Human hepatic fibrosis and liver cancer risk gene TNF-alpha polymorphism detection kit and preparation method and application thereof
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, high equipment requirements, improve detection efficiency, avoid false negative and false positive problems, and ensure The effect of accuracy
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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
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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