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A human liver fibrosis, liver cancer risk gene tnf-alpha polymorphism detection kit and its preparation method and application

A detection kit and detection reagent technology, applied in the field of biomedical clinical detection, can solve the problems of low detection trace sensitivity, unfavorable clinical promotion, low sensitivity, etc., to improve detection efficiency, avoid false negative and false positive problems, and ensure The effect of accuracy

Active Publication Date: 2019-08-09
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most common method is the sequencing method, which is low in cost, but takes a long time and is not sensitive; the high-resolution melting curve method requires relatively high equipment requirements, which is not conducive to clinical promotion; the traditional fluorescent PCR method is widely used in clinical practice. However, the detection sensitivity is not high

Method used

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  • A human liver fibrosis, liver cancer risk gene tnf-alpha polymorphism detection kit and its preparation method and application
  • A human liver fibrosis, liver cancer risk gene tnf-alpha polymorphism detection kit and its preparation method and application
  • A human liver fibrosis, liver cancer risk gene tnf-alpha polymorphism detection kit and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A kit for rapid detection of human liver fibrosis and liver cancer risk gene TNF-α gene polymorphism, the detection reagent is aimed at the rs1800629 polymorphism site of the TNF-α gene, and the detection reagent includes: the sequence is SEQ ID NO. 1, the sequence is the reverse primer of SEQ ID NO.2, the sequence is the identification primer of SEQ ID NO.3 (the 5' end carries the VIC fluorescent group), the sequence is the identification primer of SEQ ID NO.4 ( The 5' end carries the FAM fluorescent group), the sequence is the PNA of SEQ ID NO.5 (the 3' end carries the BHQ quenching group), the sequence is the PNA of SEQ ID NO.6 (the 3' end carries the BHQ quenching group ), the sequence is the internal reference forward primer of SEQ ID NO.7, the sequence is the internal reference reverse primer of SEQ ID NO.8, the sequence is the internal reference identification primer of SEQ ID NO.9 (the 5' end carries the ROX fluorescent group), the sequence It is the internal re...

Embodiment 2

[0051] A human liver fibrosis, liver cancer risk gene TNF-α polymorphism detection kit, including detection reagents, positive control and negative control; 2 shows:

[0052] Table 1 detection reagent composition

[0053] components Proportion Premix qPCR MIX 25 μL SEQ ID NO.1 100~400nM SEQ ID NO.2 100~400nM SEQ ID NO.3 50~100nM SEQ ID NO.4 50~100nM SEQ ID NO.5 50~100nM SEQ ID NO.6 50~100nM SEQ ID NO.7 100~400nM SEQ ID NO.8 100~400nM SEQ ID NO.9 50~100nM SEQ ID NO.10 50~100nM TE buffer Make up to 30 μL

[0054] Table 2 Kit Composition

[0055]

Embodiment 3

[0057] The minimum detection limit of the sample detected using the kit prepared in Example 2 specifically includes the following steps:

[0058] (1) Use Tiangen Biochemical Biotechnology Company Whole Blood DNA Extraction Kit to extract 5 human peripheral blood genomic DNAs. The specific operation steps are strictly in accordance with the kit instructions to obtain human genomic DNA samples to be tested, and dilute the samples to 2ng / μL , 0.5ng / μL, 0.1ng / μL, 0.05ng / μL.

[0059] (2) Take PCR eight-tubes and add 20 μL of gradient dilution samples to each well; add 30 μL of well-mixed detection reagents to each well; oscillate and centrifuge the eight-tubes to mix the reaction solution;

[0060] (3) Put the eight tubes into the ABI 7500 fluorescent PCR instrument for amplification detection. The PCR reaction conditions are shown in Table 3:

[0061] Table 3 PCR reaction program

[0062]

[0063] (4) Analysis results:

[0064] Analysis of 4 concentration gradient detection ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a human hepatic fibrosis and liver cancer risk gene TNF-alpha polymorphism detection kit and a preparation method and application thereof. The kit comprises a detection reagent, a positive control product and a negative control product which are used for amplifying risk gene TNF-alpha polymorphism related to hepatic fibrosis and liver cancer risk, and the detection reagent contains a primer sequence, a PNA sequence and a PCR reaction solution used for amplifying the rs1800629 site of TNF-alpha gene. The kit isused for detecting human hepatic fibrosis and liver cancer risk gene TNF-alpha polymorphism, has the advantages of being high in sensitivity, high in specificity, simple and convenient in operation,reliable in result and the like, can complete detection in one hour, and is simple and objective in result interpretation.

Description

technical field [0001] The invention belongs to the technical field of biomedical clinical detection, and specifically relates to a human liver fibrosis and liver cancer risk gene TNF-α polymorphism detection kit and a preparation method and application thereof. Background technique [0002] Liver fibrosis refers to the pathological process in which the proliferation and degradation of extracellular matrix are out of balance when liver cells are stimulated by inflammation or necrosis, which leads to abnormal deposition of connective tissue in the liver. Primary liver cancer ranks fifth among common malignant tumors, and more than 600,000 people die from liver cancer every year, ranking third in global tumor-related deaths. my country's liver cancer cases account for more than 50% of the world's total. Timely diagnosis of liver cancer And treatment is of great significance to improve the survival rate of patients. [0003] The process of liver fibrosis involves a variety of c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6886C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q2600/156
Inventor 马晓晶李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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