Detection of Vibrio parahaemolyticus in Edible Crustaceans by Real-time Fluorescence Saltatory Rolling Circle Amplification Combined with Propidium Monoazide

Objective: A novel and rapid method for the detection of Vibrio parahaemolyticus in shrimp products was established by combined use of real-time fluorescent saltatory rolling circle amplification (SRCA) and propidium monoazide(PMA). Methods: Primers were designed and screened based on the V. parahaemolyticus-specific gene toxR. The specificity,sensitivity and detection limit of the PMA-RF-SRCA method were analyzed and compared with those of PMA combined with real-time polymerase chain reaction (PMA-real-time PCR). To evaluate its practicability, the relative sensitivity, relative specificity and relative accuracy of PMA-RF-SRCA were calculated using the national standard method GB 4789. 7-2013 as a reference standard when it was used to detect 76 samples. Results: The specificity of the designed primers was excellent, giving positive results for 12 V. parahaemolyticus strains and negative results for 18 non-V. parahaemolytic strains. PMA-RF-SRCA was 100-fold more sensitive than PMA-RF-SRCA with a sensitivity of 3.2×100 CFU/mL and a detection limit of 2.08 × 101 CFU/g. Compared with the GB 4789. 7-2013 method, the relative sensitivity, relative specificity and relative accuracy of PMA-RF-SRCA were 100.00%, 96.00% and 98.68%, respectively. Conclusion: The PMA-RF-SRCAmethod has the advantages of strong specificity and high sensitivity, and is suitable for the detection and screening of live V. parahaemolyticus in edible crustaceans. © 2021, China Food Publishing Company. All right reserved.