Construction of standard plasmids for detecting Salmonella in meat products with real-time fluorescence quantitative polymerase chain reaction

ObjectiveTo construct standard plasmids for detecting Salmonella in meat products with real-time fluorescence quantitative polymerase chain reaction(PCR).

MethodsPrimers directed at Salmonella invA gene were designed. Specific fragments were amplified and cloned into plasmid vectors to construct recombinant plasmids. The constructed recombinant plasmids were used as standards for implementing the real-time fluorescence quantitative PCR after optimization, establishing standard curves and examining the sensitivity and stability of these standards.

ResultsThe plasmid standard containing the invA gene in Salmonella was successfully constructed. The cycle threshold value(Ct value) of the standard curve established by means of the plasmid standard and the number of template copies exhibited good linear relationship(r²=0.9979).The minimum that could be detected by means of this method was 10 copies/reaction. The standard plasmid was proved to have good stability. This standard plasmid was used to detect Salmonella in meat products. After two hours enrichment, sample detection could be completed within seven hours.

ConclusionThe constructed plasmid standard can be used for detecting Salmonella in meat products by means of the fluorescence quantitative PCR, which can provide reliable reference bases for examining quality of relevant experiments.