Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

  • Wu, Jiong (Department of Laboratory Medicine, Zhongshan Hospital, Fudan University) ;
  • Zhou, Yan (Department of Laboratory Medicine, Zhongshan Hospital, Fudan University) ;
  • Zhang, Chun-Yan (Department of Laboratory Medicine, Zhongshan Hospital, Fudan University) ;
  • Song, Bin-Bin (Department of Laboratory Medicine, Zhongshan Hospital, Fudan University) ;
  • Wang, Bei-Li (Department of Laboratory Medicine, Zhongshan Hospital, Fudan University) ;
  • Pan, Bai-Shen (Department of Laboratory Medicine, Zhongshan Hospital, Fudan University) ;
  • Lou, Wen-Hui (Surgical Department, Zhongshan Hospital, Fudan University) ;
  • Guo, Wei (Department of Laboratory Medicine, Zhongshan Hospital, Fudan University)
  • Published : 2015.01.22


Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.


Supported by : Ministry of Education


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