Primer, probe and reagent kit for detecting mutation of 2312nd-2313rd sites of EGFR (epidermal growth factor receptor) gene

[0020] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the examples are in accordance with the conventional conditions well known to those skilled in the art, or in accordance with the conditions recommended by the manufacturer.

[0021] 1. Principle

[0022] After the specific primers and probes of the present invention are combined with the DNA template, the Taq DNA polymerase uses deoxynucleotide (dNTP) as a substrate to expand the mutant genes of the internal reference gene (IR) and EGFR gene in vitro. increase. The detection adopts fluorescent PCR technology, which releases fluorescence through hydrolysis of specific probes, monitors the progress of the PCR reaction, and determines the EGFR gene mutation.

[0023] The kit of the present invention is separately provided with an internal reference gene detection system. The reference gene is a housekeeping gene that is different from the EGFR gene to be tested. By detecting the amplification of the internal reference gene (FAM channel), it is possible to analyze whether the DNA to be tested can be amplified normally, so as to rule out the failure of PCR detection due to poor DNA purity, concentration, or PCR inhibitors.

[0024] The kit of the present invention simultaneously sets an internal control gene (InternalControl, IC) detection system in the EGFR gene mutation detection system. The two systems react simultaneously in the same PCR tube. The internal control gene is also a housekeeping gene that is different from the gene to be tested EGFR. The probe that recognizes the EGFR gene mutation template is modified into a FAM fluorescent group, and the probe that recognizes the internal control gene template is modified into a HEX fluorescent group. By detecting the internal control gene amplification (HEX channel), it is possible to analyze whether the DNA to be tested can be amplified normally, so as to rule out the failure of PCR detection due to missing reagents or samples, or samples containing PCR inhibitors.