the supernatant is ready for use HBVDNA template.

[0057] (2) PCR amplification detection: use the HBV DNA extracted in step (1) as a template, and use the kit provided by the present invention to perform corresponding amplification detection.

[0058] Mix PCR reaction premixes 1, 2 and 3 with the enzyme mixture in ...

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Abstract

The invention provides a kit for detecting the drug-resistant mutation in the YMDD motif region of hepatitis B virus, and belongs to the technical field of molecular biology. Specifically, we designedspecific primers and probes for rtM204I/V (C region) +/- rtL180M (B region) mutation of HBV YMDD resistance gene by using RNase H2 and Taq DNA polymerase biphasic enzyme system to detect drug resistance mutation of HBV YMDD motif region. The kit provided by the invention has the advantages of high sensitivity, fast detection, good stability and closed detection, and reduces the possibility of later pollution.

Description

technical field [0001] The invention relates to the technical field of molecular biology, belongs to a drug-resistant mutation detection method in the field of in vitro diagnostic reagents, in particular to a kit for drug-resistant mutations in the YMDD motif region of hepatitis B virus. Background technique [0002] At present, the treatment of chronic hepatitis B mainly uses interferon or nucleoside analogs, such as lamivudine, adefovir, ganciclovir, etc., to interfere with HBV replication to achieve the purpose of treatment. Lamivudine, Chinese name Hepting, is an oral cytosine nucleoside analogue, which was successfully developed and produced by GlaxoSmithKline in 1992. It entered China in 1998 and was approved for clinical use. Mivudine is convenient to take, has few adverse reactions, and is easily accepted by patients. It is currently recognized as the most effective and safe nucleoside analogue drug for inhibiting virus replication. Treatment with alpha-interferon o...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/70C12N15/11
CPCC12Q1/6858C12Q1/706C12Q2521/327C12Q2521/101C12Q2563/107C12Q2531/113
Inventor 徐志勇秦伟
Owner WUHAN CMLABS CO LTD
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