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Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit

A hepatitis B virus and detection kit technology, which is applied in material stimulation analysis, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problem that enzyme cleavage sites are easily affected by gene variation, and the sensitivity is lower than that of specific probes , complex bands and other problems, to achieve the effect of continuing fast and simple performance, improving typing sensitivity, and eliminating interference

Active Publication Date: 2012-07-18
泰普生物科学(中国)有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

Although the results of sequence determination are accurate and reliable, it is difficult to be used in clinical routine due to its complicated technology, long experimental process, high experimental conditions, time-consuming and expensive costs. Currently, it is only used in laboratory research; Points are 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; multiple amplification tubes are required for PCR amplification using the SSP method, and electrophoresis of products after conventional PCR is used The method is also prone to contamination, and its sensitivity is lower than that of specific probes; the SSO method can be used to detect the PCR product of a single tube to type, so it has the characteristics of relatively simple operation and low cost, and has practical value

Method used

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  • Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit
  • Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit
  • Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit

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Effect test

preparation example Construction

[0086] 2. Preparation of working reference products

[0087] The sources of working reference materials are HBV-positive and HBV-negative clinical serum samples collected from "Fujian Provincial Hospital", which were inactivated at 100°C for 5 minutes before the experiment (according to the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B"). Since any reagent or method cannot guarantee 100% accuracy, potential biological hazards cannot be ruled out, and care should be taken during operation. Use foam boxes and ice packs for sealed transportation, and the transit time limit does not exceed 48 hours. The composition of the working reference is shown in Table 3.

[0088] table 3

[0089]

[0090] In addition to this product kit, reagents used include:

[0091] National reference product: "National reference product of hepatitis B virus PCR nucleic acid detection reagent (non-human use)", approval number: (2001) Guosheng Shenzi 0009, product batch number 0...

Embodiment 1

[0144] The preparation of embodiment 1 kit of the present invention

[0145] 1. Preparation and subpackage of PCR reaction solution of the present invention

[0146] In the present invention of the laboratory, the dosing volume of the PCR reaction solution is about 20 people each time, and the preparation method is as follows:

[0147] 1.1 Dosing preparation

[0148] 1.1.1 dN(U)TP preparation

[0149] Mix the purchased 100mM dATP, dUTP, dGTP, and dCTP according to 1:2:1:1 (volume ratio). Fill each tube with 50 μL of dN(U)TP stock solution.

[0150] Before use, add 50 μL of pure water to the above dN(U)TP stock solution and dilute to 100 μL of dN(U)TP use solution with a concentration of 10 mM.

[0151] 1.1.2 Primers

[0152] Centrifuge the synthesized primers at 13000 rpm for 1 min, add an appropriate amount of pure water (calculated according to the amount of synthesis) to dissolve the primers to 100 μM, take 0.5 mL centrifuge tubes, and aliquot them into 40 μL primer st...

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Abstract

The invention aims at fully utilizing the advantages of a Taqman probe technology, designing specific probes against various genotypes of HBV (hepatitis B virus), using the probes to mark different fluorescent dyes, detecting at different wavelengths and further achieving the genotyping purpose. A kit disclosed by the invention can detect B, C and D type hepatitis B virus in samples and detect B,C and D type single infection and mixed infection by designing primers and the specific probes against all the genotypes of the HBV. The B (or D) type hepatitis B virus is detected by adopting a double-color probe at FAM wavelength and the C-type hepatitis B virus is detected at HEX wavelength. The kit has the advantages that the B, C and D type hepatitis B virus can be simultaneously detected inone experiment, the operation is simple, dUTP and UNG enzymes are used in the kit, and the interference of a polluted amplified product on a detection result can be effectively eliminated.

Description

technical field [0001] The invention relates to a disease pathogen gene detection technology, in particular to a hepatitis B virus genotyping detection kit (PCR-fluorescent probe method). Background technique [0002] Hepatitis B virus (HBV) is one of the hepatitis viruses that seriously endanger human health and is an important pathogenic factor of liver diseases. There are 2 billion people in the world who have been infected with HBV. At present, there are at least 380 million patients with chronic HBV infection in the world. Among them, 75% of chronic HBV carriers are distributed in Southeast Asia and the sub-Saharan region, and my country accounts for 150 million people, of which 20 million people are Chronic hepatitis B patients, nearly 500,000 people die from the disease every year. It is estimated that 1-2 million people worldwide die directly from persistent HBV infection every year. [0003] HBV belongs to the Hepadnaviridae family and is a DNA virus. Its genome is...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 阳卫超陈宁
Owner 泰普生物科学(中国)有限公司
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