Primer, method and application based on next-generation sequencing technology to detect HBV drug-resistant mutation site

A technology for the detection of drug resistance mutation sites and techniques, which is applied in the fields of genomics and molecular biology, and can solve the problems of difficult separation, insignificant differences in fragment lengths, and large numbers, and achieves the effect of high stability.

Inactive Publication Date: 2016-02-10
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventors found in practice that after adding Adaptor to many nested PCR products, the fragment length difference is not obvious, and the number of fragments is large, which is difficult to separate in agarose gel electrophoresis, which seriously affects the detection of HBV drug-resistant mutation sites by next-generation sequencing application in

Method used

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  • Primer, method and application based on next-generation sequencing technology to detect HBV drug-resistant mutation site
  • Primer, method and application based on next-generation sequencing technology to detect HBV drug-resistant mutation site
  • Primer, method and application based on next-generation sequencing technology to detect HBV drug-resistant mutation site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A method for detecting HBV drug-resistant mutation sites based on next-generation sequencing technology, a round of PCR comprises the following steps:

[0058] 1. Design and synthesize the primers shown in SEQUENCE NO.1 and SEQUENCE NO.2;

[0059] 2. Amplify the HBV RTDNA fragment (HBV552~HBV992), including 10 drug resistance mutation sites

[0060] Configure the PCR system as follows (the DNA template is DNA extracted from 200ul serum of HBV patients, and the extraction kit uses the bloodminikit of qiagen):

[0061]

[0062] The configured system was subjected to PCR reaction according to the following procedures:

[0063]

[0064]

[0065] After the PCR reaction, use 1.5% agarose gel electrophoresis to verify the size of the enriched fragment of the PCR product, select the 441bp target fragment to recover by gel cutting, and purify to 34 μL. The purification steps are as follows:

[0066] 1) Add 3 times the volume of BufferQG (conventional DNA recovery buff...

Embodiment 2

[0076] A method for detecting HBV drug-resistant mutation sites based on next-generation sequencing technology, the steps of a round of PCR include: except that the PCR program is different from that of Example 1, other steps are the same as in Example 1, wherein the steps of this Example 2 The PCR program is as follows:

[0077]

[0078] Example 1 and Example 2 used the same primer pair.

[0079] After the PCR product was purified, an equal volume of the product was taken for electrophoresis, and the results were as follows: figure 1 shown. exist figure 1 Among them, A-1 is the result after purification of the PCR product of Example 1, and A-2 is the result after purification of the PCR product of Example 2. Depend on figure 1 It can be seen that the effect of A-1 is better than that of A-2 in terms of specificity and product quantity.

Embodiment 3

[0081] A method for detecting HBV drug-resistant mutation sites based on next-generation sequencing technology, the steps of one round of PCR include: except that the PCR primer pair is different from that of Example 1, other steps (i.e. conditions such as a round of PCR reaction system and procedures) are all Same as embodiment 1, wherein, the PCR primer pair of present embodiment 3 is as shown in SEQUENCENO.3 and SEQUENCENO.4 as follows:

[0082] SEQUENCE NO.3: CHTGTTGCTGTACAAAACCT

[0083] SEQUENCE NO.4: GCAGGATAWCCACATTG

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Abstract

The invention provides a primer, method and application based on next-generation sequencing technology to detect HBV drug-resistant mutation sites. A primer pair designed specially for a next-generation sequencing platform only needs a round of PCR, so that compared with existing detection methods based on nested PCR, the technical scheme has high stability, specificity and sensitivity. The technical scheme is especially suitable for adopting the next-generation sequencing platform to detect the HBV drug-resistant mutation sites, and especially suitable for Illumina sequencing platforms.

Description

technical field [0001] The invention belongs to the field of genomics and molecular biology, and in particular relates to a primer, method and application for detecting HBV drug-resistant mutation sites based on next-generation sequencing technology. Background technique [0002] During the replication process of the HBV genome, during the RNA reverse transcription replication process, due to the lack of strict correction mechanism of RNA polymerase and reverse transcriptase, the viral gene replication process is prone to nucleotide mismatches. Therefore, HBV It is a highly variable virus. Under the multiple effects of the body itself, drugs and viruses, a variety of drug-resistant mutations will occur in the reverse transcription active region (RT region) of HBV DNA polymerase. Drug-resistant mutations will not only reduce the effect of antiviral therapy, but also make hepatitis B further progress and worsen. HBV gene mutation and the resulting substitution of drug target...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李改玲葛良进徐国伟刘松邓力蔚林群婷刘丽春曾立董曾健明
Owner GENEMIND BIOSCIENCES CO LTD
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