Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxx-qPCR. (April 2020)

• Record Type: Journal Article
• Authors: Zhao, Lichao; Lv, Xinrui; Cao, Xiao; Zhang, Jingfeng; Gu, Xiaokui; Zeng, Haiyan; Wang, Li
• Is Part Of: Food control. Volume 110(2020)
• Publication Date: 2020-04
• Subjects: Immunomagnetic separation (IMS) -- Vibrio parahaemolyticus -- Viable but non-culturable (VBNC) -- Real-time (quantitative) polymerase chain reaction PCR (qPCR) -- Improved propidium monoazide (PMAxx); 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.2019.106962
• Languages: English
• ISSNs: 0956-7135

Abstract

Abstract: Immunomagnetic separation (IMS) is an effective method for specific enrichment and purification of target food-borne pathogens from complex food samples. To detect viable but non-culturable (VBNC) Vibrio parahaemolyticus ( V. parahaemolyticus ) with greater accuracy and sensitivity, we used an improved propidium monoazide (PMAxx) dye to eliminate dead cell interference in an IMS-PMAxx-real-time (quantitative) polymerase chain reaction (IMS-PMAxx-qPCR) assay. We prepared immunomagnetic beads (IMBs) using streptavidin-conjugated magnetic nanoparticles and biotinylated polyclonal antibodies, and optimized the reaction conditions to establish an IMS method for VBNC V. parahaemolyticus . We determined the optimal antibody amount (30 μg), IMBs volume (150 μL), incubation time (45 min), immunomagnetic separation time (4 min), and separation temperature (25 °C). The IMS-PMAxx-qPCR method could detect VBNC V. parahaemolyticus in raw shrimp samples at levels as low as 1.85 CFU/g without any pre-enrichment. The IMS-PMAxx-qPCR assay is highly sensitive, selective, simple, and rapid (<4 h), and outperformed the conventional PCR based assays. Thus, this method can potentially improve rapid detection of VBNC V. parahaemolyticus in raw shrimp. Highlights: Optimized IMS method to enrich for VBNC Vibrio parahaemolyticus in actual samples. LOD using IMS-PMAxx-qPCR in actual samples was determined to be 1.85 CFU/g. IMS-PMAxx-qPCR is suitable for the detection of trace VBNC microbial contamination.