Specific multiplex-PCR method for rapid detection of Vibrio parahaemolyticus

Abstract:

[Objective] This study aims to establish a rapid specific multiplex-PCR detection of Vibrio parahaemolyticus by targeting gyrase, tdh, trh genes simultaneously. [Methods] Three pairs of reported primers were mixed in a single PCR tube. Through optimizing the concentration of primers and anneal temperature, the best reaction condition and primers ratio were determined. This method was validated by specificity test, sensitivity test and comparison test. The PCR-amplified products were analyzed by automatic capillary electrophoresis device. [Results] The predicted DNA amplified bands exhibited at sequences of 91, 269 and 485 bp, indicating high specificity. Assay on sensitivity of cell concentration in pure culture condition showed that the detection limit of amplified gyrase, tdh and trh genes were 6.6×101, 6.6×102 and 6.6×101 CFU/mL respectively. When background interference existed, the detection limit of cell concentration of amplified gyrase, tdh and trh genes were 6.6×103, 6.6×104 and 6.6×103 CFU/mL respectively. The sensitivity experiment for testing genomic DNA template concentration showed that the detection limit was 1.36 μg/L. This method was applied to the test for imported seafood, the results matched the FDA 2004 standard. The amplified bands can be distinguished from each other more easily, comparing with those of FDA 2004 standard. [Conclusion] The multiplex-PCR method was successfully established. It is accurate, rapid and convenient. It can be applied to detect Vibrio parahaemolyticus or pathogenic Vibrio parahaemolyticus with tdh gene or trh gene.