Detection Of KRAS Gene In Colorectal Cancer By Using Allele Primers And Taqman Probe-based QPCR

The KRAS gene of colorectal cancer is located on the second exon of human chromosome 12 and plays the vital founction in signal transduction from membrane receptor to adenylate cyclase.In recent years,the correlation between KRAS gene and anti-EGFR antibody efficacy is the most prominent result in the research field of colorectal cancer.The results showed that anti-EGFR antibody was recommended for treatment in patients with KRAS unmutations.Therefore,it is particularly important for the detection of gene mutations before targeted drug utilization.Therefore,this study intends to establish an economical,simple and sensitive KRAS gene detection technology for colorectal cancer.Seven KRAS gene mutants were successfully constructed by gene cloning,and seven allelic primers and a Taqman probe were designed.Single-pair primers were used to detect the specificity of mutant templates and allele primers and Taqman probes.Mixed 7 pairs of primers and then add probes,and the qPCR detection system was used to detect 7 common KRAS gene mutation sequences to construct a stable detection system.Analytical performance testing of constructed mixed allele primers and Taqman probe qPCR detection system:(1)KRAS wild-type tissue DNA of Colorectal cancer as background,7 KRAS mutation sequences were prepared to correspond to 10%,1%,0.1% and 0.01% mutant concentrations,the minimum detection limit was 0.1%.(2)In the background of colorectal cancer KRAS wild-type tissue DNA,samples containing 5% KRAS three homologous gene sequences(codon 12/13 pseudogene sequence of KRAS,the second exon of NRAS and the third exon of HRAS)were detected,the results was undetermined.The wild-type tissue DNA of KRAS in colorectal cancer was mixed with 5% of 7 KRAS mutant sequences,and detected in the presence of 5% homologous gene sequences.The results showed that no cross-reaction occurred.Microorganisms were added to the KRAS wild-type tissue DNA of colorectal cancer,and then extracted DNA.Any of the seven KRAS mutant sequences were selected and prepared to detect samples corresponding to the 0.1% mutation level.The results showed that in the presence and absence of microorganisms,the test results were positive at 0.1% mutation concentration.(3)In the presence of triglycerides and bovine hemoglobin,the wild-type tissue DNA of colorectal cancer KRAS was extracted,and 0.1% of 7 KRAS mutant sequences were mixed.The results showed that in the presence and absence of triglyceride or bovine hemoglobin,the test results at 0.1% mutation concentration were positive,which did not affect the determination of the experimental results.Eighteen patients with colorectal cancer were enrolled in paraffin-embedded tissues.Among them,13 KRAS gene mutations and 5 KRAS genes were wild-type,and methodological comparisons were performed.(1)Consistency evaluation was performed with Sanger sequencing: 13 cases were positive and 5 cases were negative in both methods,and the total detection rate was 100%(13/13),indicating that the two methods were completely consistent.(2)Repeatability assessment.An example of a KRAS gene mutant FFPET sample was tested for intra-and inter-assay.The CV values were 1.6% and 2.0%,respectively,indicating that the protocol has good stability.The detection method constructed in this study has the characteristics of high specificity,high sensitivity and short time consumption in the detection of colorectal cancer KRAS gene,and it is worthy of application in clinical sample detection.