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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

AI Technical Summary

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 by drinking and drugs, extrahepatic cholestasis, etc. Further examinations are needed to clarify the cause of ALT increase

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
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: 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, Ⅲ:

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

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

[0054] (1) Weigh 1 gram of agarose, put it in a clean Erlenmeyer flask, add 100ml of 1×TBE buffer solution, 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 ...

Embodiment 2

[0067] Embodiment 2: Extraction and PCR amplification of DNA in the sample

[0068] (1) Extraction of DNA from serum samples of patients with hepatitis B and liver cancer, brief description of DNA extraction by boiling method:

[0069] 1) Add 200ul of serum to a 1.5ml centrifuge tube, add 400ul of PEG, shake and mix well, and 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, remove the precipitate, and use the supernatant as a template for later use;

[0073] (2) Nested PCR amplification, detecting A31T, T49A, A52C site PCR reaction 50ul system:

[0074]

[0075] Amplification conditions: 94°C for 3min, 94°C for 1min, 66°C for 1min, 72°C for 1min, 72°C for 10min, 30 cycles. The amplified PCR product was electrophoresed, 1% agarose electrophoresis, and ethidiu...

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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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