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Primer, probe and reagent kit for predicting liver cancer susceptibility of patient suffering from hepatitis B

A susceptibility, kit technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of reduced HBeAg synthesis and secretion, inhibition of HBeAg synthesis, abnormal HBeAg expression, etc. Easy to judge, high specificity, accurate and reliable results

Active Publication Date: 2019-08-30
阿吉安(福州)基因医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in the C gene region lead to abnormal expression of HBeAg. The most common mutation is the double mutation of 1762T and 1764A in the core promoter (BCP) region of the C region.
Studies have shown that the mutation of 1896A in the preC region can also cause the synthesis inhibition or even termination of HBeAg

Method used

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  • Primer, probe and reagent kit for predicting liver cancer susceptibility of patient suffering from hepatitis B
  • Primer, probe and reagent kit for predicting liver cancer susceptibility of patient suffering from hepatitis B
  • Primer, probe and reagent kit for predicting liver cancer susceptibility of patient suffering from hepatitis B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A test kit for predicting susceptibility to liver cancer in patients with hepatitis B, comprising the components shown in Table 3;

[0045] table 3

[0046] serial number components Element quantity Specification 1 PCR reaction solution 1 Primers, probes, Taq enzymes, dNTPs, UNG enzymes, water 1 960μl / tube 2 PCR reaction solution 2 Primers, probes, Taq enzymes, dNTPs, UNG enzymes, water 1 960μl / tube 3 Positive quality control 1 Serum samples of inactivated HBV1896A 1 1.6mL / tube 4 Positive quality control 2 Serum samples of inactivated HBV1762T, 1764A 1 1.6mL / tube 5 Positive Control 3 Serum samples of inactivated HBV1762T, 1764G types 1 1.6mL / tube 6 Positive Control 4 Serum samples of inactivated HBV1762A, 1764A 1 1.6mL / tube 7 negative control Sterilized purified water 1 1.6mL / tube

[0047] Wherein, the PCR reaction solution 1 includes primers and probes for detecting G1896A...

Embodiment 2

[0052] 1. Sample DNA extraction

[0053] Use a commercial viral genome extraction kit to extract sample DNA, and positive quality control products need to participate in the extraction and preparation of HBV genomic DNA.

[0054] 2. PCR reaction system preparation and sample addition

[0055] Prepare PCR reaction system copies n according to the number of samples (n = number of samples + positive control × 4 + negative control), and fill each fluorescent quantitative PCR tube with 20 μL DNA template (DNA sample, negative control, positive control) of the PCR reaction system 10 μL, mixed well and centrifuged briefly, ready to go on the machine.

[0056] Using the kit of Example 1, prepare n tubes of PCR reaction system 1-2 according to Table 4.

[0057] Table 4

[0058]

[0059] 3. PCR amplification detection

[0060] Put the above fluorescent quantitative PCR tube into the fluorescent PCR instrument, and the program settings are shown in Table 5:

[0061] table 5

[0...

Embodiment 3

[0072] The method of Example 2 was used to test 18 samples, and the test results are shown in Table 7. Compared with the sequencing results, the consistency was 100%.

[0073] Table 7

[0074]

[0075]

[0076] In summary, the advantages of the primers and probes provided by the present invention are: using MGB modification to improve detection specificity, the accuracy of detection results and sequencing results is as high as 100%; the wild type and mutant type of the site can be detected at the same time; The judgment result of the ΔCt value method is easier to judge, and the result can be accurately typed by the typing software, and can also be confirmed and checked by the fluorescent quantitative PCR curve, and the result is accurate and reliable; Satisfies the needs of gene detection with low virus concentration in the market; not limited by the number of samples, the number of test samples can be allocated, when the number of samples is small, there is no need to s...

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Abstract

The invention discloses a primer, probe and reagent kit for predicting liver cancer susceptibility of a patient suffering from hepatitis B, and belongs to the field of in vitro nucleic acid detection.The reagent kit for predicting liver cancer susceptibility of the patient suffering from hepatitis B comprises the primer and the probe for detecting three mutation sites including G1896A, A1762T andG1764A. According to the primer and the probe provided by the invention, the precision rates of the detection result and the sequencing result can be as high as 100%; the primer and the probe can beused for simultaneously detecting the wild type and the mutant type of the sites, and the result is accurate and reliable; the detection lower limit of the DNA concentration of a detected HBV genome is 100IU / mL, so that gene detection requirement of low-virus concentration in the market can be met; and the reagent kit provided by the invention is free from the limit of sample size, the detection sample size can be distributed, when the sample size is low, accumulating samples is not needed, and the experimental result is quick and convenient to present.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a primer, a probe and a kit for predicting the susceptibility of liver cancer in patients with hepatitis B. Background technique [0002] Hepatitis B virus (HBV) infection is one of the important factors leading to the occurrence of liver cancer, and the liver cancer caused by hepatitis B virus (HBV) infection accounts for more than 60%. After 5 years of chronic hepatitis B infection, 10-20% of patients will develop liver cirrhosis, and 6-15% of patients with chronic liver cirrhosis and hepatitis B will develop liver cancer. Liver cancer is one of the most important cancers that seriously endanger the life and health of Chinese people, and it is a major public health problem that needs to be solved urgently. [0003] Clinically, hepatitis B E antigen (HBeAg) transfer and hepatitis B E antibody (HBeAb) positive are often considered to have produced antiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2561/113C12Q2563/107C12Q2545/114Y02A50/30
Inventor 薛怡婷
Owner 阿吉安(福州)基因医学检验实验室有限公司
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