Kit for detecting pre-S region variation of advanced liver disease related hepatitis B virus

A hepatitis B virus and kit technology, applied in the field of medical biological detection, can solve problems such as increase, and achieve the effect of strong sensitivity and high specificity

Inactive Publication Date: 2015-06-10
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the increase of ALT may be caused by many reasons, such as acute and chronic viral hepatitis, liver damage caused

Method used

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  • Kit for detecting pre-S region variation of advanced liver disease related hepatitis B virus
  • Kit for detecting pre-S region variation of advanced liver disease related hepatitis B virus
  • Kit for detecting pre-S region variation of advanced liver disease related hepatitis B virus

Examples

Experimental program
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Example Embodiment

[0049] Example 1: The kit of the present invention

[0050] The specific preparation steps of the components of the kit are as follows:

[0051] 1. Preparation of PCR primers I, II and III:

[0052] Artificially synthesized primers Ⅰ, Ⅱ, and Ⅲ. The detailed sequences are shown in Table 1. The synthesized primers are diluted to 5μM. The primer concentration after the upstream primer and the downstream primer are mixed in equal amounts is 10μM, P1 and P2 are mixed in equal amounts to form primer I, P3 and P4 are mixed in primer II, and P7 and P8 are mixed in equal amounts to form primer III.

[0053] 2. Preparation and electrophoresis of agarose gel:

[0054] (1) Weigh 1 gram of agarose, place it in a clean Erlenmeyer flask, add 100ml of 1×TBE buffer, and heat it in a microwave oven to melt it completely. After that, add 5ul EB stock solution and mix (final concentration 0.5mg / ml) to make 1% agarose solution.

[0055] (2) Prepare the gel mold, put a sample comb on one end of the mold, 0....

Example Embodiment

[0067] Example 2: Extraction of DNA from samples and PCR amplification

[0068] (1) Brief description of DNA extraction from serum samples of hepatitis B patients and liver cancer patients, and the boiling method for DNA extraction:

[0069] 1) Take 200ul serum into a 1.5ml centrifuge tube, add 400ul PEG, shake and mix well, centrifuge at 1300rpm / min for 10min;

[0070] 2) Remove the supernatant, add 80ul lysate, shake and mix well, until the bottom of the tube is precipitated and mixed;

[0071] 3) Boil in boiling water for 10 minutes;

[0072] 4) Centrifuge at 1300rpm / min for 10min to remove the precipitate and use the supernatant as a template for later use;

[0073] (2) Nested PCR amplification, detection of A31T, T49A, A52C sites PCR reaction 50ul system:

[0074]

[0075] Amplification conditions: 94℃3min, 94℃1min, 66℃1min, 72℃1min, 72℃10min, 30 cycles. Electrophoresis the amplified PCR products, 1% agarose electrophoresis, and develops the color with ethidium bromide. The results ...

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Abstract

The invention relates to the technical field of biomedical detection, and particularly relates to a kit for detecting pre-s region variation of advanced liver disease related hepatitis B virus. The kit is used for detecting the variation sites of the pre-S region of hepatitis B virus, in particular A31T, T49A, A52C, G105C, C109A, A135C and G147C. The kit is simple, convenient and practical and has high specificity and strong sensitivity.

Description

technical field [0001] The invention relates to the technical field of medical biological detection, in particular to a kit for detecting variation of hepatitis B virus, in particular to a kit for detecting variation of preS of hepatitis B virus associated with progressive liver disease. Background technique [0002] About more than 3 billion people are infected with hepatitis B virus in the world. The mutation of hepatitis B virus is one of the important risk factors of hepatitis B virus positive liver disease (including hepatitis B virus, liver cirrhosis and hepatocellular carcinoma). As the infection persists, the mutation rate of the virus gradually increases during the progression of liver disease from hepatitis B to cirrhosis and liver cancer. [0003] Hepatitis B virus (HBV) belongs to the orthohepadnavirus genus in the family hepadnaviridae. HBV DNA is composed of incomplete circular double-stranded DNA, the long one is negative strand, and the short one is It is a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 曹广文殷建华蒲蕊张琪张宏伟
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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