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Gene-sequencing-method-based hepatitis B virus genotyping kit

A hepatitis B virus and gene sequencing technology, applied in the biological field, can solve problems such as complex bands, low sensitivity, and high cost of gene chip methods, and achieve high automation, simple and safe operation, and good specificity

Inactive Publication Date: 2011-09-14
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Restriction fragment length polymorphism technology is relatively simple, but the enzyme cutting site is easily affected by gene variation, and in case of mixed infection or incomplete enzyme digestion, complex bands will appear, which will affect the judgment of typing results; type-specific probe operation Complicated and low sensitivity; the gene chip method is costly and cumbersome to make; while the gene sequencing method has high sensitivity and specificity, relatively simple operation, and high-throughput clinical sample detection can be completed
At present, the methods for HBV genotyping by gene sequencing mainly include whole gene sequence determination and S gene sequence determination, etc. Although these methods are accurate and reliable for HBV genotyping, they are time-consuming and labor-intensive.
[0005] At present, there is no literature report on kits for detecting different genotypes of hepatitis B virus (HBV) using gene sequencing technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The kit of the present invention consists of the following:

[0027] ①HBV DNA extraction reagent: 10mmol / L Tris-HCl (pH8.0), 5mmol / LEDTA, 0.5% SDS, 150μg / ml proteinase K.

[0028] ② Primers:

[0029] The upstream primer sequence of the HBV DNA S gene is: 5'-CTAGGACCCCTGCTCGTGTT-3' (as shown in SEQ ID NO: 1)

[0030] The downstream primer sequence of the HBV DNA S gene is: 5'-GACAAAAGAAAATTGGTAAC-3' (as shown in SEQ ID NO: 2)

[0031] The above primer sequences were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0032] ③ Negative control and positive control: Deionized water was used as negative control, and samples containing HBV DNA were used as positive control.

[0033] ④PCR reaction solution: 10×PCR Premix, 0.25pmol / μl primer, 2.5-4.0mM MgCl 2 , 2U Taq enzyme, 0.2-0.4mM dNTPs, 0.3-0.6mM dUTP, usually take 1-2μl template. (All the above concentrations refer to the final concentration)

[0034] ⑤PCR amplification program setting: on...

Embodiment 3

[0052] Embodiment 3 Evaluation of the detection ability of the kit of the present invention

[0053] The "restriction fragment length polymorphism method" used in this embodiment for comparison (see literature: Peng Liang, Ding Jingjuan. The relationship between upper gastrointestinal diseases and hepatitis B virus infection and its genotype, Fudan Journal (Medical Edition). 2004; 31(5): 534-537) and the detection ability of this kit, it was found that the sensitivity, specificity and sensitivity of this kit were compared with the restriction fragment length polymorphism method, and this kit was more accurate and completely It meets the practical requirements of current clinical diagnosis and treatment, and the comparison results are as follows:

[0054]

[0055] in:

[0056] ① Specificity: 100%;

[0057] ②Sensitivity: 95%;

[0058] ③ Positive predictive value: the positive predictive value reaches 100%;

[0059] ④Negative predictive value: The negative predictive value...

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Abstract

The invention relates to the field of biotechnology. At present, determined genotypes of hepatitis B virus (HBV) comprise eight types A, B, C, D, E, F, G and H, and the pathogenicity, the medicine resistance and the treatment effects of antiviral medicaments vary with the genotypes. In order to identify the HBV genotypes of clinical patients and patients to be tested, the invention provides the gene-sequencing-method-based hepatitis B virus genotyping kit, and a method for identifying the genotypes comprises the following steps of: (1) extracting an HBV deoxyribonucleic acid (DNA) genome fromserum or blood plasma; (2) designing genotyping primers according an HBV S area; (3) performing amplification on a target DNA fragment by a polymerase chain reaction (PCR); (4) detecting a gene sequence of the target DNA by a gene sequencing instrument; and (5) judging the genotype of HBV according to the gene sequence of the target DNA of the HBV. The kit is accurate in result and high in sensitivity, can be operated with high flux, is convenient to popularize and use, and provides a basis for the clinical treatment and rational administration of medicaments for HBV patients.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting different gene subtypes of hepatitis B virus (HBV) by using gene sequencing technology. Background technique [0002] Hepatitis B is a disease that seriously endangers human health worldwide. About 2 billion people are infected with hepatitis B virus (HBV), of which 350 million are chronically infected. Leading cause of failure and liver cancer. There are currently eight HBV genotypes identified: A, B, C, D, E, F, G, and H, and two new genotypes I and J have recently been reported (Olinger CM, Jutavijittum P, Hubschen JM, et al . Possible new hepatitis B virus genotype, southeast Asia. Emerg Infect Dis. 2008; 14: 1777-80). The Chinese are mainly genotypes B and C. Different genotypes of HBV infection have different effects of antiviral drug treatment. The research report pointed out that the genetic characteristics of HBV, including HBV genotype and gene varia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 刘辉刘亚莉张敬磊周伟平
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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