Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxx-qPCR

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 mu g), IMBs volume (150 mu L), incubation time (45 min), immunomagnetic separation time (4 min), and separation temperature (25 degrees 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.