Identification of Tps2 Used as an Endogenous Reference Gene in Qualitative and Real-time Quantitative PCR Detection of Flammulina velutipes

Among the methods of detecting food adulteration, the PCR amplification techniques of the endogenous reference gene are increasingly significant. In this article, the improved SDS-CTAB is chosen for the DNA extraction of Flammulina velutipes, the tre6P Phosphatase gene (Tps2) is selected an endogenous reference gene suitable to identify agricultural important fungus F. velutipes and by-products via PCR methods. The Tps2 gene was assayed in 2 varieties of F. velutipes and 9 non-F. velutipes species by both qualitative and quantitative PCR, no amplicon was captured in the non-F. velutipes species. By SYBR Green quantitative PCR, the melting curve showed that the target Tps2 sequence displayed high homogeneity in different F. velutipes varieties. In Taqman probe quantitative PCR analysis, the detection limit was as low as 3 pg/uL of DNA template. From what is said in the foregoing, we can conclude that the Tps2 gene is a reliable, convenient, and accurate endogenous reference gene for detecting F. velutipes component, even in the processed food products, such as sauce and biscuits.