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.

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 19.4 s 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.

The NT5E gene variant strongly affects the degradation rate of inosine 5'-monophosphate under postmortem conditions in Japanese Black beef.

Inosine 5'-monophosphate (IMP) contributes to the umami taste in Japanese Black beef. In a previous study, it was suggested that single nucleotide polymorphisms (SNPs) in the ecto-5'-nucleotidase (NT5E) gene affect the concentration of IMP under postmortem conditions by regulating NT5E enzymatic activity in Japanese Black beef. The present study showed that the degradation rate of IMP in Japanese Black beef was significantly different among NT5E genotypes in the middle stage of postmortem, indicating a significant difference in the concentrations of IMP and its degradation products among NT5E genotypes. In addition, no significant difference was observed among NT5E genotypes in the concentrations of IMP precursors or other taste-active compounds. These results indicate the significant effect of the interaction between the NT5E genotype and the aging period on the degradation rate of IMP in beef.