Development, evaluation and application of a polymerase spiral reaction (PSR) based assay on accurate detection of Vibrio parahaemolyticus viable cells and virulence factors from rice product
Vibrio parahaemolyticus is a predominate cause of microbiological food safety. This study aimed to develop a polymerase spiral reaction (PSR) based assay for rapid and visual detection on V. parahaemolyticus . Firstly, the specificity and conservativity of species-specific target tlh gene were determined. Secondly, targeting tlh and virulence factors tdh and trh , PSR assay was developed and optimized using constructed positive plasmid controls and standard strain. Visualized result determination using fluorescent dye was applied to avoid false positive issues. Thirdly, 44 strains were used for specificity evaluation. Fourthly, pure and mixed species artificial contamination systems were included to test its applicability. In addition, a propidium monoazide (PMA)-PSR assay was set up to determine viable but non-culturable (VBNC) cells. Target tlh gene acquired high specificity and PSR assay was optimized to carry out at 65ᵒC for 1 h. Detection limit of PSR assay in purified genomic DNA, plasmid and artificially contaminated food samples were 5.31–53.1 pg/μL, 100 copies, and 10 3 CFU/mL, respectively, demonstrating the sensitivity as 100-fold higher than regular PCR. With PMA-PSR assay, VBNC cells were accurately determined without the disruption of dead cells and food compositions. Thus, PSR is applicable in detection of V. parahaemolyticus viable cells and virulence factors.
危险品化学品经营许可证（带存储）