Establishment and application of a multiplex real-time PCR assay coupled with propidium monoazide for the simultaneous detection of viable Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus

Abstract

A propidium monoazide (PMA) - multiplex real-time PCR (mRT-PCR) assay was established for the simultaneous detection of viable Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus. According to the unique sequences of the target genes identified by our laboratory for the above pathogenic bacteria, an efficient and specific mRT-PCR method based on TaqMan probes was developed, and then PMA was introduced into the mRT-PCR system. The optimization of the PMA treatment conditions successfully achieves the purpose of distinguishing viable and dead bacteria in the cell suspension, and effectively eliminates false positive results caused by dead cells. The method's specificity was verified by 54 strains, exhibiting 100% specificity without cross-reactivity with other pathogens. Furthermore, the developed method could accurately detect the target bacteria in the presence of non-target bacteria at a concentration range of 100-107 CFU/mL, demonstrating excellent anti-interference ability. The detection limits of V. parahaemolyticus, V. vulnificus, and V. alginolyticus were 2.0 × 101 CFU/mL, 3.2 × 101 CFU/mL, and 3.75 × 101 CFU/mL, respectively. To reflect the practicability of the method, the established method was developed as a detection kit, and the evaluation results showed that the kit has good stability and good reproducibility. The applicability of this kit was satisfying after evaluating in artificially spiked aquatic products (shrimp, yellow croakers and oysters) with recoveries ranging from 90.22% to 109.35%. The PMA-mRT-PCR detection assay is a feasible method for the rapid, sensitive, and quantitative detection of bacterial pathogens. And the novel kit developed based on this method has significant application value for the large-scale screening and identification of harmful Vibrios in food matrices.