The devolopment of duplex real-time PCR based on SYBR Green florescence for rapid identification of ruminant and poultry origins in foodstuff

摘要

SDRT-PCR (SYBR duplex real-time polymerase chain reaction) was designed for an assay that can combine the advantages of real-time PCR and multiplex PCR to identify animal genes more quickly. SDRT-PCR based on melting temperature (tm) discrimination by using SYBR Green fluorescence dye was developed for the analysis of ruminant and poultry contained in foodstuffs. Primers specific to ruminant and poultry were designed for species-specific gene-amplification. Appropriate mixtures of poultry and ruminant meat DNAs were used to develop the assay. Gene products of ruminant and poultry were represented in two melting peaks generated simultaneously at temperatures of 83.2 and 86.5 ℃, respectively. Duplexing results obtained with one of the multiplex polymerase mixes correlated extremely well with the singleplex reference. The objective of the study was to design a rapid, specific and accurate duplex real-time PCR assay by using SYBR Green fluorescence dye that is cheaper than double labelled probes to detect a group of mixed meats simultaneously.