Ultra-rapid real-time polymerase chain reaction assay for genotyping of the NT5E gene in Japanese black beef using a mobile PCR device, PCR 1100.

Abstract

The concentration of inosine 5'-monophosphate (IMP) in beef is an important factor contributing to beef palatability. A previous study suggested that single nucleotide polymorphisms (SNPs) in the ecto-5'-nucleotidase (NT5E) gene strongly affect the concentration of IMP under postmortem conditions by regulating NT5E enzymatic activity in beef. Genotyping of the NT5E gene is performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR assay. However, these conventional laboratory assays require large installed instruments. They also involve complicated procedures and are time-consuming. Here, the PCR primers and probes for the NT5E gene (rs42508588 SNP) were designed and synthesized, and we examined the rapid genotyping of the NT5E gene using a PicoGene PCR 1100 mobile PCR device. The results showed that this system enabled rapid amplification of each allele at approximately 19.4s per cycle, with a total run time of 13 min 36 s. This device is portable and does not require a power supply, which facilitates its use not only in specific laboratories but also in meat production farms and distribution stages of beef.