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

The detection and identification of single nucleotide polymorphism (SNP) is an important basis for the evaluation of individual medicine and the judgement of disease susceptibility. At present, SNP genotyping technology includes the Sanger sequencing, TaqMan probe quantitative polymerase chain reaction (qPCR), amplification-refractory mutation system (ARMS)-PCR, Kompetitive allele specific PCR (KASP), and next-generation sequencing (NGS), etc. However, there are some disadvantages such as high cost of development and detection, long detection period and easily occurring false-positive results to these technologies. Focusing on these limitations, we proposed a new SNP detection method named as universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR). In this method, only two types of fluorescence-labeled probes were used for all SNP genotyping, thus, greatly reducing the cost of development and detection for SNP genotyping. In the amplification process, unlabeled intermediate primers with template specific recognition function were able to trigger probe hydrolysis and specific signal release. 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%. This novel technology gave rise to the low cost and high accuracy of SNP genotyping, and provided a new and reliable SNP genotyping method for the development of precision medicine.