Rapid Detection of Salmonella in Shrimp by Immunomagnetic Separation Combined with Real-time PCR
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Abstract:
A rapid and sensitive detection method of immunomagnetic separation (IMS) combined with real-time PCR for Salmonella in shrimp was established. The immunomagnetic beads were prepared by coupling anti-Salmonella polyclonal antibody to magnetic beads and the reaction conditions were optimized to establish the IMS method, and a probe and primers were synthesized for ttr gene of Salmonella to construct the real-time PCR system. The specificity and sensitivity of four representative Salmonella strains were examined. The results showed that the optimal dosage of immunomagnetic beads was 100 μL, and the optimal reaction time of immunomagnetic beads with sample bacteria was 30 min. The proposed IMS-real time PCR had a high specificity and the limits of detection were 5×101 CFU/25 g for Salmonella typhimurium, 5×102 CFU/25 g for Salmonella choleraesuis and 1×102 CFU/25 g for both Salmonella enteritidis and Salmonella paratyphi A, respectively, and the detection process could be completed within 6 h. The test results of IMS-real time PCR were same with those of the national standard for 40 collected shrimp samples. The established IMS-real time PCR need less time and had a high sensitivity, which provided technical support for the rapid detection of Salmonella in aquatic products as well as the practical significance for the prevention and control of foodborne diseases caused by Salmonella.
