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Method and kits for detecting genotype of hepatitis B virus

A hepatitis B virus and genotyping technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/testing, can solve problems such as missed detection and affecting detection accuracy, so as to avoid missed detection, Effects of improving binding efficiency and increasing signal strength

Inactive Publication Date: 2009-11-25
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Hepatitis virus genotype method, but this method only designs one probe for some genotypes, because HBV has the characteristics of high mutation rate, one probe will miss some of the mutated genotypes, thus affecting the accuracy of detection sex

Method used

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  • Method and kits for detecting genotype of hepatitis B virus
  • Method and kits for detecting genotype of hepatitis B virus
  • Method and kits for detecting genotype of hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Extracting HBV Genomic DNA from Serum

[0050] Take 200ul virus serum + 200ul 2x lysis buffer (0.02mol / L TrisHCl, 0.01mol / L EDTA, 1% SDS) + 10ul proteinase K (20mg / ml), digest at 40°C for 1.5 hours. Use phenol and phenol : Chloroform (1:1), each extracted once with chloroform, after mixing, 12000gx3min, recover the water phase, ie the upper layer. Add 1 / 10 volume of 3mol / L sodium acetate and 2 volumes of pre-cooled absolute ethanol to precipitate DNA, 12000g x 10 minutes, recover DNA, wash DNA once with 75% ethanol, discard supernatant, after drying, add 20μl pure Dissolve DNA in water, take 5 μl as template for PCR reaction, and store the rest at -20°C.

Embodiment 2

[0051] Embodiment 2 PCR reaction amplifies target DNA fragment

[0052] External amplification 50μl PCR system is as follows:

[0053] 10×Taq amplification buffer (TaKaRa) 5 μl

[0054] MgCl 2 (25mM) 4μl

[0055] dNTP Mixture (2.5mM each) 4μl

[0056] Upstream primer (20um) 1μl

[0057] Downstream primer 1 (20um) 1μl

[0058] Taq DNA polymerase 5U / μl (TaKaRa) 0.25μl

[0059] Autoclaved distilled water 29.75μl

[0060] Template 5 μl purified HBV DNA, or 5 μl distilled water (negative control)

[0061] Put each reaction tube into the calibrated PCR machine. Start to perform the following external amplification program: 94°C pre-denaturation for 4 minutes, then 40 cycles of 94°C for 30 seconds, 56°C for 40 seconds, 72°C for 50 seconds, and finally 72°C for 10 minutes.

[0062] After completing the above procedures, the samples should be used for electrophoresis immediately, and if there is no positive band, perform internal amplification, and store the rest at -20°C or p...

Embodiment 3

[0076] The preparation of embodiment 3 hybridized carrier nylon film strips

[0077] Dilute each artificially synthesized probe to a final concentration of 10 pmol / μl, and fix it on the appropriate position of the membrane strip. The preparation of the membrane strip is as follows:

[0078] (1) Add the DIG-labeled PCR product and 10 probes into the No. 1 pump and No. 2 pump of the automatic film spraying machine respectively. After spraying, clean the two pipes with distilled water and replace them again. The other two probes are sprayed with film.

[0079] (2) Set working parameters: spray volume 2 μl / cm, rail speed 6.0 cm / s, nozzle interval 5 mm.

[0080] (3) Fixing the probe: After spraying, let the film dry naturally at room temperature, perform ultraviolet cross-linking, and then bake at 120°C for 30 minutes.

[0081] (4) Cutting: After fixing, cut the film into 3mm×7cm film strips with a strip cutter along the marking line.

[0082] (5) Pre-hybridization: Add 2ml of p...

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Abstract

The invention provides a method and kit for detecting genotype of hepatitis B virus, the method includes following steps: 1, extracting DNA genome of HBV from blood serum or blood plasma; 2, designing typing premer and probe according to conserved sequence of HBV genome; 3, using micromolecule marked oligonucleotide primer to proceed PCR reaction to amplify target DNA sequence; 4, oligonucleotide probe is added with a tail chain poly dC, then UV cross-linking on substrate and baking at a temperature of 120 DEG for fixing; 5, hybridizing amplication product of the marked target DNA with specific oligonucleotide probe on substrate; 6, detecting bybridization conjugates to judge different genotypes of HBV. The invention designs specific probe with high conservative and PCR amplication primer in different base position, and improves detection sensitivity, such that HBV genotypes can be detected quickly, conveniently and accurately.

Description

technical field [0001] The invention relates to a method for detecting hepatitis B virus gene, in particular to a method for detecting hepatitis B virus genotype in clinical blood samples by DNA reverse linear hybridization technology and a kit thereof. Background technique [0002] Hepatitis B virus (hepatitis B virus, HBV) infection is widespread worldwide. There are nearly 400 million chronic HBV carriers in the world, of which more than 100 million are chronic HBV carriers in my country. HBV infection is harmful to people's health and causes national One of the most costly diseases. HBV is a double-stranded DNA virus consisting of two strands, plus and minus. According to the heterogeneity of the complete HBV nucleotide sequence ≥ 8% or the nucleotide difference of the S gene sequence ≥ 4.1%, the hepatitis B virus strains are divided into A-H8 genotypes. A large number of studies have shown that different HBV genotypes are closely related to the severity, prognosis and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 黄爱龙张文露胡源赖国旗赵丽刘彦辰
Owner CHONGQING MEDICAL UNIVERSITY
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