Salmonella is a kind of food-borne pathogen which belongs to Enterobacteriaceae. However, the content of Salmonella in food is usually low, the routine test is time-consuming, so the detection with high sensitivity, repeatability and accuracy is needed. The nucleic acid reference material of Salmonella was used to verify the real-time fluorescence quantitative PCR (qRT-PCR) method for detecting Salmonella, the applicability, uniformity and stability of nucleic acid reference material of Salmonella were investigated in this study. To optimize the nucleic acid reference materials for verifying qRT-PCR method in detection of Salmonella, various enzyme systems for PCR and protective agents for nucleic acid reference materials were studied. The homogeneity, short-term stability and repeated freeze-thaw stability of nucleic acid reference materials were analyzed based on the linear graph and parameter table. The results showed that qRT-PCR method with relevant nucleic acid reference materials in this study was specific to Salmonella amplification, it was applicable to commonly used PCR instruments and various enzyme systems. The protective agent in reference materials did not inhibit the fluorescence amplification of Salmonella. The quality evaluation of reference material indicated that Salmonella nucleic acid samples were uniform, it remained stable within 6 days in the stability test without dilution. The quantity was stable after repeated freezing and thawing for 10 times. And the stability of diluted Salmonella nucleic acid sample was enhanced after adding protective agent. However, the short-term stability and quantity of diluted Salmonella nucleic acid samples were unstable in repeated freezing and thawing tests. The RT-PCR with reference material has high accuracy and stability in detecting Salmonella. It can be used to verify the detection ability of laboratories in rapidly and accurately detecting microbial nucleic acid with traceability.
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