Rapid and accurate detection of viable Vibrio parahaemolyticus by sodium deoxycholate-propidium monoazide-qPCR in shrimp

Abstract

Vibrio parahaemolyticus is an important human pathogen causing a variety of life-threatening diseases and is widely distributed in marine and estuarine environments. The objective of this study was to develop a sensitive, specific, and accurate method by using sodium deoxycholate (SD)-propidium monoazide (PMA)-qPCR (SD-PMA-qPCR) for selective detection of viable V. parahaemolyticus cells in shrimp. A qPCR assay was developed by targeting a unique fragment in the toxR gene in V. parahaemolyticus. The qPCR assay demonstrated superior specificity (100%) on V. parahaemolyticus strains (n = 70) and non-V. parahaemolyticus strains (n = 37) examined in the inclusivity and exclusivity tests; and the limit of detection (LOD) of the assay reached 5 × 101 CFU/ml. To remedy the drawback of PCR, SD-PMA treatment was incorporated with the qPCR assay. The optimized PMA treatment conditions were determined as follows, 40 μM PMA and 3-min light exposure at 40 w. The maximum removal efficiency of non-viable cell DNA was achieved by an optimal amplicon (262 bp) of qPCR for PMA treatment with SD at an optimal concentration (0.02% wt/vol). Furthermore, we have applied the SD-PMA-qPCR assay for detection of viable V. parahaemolyticus cells in shrimp. Consequently, the SD-PMA-qPCR assay could accurately detect as low as 5 × 101 CFU/g of V. parahaemolyticus in the presence of a large number of non-viable cells (5 × 107 CFU/g) in spiked shrimp with a 4-h enrichment. In summary, the qPCR assay based on the target gene, toxR, is sensitive and specific; treatment of non-viable cells with SD and PMA improved the removal efficiency of DNA of non-viable cells; and the SD-PMA-qPCR assay developed in this study is a specific and accurate detection method for viable V. parahaemolyticus, providing an effective and rapid means for detection of viable V. parahaemolyticus in food.