[Establishment of a novel TB-ARMS qPCR method for kras mutation detection]

A simple, robust and highly sensitive TB-ARMS method based on qPCR technique was developed to detect kras mutations. The technique was evaluated, and its clinical application was investigated. Mutation specific primers for eight common kras mutations and wild type gene targeted blockers were designed and optimized. Moreover, a mutant-enriched condition was used in to improve the sensitivity and specificity of mutation detection. Constructed plasmids carrying mutant kras genes, as well as confirmed wild type genomic DNA, were used as standard samples for evaluation of the methodology. The performance of our new method was validated by comparing the results of our method with that of a commercial kras kit in testing 40 clinical samples. Preoperative plasma samples, as well as paired tissue samples, were tested in parallel for evaluation of its clinical application. We have developed a new TB-ARMS method for kras mutation detection that can detect minor mutant alleles with a frequency as low as 0.01% in a heterogeneous sample. We have successfully demonstrated its 0.01% detection sensitivity with highly specific mutant amplification in conjunction with selective wild type suppression by blocker under a mutant-enriched reaction condition. We also showed that our TB-ARMS method was more accurate than the commercial kras kit, which is widely used presently. Furthermore, we have validated our method as an efficient liquid biopsy method, and the results of the plasma DNA detection with our TB-ARMS method were in consistent with the sequencing results of paired tissue samples. In conclusion, our TB-ARMS qPCR method could be effectively applied in kras mutation test for clinical tissue samples, as well as for liquid biopsy samples such as plasma.