Detection of viable but nonculturable Vibrio parahaemolyticus in shrimp samples using improved real-time PCR and real-time LAMP methods. (September 2019)

• Record Type: Journal Article
• Authors: Cao, Xiao; Zhao, Lichao; Zhang, Jingfeng; Chen, Xun; Shi, Lei; Fang, Xiang; Xie, Hui; Chang, Yanlei; Wang, Li
• Is Part Of: Food control. Volume 103(2019)
• Page Start: 145
• Page End: 152
• Publication Date: 2019-09
• Subjects: Improved propidium monoazide (PMAxx) -- Vibrio parahemolyticus -- Viable but nonculturable (VBNC) -- Real-time PCR (qPCR) -- Real-time loop-mediated isothermal amplification (qLAMP) -- Quantitative detection; Food -- Quality -- Periodicals; Food -- Analysis -- Periodicals; Food handling -- Periodicals; Food industry and trade -- Quality control -- Periodicals; Aliments -- Industrie et commerce -- Qualité -- Contrôle -- Périodiques; Aliments -- Qualité -- Périodiques; Aliments -- Analyse -- Périodiques; Hygiène alimentaire -- Périodiques; Food -- Analysis; Food handling; Food -- Quality; Periodicals; Electronic journals; 664.07
• DOI: 10.1016/j.foodcont.2019.04.003
• Languages: English
• ISSNs: 0956-7135

Abstract

Abstract: Accurate and highly sensitive detection of viable but nonculturable (VBNC) Vibrio parahaemolyticus is crucial for preventing seafood-related outbreaks. The VBNC state may occur due to bacterial susceptibility to cold shock during food storage. Propidium monoazide (PMA) has been widely applied to detect VBNC foodborne pathogens. In this study, we developed and compared real-time PCR (qPCR) and real-time LAMP (qLAMP) methods combined with an improved propidium monoazide (PMAxx) to detect pathogenic VBNC V. parahaemolyticus . The designed primers and probes were determined to be highly selective for V. parahaemolyticus strains. The combined PMAxx-qPCR method, requiring 100 min, demonstrated a quantification limit of 10.5 colony-forming units (CFU)/mL in pure culture and 28 CFU/g in raw shrimp respectively, which were 10-fold lower than the PMAxx-qLAMP method (45min). When testing mixtures containing different ratios of VBNC to dead V. parahaemolyticus, PMAxx-based methods were notably superior at distinguishing between VBNC and dead bacteria when VBNC cell concentrations were low. Therefore, PMAxx is an effective means for improving the detection and quantification of VBNC V. parahaemolyticus by qPCR and qLAMP. Pretreatment with PMAxx was shown to be suitable for detection of VBNC V. parahaemolyticus in complex food samples, including raw shrimp. Highlights: PMAxx-qPCR and PMAxx-qLAMP were developed and compared to detect VBNC Vibrio parahaemolyticus. Both methods had good specificity, but PMAxx-qPCR had higher sensitivity. Respective LoD in pure culture and raw shrimp were 10.5 CFU/mL and 28 CFU/g without a prior enrichment step. PMAxx exhibited higher accuracy than PMA in quantitative detection.