Rapid Detection of K-ras Mutations in Bile by Peptide Nucleic Acid-mediated PCR Clamping and Melting Curve Analysis: Comparison with Restriction Fragment Length Polymorphism Analysis

摘要: Background: Current methods for detection of K- ras gene mutations are time-consuming. We aimed to develop a one-step PCR technique using fluorescent hybridization probes and competing peptide nucleic acid oligomers detect in bile compare the efficacy with restriction fragment length polymorphism (RFLP) analysis. Methods: Bile samples were obtained from 116 patients biliary obstruction, including gallstones (n = 64), benign strictures 6), pancreatic cancer 20), cholangiocarcinoma 26). The DNA was extracted subjected mutation analysis by real-time RFLP analysis. Mutations confirmed direct sequencing. sensitivity specificity calculated according clinical results. Results: time <1 h, whereas took more than 2 days. With sensor probe designed GAT (G12D) mutant codon 12 gene, method also detected GTT (G12V) mutant. In contrast, specific TGT (G12C) (G12D), AGT (G12S), mutants addition assay allowed 3000-fold excess wild-type DNA. bile, 16 46 malignant cases 15 All wild type. Conclusion: Real-time cysteine-specific (TGT) can rapidly diagnose obstruction high specificity.