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A method and kit for detecting hepatitis B virus genotype

A hepatitis B virus and genotyping technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/examination, etc., can solve the problems of affecting the typing results, unable to identify specimens, complex band types, etc. To achieve the effect of enhancing detection sensitivity, improving binding efficiency, and improving signal strength

Inactive Publication Date: 2011-11-30
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The whole HBV gene sequencing method is the gold standard of genotyping and the basis of genotyping, but the sequencing method cannot detect the mixed infection of multiple subtypes, and the technology is complex, time-consuming and laborious, and it is not suitable for epidemiological investigations and large-scale clinical trials. Scale promotion and use
The PCR-RFLP method can distinguish A to F6 genotypes, and the enzyme digestion map is concise and intuitive, but the PCR RFLP typing method established at present cannot identify 100% of the specimens, and this typing method requires a long time and many steps. And in the case of mixed infection or incomplete enzyme digestion, complex band patterns will appear, which will affect the judgment of typing results
Monoclonal antibody enzyme-linked immunoassay is a method for genotyping monoclonal antibodies against genotype-specific epitopes in the pre-S2 region and using ELISA. This method is simple and suitable for large-scale epidemiological investigations, but It is relatively difficult to screen a set of monoclonal antibodies, and strains with mixed infection of different genotypes or strains with genotype-specific episite mutations cannot be typed
The gene chip method is a new technology developed in recent years, which can also be used for genotyping of hepatitis B virus, but the cost is high, and an expensive chip scanner is required for detection
Chinese patent 200710026605.8 provides a method for detecting hepatitis B virus genotypes using reverse dot hybridization technology, but only one probe is designed for some genotypes in this method. Because HBV has the characteristics of high mutation rate, one probe is Some of the mutated genotypes will be missed, which will affect the accuracy of detection

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  • A method and kit for detecting hepatitis B virus genotype
  • A method and kit for detecting hepatitis B virus genotype
  • A method and kit for detecting hepatitis B virus genotype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Extracting HBV Genomic DNA from Serum

[0047] 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

[0048] Embodiment 2PCR reaction amplifies target DNA fragment

[0049] Externally amplify 50 μl PCR system as follows:

[0050] 10×Taq amplification buffer (TaKaRa) 5 μl

[0051] MgCl 2 (25mM) 4μl

[0052] dNTP Mixture (2.5mM each) 4μl

[0053] Upstream primer (20um) 1μl

[0054] Downstream primer 1 (20um) 1μl

[0055] Taq DNA polymerase 5U / μl (TaKaRa) 0.25μl

[0056] Autoclaved distilled water 29.75μl

[0057] Template 5 μl purified HBV DNA, or 5 μl distilled water (negative control)

[0058] Put each reaction tube into the calibrated PCR instrument. 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.

[0059] 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 perfo...

Embodiment 3

[0073] The preparation of embodiment 3 hybridized carrier nylon film strips

[0074] 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:

[0075] (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.

[0076] (2) Set working parameters: spray volume 2 μl / cm, rail speed 6.0 cm / s, nozzle interval 5 mm.

[0077] (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.

[0078] (4) Cutting: After fixing, cut the film into 3mm×7cm film strips with a strip cutter along the marking line.

[0079] (5) Pre-hybridization: Add 2ml of p...

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Abstract

The invention provides a method and a kit for detecting the genotype of hepatitis B virus, the method comprising the following steps: (1) extracting the HBV DNA genome from serum or plasma; (2) designing according to the conserved region of the hepatitis B virus genome Typing primers and probes; (3) Use small molecule labeled oligonucleotide primers to perform PCR reaction to amplify target DNA fragments; (4) Add poly dC tails to oligonucleotide probes, and spot them on the substrate for ultraviolet radiation After linking, bake and fix at 120°C; (5) Use the labeled target DNA amplification product to hybridize with the specific oligonucleotide probe on the substrate; (6) Detect the hybridized conjugates to determine the different genotypes of HBV. The invention designs highly conserved type-specific probes and PCR amplification primers at different base positions, improves detection sensitivity, and can perform HBV genotyping quickly, conveniently and accurately. The invention also provides a test kit for HBV genotyping, which is clinically used for detecting the genotype of hepatitis B virus, can provide reliable basis for clinical treatment and rational drug use, and is worthy of popularization and use.

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 Patents(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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