Rapid Detection and Identification of Salmonella in Proficiency Testing Samples by TaqMan Real-Time Fluorescent PCR
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Abstract:
According to GB 4789.4-2016 standard, the conventional culture method was used to test the ACAS-PT526 proficiency test sample for Salmonella, and TaqMan real-time fluorescence PCR was used to detect and identify rapidly the pre-enriched culture. In this study, specific primers and probes were designed and synthesized through using the Salmonella specific gene hut gene as the target gene. The nucleic acid DNA of various foodborne strains was extracted for real-time fluorescent PCR reaction. Only Salmonella showed positive amplification, while non Salmonella species, negative control and blank control had no amplification signal, confirming thee relatively high specificity of the designed and synthesized primer probes. Secondly, after pre-enrichment, enrichment, isolation, purification, biochemical tests and serological identification of the proficiency test samples and spiked samples, as well as real-time fluorescent PCR detection of pre-enriched culture, the 18-D319 and spiked samples exhibited significant S-type amplification with the Ct value being 24.34 and 26.21, respectively (which were positive for Salmonella), whilst 18-M906 had no significant fluorescence signal with the Ct value higher than 40 (which was negative for Salmonella). Based on the determination using API20E reagent strips, 18-D319 was a subspecies of Salmonella cholerae in Arizona, with the percentage of identification as 99.90% and T value as 0.97. 18-m906 was Escherichia coli, with the percentage of identification as 99.80% andT value as 0.94. The results of real-time fluorescence PCR were consistent with those obtained by the conventional culture method, and the PCR method was simpler and more rapid, requiring only 12 hours from pre-enrichment to result determination, and leading to results with high accuracy and ability to detect multiple samples in one batch. This method allows rapid screening of Salmonella in a large number of samples and detection and verification of proficiency testing samples.
