A novel asymmetric and competitive allele specific real-time fluorescent PCR with MGB universal probes for detection of SNPs and point mutations

Background: Current technologies for detecting gene mutations have some disadvantages in terms of equipment requirements, flexibility, cost, technical difficulties and assay performance problems including sensitivity, reliability, reproducibility and accuracy. In this study, we developed an asymmetric and competitive allele specific PCR system with minor groove binder (MGB) universal probes (F-CAUM PCR) for detection of single nucleotide polymorphisms (SNP) and point mutations. Methods: By combining characteristics of competitive allele specific PCR (Kompetitive Allele Specific PCR, KASP) and asymmetric PCR, there are three essential components contributing to the performance of this F-CAUM PCR assay, including the unique composition of the forward primers, the ratio between the forward and the reverse primers, and the structure of MGB-labeled universal probes. The 3' ends of the two forward primers are designed complementary to the target sequences of the wild type and the mutant type respectively; each forward primer is comprised of target sequence specific region, universal tag region and the polymerase-binding region. Furthermore, the quantity of forward primers is less than the reverse primer, which minimizes the interference caused by its competitive binding to the universal probe. Lastly, MGB is attached to the quenching molecules, which further improves the assay specificity. We assessed the specificity, sensitivity and reproducibility of F-CAUM PCR. Results: In specificity assay, F-CAUM PCR could identify KRAS G12D mutation with high specificity, without interference by other KRAS mutations at the same locus. In sensitivity assay, the lowest detectable concentration by F-CAUM PCR was 10-1 copies/mu l for brain derived neurotrophic factor gene (BDNF) rs6265 and KRAS G12D mutation. The lowest detectable percentage of the KRAS G12D mutation was 1% for KRAS G12D mutation. In reproducibility assay, for detecting the BDNF human genomic DNA standards and the KRAS plasmid mixture, the Ct values and Delta Ct values among six replicates showed minimal variance, with standard deviation (SD) ranging from 0.15 to 0.48, and coefficient of variation (CV) ranging from 3.22% to 6.15%. For clinical samples, the genotyping results of BDNF rs6265 in 30 oral epithelium mucosa specimens and KRAS mutation detection in colorectal cancer patients by the F-CAUM assay were consistent with those from Sanger sequencing. Conclusions: Our results confirmed that F-CAUM PCR was highly specific, sensitive and reproducible for SNPs and gene mutation detection. The results of clinical samples by F-CAUM PCR were consistent with those from Sanger sequencing.