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Updated: Jun 1, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
A fully integrated, automated and rapid detection system for KRAS mutations
Norio Ureshino1, Naoko Sueoka-Aragane, Tomomi Nakamura
1Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, and Department of Medical Oncology, Saga Prefectural Hospital, Saga 849-8501, Japan.
Abstract:
KRAS mutations are detected in tumors of various organs, and they are also markers of resistance for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and monoclonal antibodies against the EGFR. Thus, the accurate and rapid detection of KRAS mutations is crucial, not only for screening, but also for the prediction of the efficacy of molecular-targeted therapy. The aim of the present study was to establish a novel automated detection system for KRAS mutations. One hundred and thirty-six lung adenocarcinoma patients were genotyped for KRAS mutations with both the conventional direct sequence (DS) method and with the newly developed quenching probe (QP) method that obtains data automatically within 60 min. The detection limit of the QP method using a control plasmid containing the KRAS mutation was 50 copies, and 10% mutant plasmid was detected in the mixture of wild-type and mutants. The results obtained by the QP and DS methods were identical in all but two of the 136 cases. The two differentially identified samples, which consisted of substantially fewer lung cancer cells, were positive according to the QP method but negative as determined by DS for KRAS mutations. These findings characterize the QP method as an accurate and rapid detection system for KRAS mutations.
Insights
A new automated quenching probe (QP) method accurately and rapidly detects KRAS mutations in lung adenocarcinoma. This system offers a faster alternative to conventional methods for targeted therapy prediction.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- KRAS mutations are prevalent in various cancers and indicate resistance to EGFR-targeted therapies.
- Accurate and rapid KRAS mutation detection is vital for cancer screening and predicting treatment efficacy.
Purpose of the Study:
- To develop and validate a novel, automated quenching probe (QP) method for KRAS mutation detection.
- To compare the performance of the QP method against the conventional direct sequencing (DS) method.
Main Methods:
- Genotyping of KRAS mutations in 136 lung adenocarcinoma patients using both direct sequencing (DS) and the automated quenching probe (QP) method.
- Assessing the detection limit and sensitivity of the QP method using control plasmids.
Main Results:
- The QP method achieved automated data acquisition within 60 minutes.
- The QP method demonstrated a detection limit of 50 copies and could detect 10% mutant plasmid in mixtures.
- The QP and DS methods yielded concordant results in 134 out of 136 cases; the QP method identified two samples as positive that were negative by DS, likely due to lower cancer cell content.
Conclusions:
- The quenching probe (QP) method is a highly accurate and rapid system for detecting KRAS mutations.
- This automated system shows potential for improving KRAS mutation analysis in clinical settings, aiding in personalized cancer therapy.
