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

A hepatitis B virus and detection kit technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem that the enzyme cleavage site is easily affected by gene variation, the sensitivity is lower than that of specific probes, and the complexity Banding and other problems, to achieve the effect of continuation of fast and easy performance, improved typing sensitivity, and simple operation

Active Publication Date: 2013-09-18
泰普生物科学(中国)有限公司
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  • Abstract
  • Description
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  • Application Information

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 materials

[0087] The sources of the working reference materials were all HBV-positive and HBV-negative clinical serum samples collected from the "Fujian Provincial Hospital", and they were all inactivated at 100°C for 5 minutes before the experiment (according to the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B"). Since no reagent or method can guarantee 100% accuracy, potential biological hazards are still not ruled out, and care should be taken during operation. Use a foam box and ice bag for airtight transportation, and the transit time should not exceed 48 hours. The composition of the work reference is shown in Table 3.

[0088] table 3

[0089]

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

[0091] National Reference Material: "Hepatitis B Virus PCR Nucleic Acid Detection Reagent National Reference Material (Non-Human Use)", approval number: (2001) Guosheng Shenzi 0009, product batch num...

Embodiment 1

[0144] Example 1 Preparation of the kit of the invention

[0145] 1. Preparation and sub-packaging of PCR reaction solution of the present invention

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

[0147] 1.1 Preparation for dosing

[0148] 1.1.1dN(U)TP preparation

[0149] Mix the purchased 100 mM dATP, dUTP, dGTP, and dCTP according to 1:2:1:1 (volume ratio). The concentrations of each component of the mixture are: 20mM, 40mM, 20mM, 20mM, and take a 0.5mL centrifuge tube. Packed into 50μL of dN(U)TP stock solution per tube.

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

[0151] 1.1.2 Primer

[0152] Centrifuge the synthesized primers at 13000 rpm for 1 min, add an appropriate amount of pure water (calculated based on the amount of synthesis) to dissolve the pr...

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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 in one 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 gene detection technology of disease pathogens, in particular to a hepatitis B virus genotyping detection kit (PCR-fluorescent probe method). Background technique [0002] Hepatitis B virus (hepatitis B virus, HBV) is one of the hepatitis viruses that seriously endanger human health and is an important pathogenic factor for 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. my country accounts for 150 million, of which 20 million are In patients with chronic hepatitis B, nearly 500,000 people die from the disease every year. It is estimated that 1 to 2 million people worldwide die directly from persistent HBV infection every year. [0003] HBV belongs to the family of hepatotropic DNA viruses. It is a D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70
Inventor 阳卫超陈宁
Owner 泰普生物科学(中国)有限公司
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