Development of a Multiplex Allele-Specific Real-time Polymerase Chain Reaction Assay for detection of KRAS gene mutations in Thai colorectal cancer tissues
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Abstract
Mutation analysis of KRAS is necessary before starting treatment with monoclonal anti-EGFR antibodies for effective and appropriate treatment for individual patients. The objective of this study is to develop a Multiplex Allele-Specific Real-time PCR assay for analysis of the mutational status of KRAS codons 12 and 13, including 7 types of KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). 160 of FFPE colorectal cancer tissues were collected from Department of Medical Services, Institute of Pathology. DNA was isolated from the FFPE tissue using AS-primers specific for mutant DNA. Moreover, the MAS-Real-time PCR analysis of samples showed good concordance (K=0.837, 95% CI 0.740-0.933) with pyrosequencing. In addition, the MAS-Real-time assay has a sensitivity of 78.26% and specificity of 100%. Our developed MAS-Real-time PCR can be applied for detection of KRAS gene mutations in FFPE tissues which is a reliable, rapid, cost-effective method and not requiring advanced instruments.
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