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Method and kit for detecting HBV genotype and/or X-region mutation, CDS standard sequence of HBx, primer and application

A standard sequence and genotype technology, applied in the field of biomedicine, can solve problems such as high cost, time-consuming and labor-intensive costs, and inability to obtain sequence information

Pending Publication Date: 2020-09-04
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whole gene sequencing is the "gold standard" of HBV genotyping, which has the highest accuracy and can obtain complete mutation site information, but it is time-consuming, laborious and expensive; S gene sequencing can obtain partial sequence information, The typing accuracy is high, but the sequence information of the X region containing many known pathogenic mutations cannot be obtained
[0006] At present, there are no related reports on methods and reagents that use X region sequencing to perform typing and simultaneously detect HBV X region mutations

Method used

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  • Method and kit for detecting HBV genotype and/or X-region mutation, CDS standard sequence of HBx, primer and application
  • Method and kit for detecting HBV genotype and/or X-region mutation, CDS standard sequence of HBx, primer and application
  • Method and kit for detecting HBV genotype and/or X-region mutation, CDS standard sequence of HBx, primer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1 Reagent of the present invention is composed as follows:

[0076] HBV DNA extraction reagent: Nucleic acid extraction or purification reagent (magnetic bead method) of Sanxiang Biotechnology Co., Ltd.

[0077] Primers: The upstream primer sequence of the X region is: 5'-CGATCCATACTGCGGAACTC-3' (as shown in SEQ ID NO: 1); the downstream primer sequence of the X region is: 5'-CAGCTTGGAGGCTTGAACA-3' (as shown in SEQ ID NO: 2 ). Primers were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0078] Negative control and positive control: deionized water was used as negative control (NC), and HBV1.3 plasmid (known as HBV-D type) was used as positive control (PC).

[0079] PCR reaction reagents: use TOYOBO's high-efficiency and high-fidelity PCR amplification reagent KOD FX (product number: KFX-101), including 2×PCR buffer for KOD FX, 2mM dNTPs and KOD FX (1.0U / μL).

[0080] PCR amplification program setting: on a PCR instrument (Eppendorf), pre-dena...

Embodiment 2 Embodiment 1

[0083] Embodiment 2 detects HBV genotype and X region mutation with the method of embodiment 1

[0084] Take the detection of HBV genotype and X region mutation in peripheral blood samples of 14 cases of hepatitis B infection as an example. Detection process: first obtain peripheral blood samples from patients with clinical hepatitis B infection, and quickly extract HBV DNA; prepare a PCR reaction system for directional amplification of the X region, recover PCR amplification products, perform sequencing reactions, and finally compare the sequencing results with the CDS standards of 10 HBx The sequences (see SEQID No.13-22 in the sequence table) are compared to determine the HBV genotype corresponding to the measured sequence and the mutation of the X region.

[0085] Specific steps are as follows:

[0086] 1) Serum HBV DNA extraction: Extract HBV DNA from 200 μL serum of HBV-infected patients according to the operation method of Sanxiang Bio-nucleic acid extraction or purifi...

Embodiment 3 Embodiment 1

[0105] Embodiment 3 detects that different HBV genotypes mix with the method of embodiment 1

[0106] The No. 4 (known as type C) and No. 14 (known as type B) sample serum were respectively 1:1 (C+B), 2:1 (2C+B) and 5:1 (5C+B) The ratio was mixed and the total volume was 200 μL. All the other operations are the same as in Example 2.

[0107] Data processing and analysis see Figure 6-12 and Table 6-7.

[0108] Judgment of results: According to whether there are scattered "set peaks" in the sequencing peak diagram corresponding to HBx CDS (there are two superimposed peaks at the same position, and the lower peak accounts for at least 1 / 5 of the height of the higher peak) and Judgment of single genotype or mixed genotype by quantity: if there is no "set peak" or the number is less than 10, it is judged as a single genotype; if the number of "set peaks" is ≥ 10, then mixed genotype is considered. According to the read main peak sequence, the homology analysis and comparison r...

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Abstract

The invention discloses a method and kit for detecting an HBV genotype and / or X-region mutation, a CDS standard sequence of HBx, a primer and application. The method comprises the following steps: selecting CDS of middle HBx of a sequence and CDS standard sequences of 10 kinds of HBx for homology analysis and comparison to obtain a result of the HBV genotypes and / or X-region mutation to be detected; further, judging whether the HBV genotype and / or X-region mutation is a single genotype or a mixed genotype through the peak nesting condition in a sequencing peak graph, and if no nested peak exists or the number of nested peaks is smaller than 10, determining the HBV genotype and / or X-region mutation as the single genotype; and if the number of the nested peaks is greater than or equal to 10,determining the HBV genotype and / or X-region mutation as the mixed genotype. The invention provides a set of convenient and high-accuracy scheme for simultaneously detecting 10 kinds of HBV genotypesand X-region mutation types, and a reference basis can be provided for HBV infected person disease progress risk assessment, antiviral crowd screening, antiviral mode selection and curative effect prediction.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a method and kit for detecting 10 HBV genotypes and X region mutations by PCR directional sequencing combined with standard sequence comparison, a CDS standard sequence of HBx, primers and applications thereof. Background technique [0002] China is the country with the highest incidence and mortality rate of liver cancer in the world. It is estimated that the number of deaths from liver cancer in China accounts for about half of the global number of deaths from liver cancer. Hepatitis B virus (hepatitis B virus, HBV) persistent infection is the most important cause of liver cancer in my country, and about 76% of liver cancer is related to it (de Martel C, Maucort-Boulch D, Plummer M, et al. World-wide relative contribution of hepatitis B and C viruses in hepatocellular carcinoma. Hepatology, 2015, 62(4):1190-1200). At present, the levels of HBV DNA and HBsAg are the key fact...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12N15/11C12R1/93
CPCC12Q1/706C12Q2600/156
Inventor 周艳文许振宇刘其摇龚国忠蒋永芳周宁肖新强马静
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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