Analytical performance of a PCR assay for the detection of KRAS mutations (codons 12/13 and 61) in formalin-fixed paraffin-embedded tissue samples of colorectal carcinoma

摘要: KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe performance a TaqMelt polymerase chain reaction (PCR) assay—the cobas® Mutation Test—designed to detect 19 mutations codons 12, 13, and 61. The limit detection was determined using DNA blends from cell lines, plasmids, formalin-fixed paraffin-embedded tissue specimens. Assay compared Sanger sequencing panel 188 Discordant specimens were subjected next generation pyrosequencing (454). repeatability assessed six A >95% correct call rate obtained all specimen types with ~5% mutant alleles at inputs 0.8–6.3 ng per PCR reaction; 100% observed recommended input 50 ng. positive percent agreement 97.5% (79/81) for 12/13 85.7% (6/7) codon Negative 94.4% (101/107) 99.4% (180/181) Nine 10 discordant yielded 454 results consistent results. With repeated testing, assay showed (192/192) operators, instruments, reagent lots, days tested. test detects 61 <5%. more sensitive specific than sequencing, highly reproducible. Test not influenced by various endogenous interfering substances or common gut microbes.