SensiScreen®KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations

• Published in: PloS one 2017-06, Vol.12 (6), p.e0178027-e0178027
• Main Authors: Riva, Alice, BØrgesen, Michael, Guldmann-Christensen, Mariann, Hauge Kyneb, Majbritt, Voogd, Kirsten, Andersen, Christina, Epistolio, Samantha, Merlo, Elisabetta, Yding Wolff, Tine, Hamilton-Dutoit, Stephen, Lorenzen, Jan, Christensen, Ulf Bech, Frattini, Milo
• Format: Article
• Language: English
• Subjects: Alleles; Assaying; Biological Assay - methods; Biology and Life Sciences; Brochures; Cancer; Cancer therapies; Cohort Studies; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - diagnosis; Colorectal Neoplasms - genetics; Deoxyribonucleic acid; Dilution; DNA; DNA methylation; DNA, Neoplasm - genetics; Epidermal growth factor; Exons; Genes; Genotype; Genotyping; Homology; Humans; Identification methods; K-Ras protein; Laboratories; Laboratory equipment; Life sciences; Lung cancer; Medicine and Health Sciences; Metastasis; Multiplex Polymerase Chain Reaction - methods; Multiplexing; Mutation; Mutation - genetics; Pathology; Patients; Polymerase chain reaction; Populations; Proto-Oncogene Proteins p21(ras) - genetics; R&D; Real time; Real-Time Polymerase Chain Reaction - methods; Research & development; Research and Analysis Methods; Sarcoma; Signal transduction; Technology; Tumor Cells, Cultured; Tumors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0178027

Activating mutations in codon 12 and codon 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene are implicated in the development of several human cancer types and influence their clinical evaluation, treatment and prognosis. Numerous different methods for KRAS genotyping are currently available displaying a wide range of sensitivities, time to answer and requirements for laboratory equipment and user skills. Here we present SensiScreen® KRAS exon 2 simplex and multiplex CE IVD assays, that use a novel real-time PCR-based method for KRAS mutation detection based on PentaBase's proprietary DNA analogue technology and designed to work on standard real-time PCR instruments. By means of the included BaseBlocker™ technology, we show that SensiScreen® specifically amplifies the mutated alleles of interest with no or highly subdued amplification of the wild type allele. Furthermore, serial dilutions of mutant DNA in a wild type background demonstrate that all SensiScreen® assays display a limit of detection that falls within the range of 0.25-1%. Finally, in three different colorectal cancer patient populations, SensiScreen® assays confirmed the KRAS genotype previously determined by commonly used methods for KRAS mutation testing, and notably, in two of the populations, SensiScreen® identified additional mutant positive cases not detected by common methods.