Method and kit for assaying hepatitis B virus DNA (deoxyribonucleic acid) sequence

Abstract

The invention relates to the field of genetic engineering, and provides a method and a kit for assaying the hepatitis B virus DNA (deoxyribonucleic acid) sequence. The method includes the following steps: (A) hepatitis B virus specific primers are utilized to amplify a plurality of target regions in a sample to be assayed, and a sequencing library is constructed on the basis of amplification products; (B) the single-molecule amplification of the sequencing library is carried out, so that a plurality of single-molecule amplification products corresponding to the target regions are obtained; (C) the high-throughput gene sequencing of the single-molecule amplification products is carried out simultaneously, so that the sequence information of the target regions is obtained. The kit comprises the hepatitis B virus specific primers and a connector element. The method and the kit can carry out deep region sequencing on a plurality of target regions of hepatitis B virus at the same time, consequently, the efficiency of assay is increased, meanwhile, the accuracy and sensitivity of assay can be increased as well, and furthermore, multi-region sequencing can be carried out on a large quantity of samples at the same time.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method and a kit for detecting the DNA sequence of hepatitis B virus. Background technique [0002] Hepatitis B is an infectious disease widely distributed in the world, showing regional distribution in many countries in the world. More than 2 billion people in the world have been infected with hepatitis B virus (hepatitis B virus, HBV), of which about 350 million people are chronic carriers. And China is the hardest-hit area of ​​chronic hepatitis B, and more than 8% of the population are chronic carriers. Chronic hepatitis B can cause serious liver diseases such as cirrhosis, liver failure, and liver cancer, and has been listed as the ninth leading cause of death by the WTO. The HBV genome is an incomplete double-stranded circular DNA molecule, about 3200bp. Since HBV has a reverse transcription process during the replication process, and its polymerase or reverse transcr...

AI Technical Summary

Problems solved by technology

It can only sequence a certain segment of the HBV gene sequence of a sample at a time, the detection efficiency is low, and the sequencing cost is high, and due to the limitation of the Sanger sequencing principle, double peaks or even double peaks will appear when detecting the signal of a sample with a heterozygote. Multiple peaks, resulting in disordered sequencing peaks, even unable to analyze the obtained sequence information, and sequencing failed

A large number of studies have confirmed that the direct sequencing method of PCR products can only detect viruses with a ratio of ≥20% in the mixed virus group, and cannot obtain the precise variation ratio of the mutation site

And there is same defect in the kit based on Sanger sequencing method in the prior art

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