Abstract
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.
Highlights
KRAS belongs to the RAS family of small GTPases involved in the coupling of signal transduction from surface receptors to many different targets that regulate diverse biological responses including cell growth, proliferation, differentiation and survival [1,2]
A central feature of SensiScreen1 is the inclusion of a BaseBlockerTM that comprises several pentabasesTM and is designed to bind strongly and to wild type (WT) DNA with little or no affinity towards mutated DNA (Fig 1)
LOD, limit of detection; WT, wild type; SPX, simplex assay; MPX, multiplex assay. * Limit of detection (LOD) determined using a ΔCt between reference and assay of 9. ^ ΔCt calculated as the lowest possible difference in Ct between reference and assay of the duplicate PCR reactions performed using the 0% dilution point. ¤ PCR efficiency calculated using the 10%, 5%, 1% and 0.5% dilution points
Summary
KRAS belongs to the RAS family of small GTPases involved in the coupling of signal transduction from surface receptors to many different targets that regulate diverse biological responses including cell growth, proliferation, differentiation and survival [1,2]. Sensitive detection of KRAS exon 2 somatic mutations using BaseBlockersTM in real-time PCR. SensiScreen products, but not the testing and analysis of patient material nor in the conclusions of the performance of SensiScreen against competing technologies. The funders had no additional role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Support for this study was provided by Danish Agency for Science Technology and Innovation [E!7034 ONSET diagnostics] and Fondazione Ticinese per la Ricerca contro il Cancro.
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