Specific probes and kits for detecting t790m, c797s and l798i sites of egfr gene
A C797S, specific technology, applied in the determination/testing of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., can solve problems such as complex operations and inapplicability to large-scale clinical promotion
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Embodiment 1
[0050] In this example, the above-mentioned specific probes T790M-P, C797S-P, L798I-P and corresponding specific primers were selected to construct a PCR reaction system.
[0051] Specifically, the selected concentration is 2×10 3 Copies / μl of artificially constructed EGFR gene mutation plasmid standard at T790M, C797S and L798I sites and 2×10 6 The copies / μl wild-type plasmid standard was used as a reaction template, and the ratio of mutant plasmid to wild-type plasmid was 1:1000. Divide 3 tubes of 10 μl PCR reaction system to detect the above 3 mutation sites respectively. The first tube contains 1 μl T790M mutant plasmid and 1ul wild-type plasmid template, 10mmol / L Tris-HCl, pH=8.3, 50mmol / L KCl, 0.5U Taq enzyme, 3.5mmol / L Mg 2+ , 10mM / L dNTP, 0.45μmol / L T790M-P probe, 0.3μmol / L T790M upstream primer, 0.3μmol / T790M downstream primer. The second tube contains 1 μl C797S mutant plasmid and 1ul wild-type plasmid template, 10mmol / L Tris-HCl, pH=8.3, 50mmol / L KCl, 0.5U Taq en...
Embodiment 2
[0054] In this example, the above-mentioned specific probes T790M-P, C797S-P, L798I-P and corresponding specific primers were selected to construct a PCR reaction system.
[0055] Select the dosage as 2×10 6 copies / ul, 2×10 5 copies / ul, 2×10 4 copies / ul, 2×10 3 copies / ul, 2×10 2 copies / ul, 2×10 1 Copies / ul, 2×100copies / ul (i.e. 10-fold serial dilution) artificially constructed EGFR gene mutant gene plasmid standards at T790M, C797S and L798I sites were used as reaction templates. 10μl of PCR reaction system was used to investigate and analyze the sensitivity of the above three mutation sites. The first tube to the seventh tube all contain 10mmol / L Tris-HCl, its pH=8.3, 50mmol / L KCl, 0.5U Taq enzyme, 3.5mmol / L Mg 2+ , 10mM / LdNTP, 0.45μmol / L T790M-P probe, 0.3μmol / L T790M upstream primer, 0.3μmol / T790M downstream primer, add 1ul of T790M mutant gene plasmid standard of the above concentration in sequence from the first tube to the seventh tube as a template. The eighth t...
Embodiment 3
[0058] In this example, the T790M site of the EGFR gene was taken as the research object, and 86 non-small cell lung cancer patient samples were tested for the T790M site mutation by using the detection method described in this article, and the human EGFR The gene mutation detection kit was used as a reference. In this example, the above-mentioned specific probe T790M-P and corresponding specific primers were selected to construct a PCR reaction system. The results of the two detection methods are shown in Table 1.
[0059] Table 1 Example of the present invention and the test result comparison table of Aide biological kit
[0060] T790M site mutation The detection method of this embodiment Ed Biological Kit yes 2 2 no 84 84
[0061] It can be seen from Table 1 that the detection results of the specific probes and kits provided by the present invention are the same as those of mature products on the market, have a good detection level, and can...
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