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
Laboratory of Molecular Pathology, Institute of Pathology, Locarno, Switzerland.
PentaBase Aps, Odense, Denmark.
PLoS One. 2017 Jun 21;12(6):e0178027. doi: 10.1371/journal.pone.0178027. eCollection 2017.
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.
KRAS（ Kirsten大鼠肉瘤病毒癌基因同源物）基因第12和13密码子的激活突变与多种人类癌症类型的发生发展有关，并影响其临床评估、治疗和预后。目前有许多不同的KRAS基因分型方法，其灵敏度、出结果时间以及对实验室设备和用户技能的要求各不相同。在此，我们展示了SensiScreen® KRAS外显子2单重和多重CE体外诊断检测方法，该方法采用基于新型实时PCR的方法，基于PentaBase专有的DNA类似物技术检测KRAS突变，并设计用于在标准实时PCR仪器上运行。通过所包含的碱基阻断技术，我们证明SensiScreen®能够特异性扩增感兴趣的突变等位基因，而野生型等位基因无扩增或扩增受到高度抑制。此外，在野生型背景下对突变DNA进行系列稀释表明，所有SensiScreen®检测方法的检测限都在0.25-1%的范围内。最后，在三个不同的结直肠癌患者群体中，SensiScreen®检测方法证实了先前通过常用的KRAS突变检测方法确定的KRAS基因型，值得注意的是，在其中两个群体中，SensiScreen®检测出了常用方法未检测到的额外突变阳性病例。
