title: Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations.
abstract: The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔCq method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Jun-Fu
authors.initials: JF
authors.lastname: Huang
authors.affiliation: Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Dong-Zhu
authors.initials: DZ
authors.lastname: Zeng
authors.affiliation: Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Guang-Jie
authors.initials: GJ
authors.lastname: Duan
authors.affiliation: Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Guo-Hong
authors.initials: GH
authors.lastname: Deng
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Han
authors.initials: H
authors.lastname: Xia
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Han-Qing
authors.initials: HQ
authors.lastname: Xu
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Na
authors.initials: N
authors.lastname: Zhao
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Wei-Ling
authors.initials: WL
authors.lastname: Fu
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Huang
mesh_terms: D003062:Codon / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000556:secondary; D004273:DNA, Neoplasm / Q000235:genetics*; D005838:Genotype; D006801:Humans; D020125:Mutation, Missense / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D003062:Codon; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)