Universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR) for SNP genotyping

Background: The detection and identification of single nucleotide polymorphism (SNP) is essential for determining patient disease susceptibility and the delivery of medicines targeted to the individual. At present, SNP genotyping technology includes Sanger sequencing, TaqMan-probe quantitative polymerase chain reaction (qPCR), amplification-refractory mutation system (ARMS)-PCR, and Kompetitive Allele-Specific PCR (KASP). However, these technologies have some disadvantages: the high cost of development and detection, long and time consuming protocols, and high false positive rates. Focusing on these limitations, we proposed a new SNP detection method named universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR). In this method, only two types of fluorescence-labeled probes were used for SNP genotyping, thus greatly reducing the cost of development and detection for SNP genotyping. Results: In the amplification process of UPIP-qPCR, unlabeled intermediate primers with template-specific recognition functions could trigger probe hydrolysis and specific signal release. UPIP-qPCR can be used successfully and widely for SNP genotyping. The sensitivity of UPIP-qPCR in SNP genotyping was 0.01 ng, the call rate was more than 99.1%, and the accuracy was more than 99.9%. High-throughput DNA microarrays based on intermediate primers can be used for SNP genotyping. Conclusion: This novel approach is both cost effective and highly accurate; it is a reliable SNP genotyping method that would serve the needs of the clinician in the provision of targeted medicine.