Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP

Abstract

One of the most challenging aspects in culture independent methods for foodborne pathogens’ detection is discrimination of dead and live microorganisms. This study aimed to determine the Vibrio parahaemolyticus in seafoods via direct plate counting (DPC) and toxR-based quantitative loop-mediated isothermal amplification (qLAMP) and to discriminate dead and live cells using propidium monoazide (PMA)-qLAMP. A total of 200 samples including finfishes (n= 100) and shrimps (n= 100), representing the Mediterranean, Black and Aegean sea were collected from supermarkets and fish markets of Konya-Turkey. qLAMP was performed in a Real-Time Turbidimeter and the time threshold (tt) values were yielded in 60 minutes. On DPC, the colonies grown on TCBS Agar was further confirmed by conventional PCR based from gyrB1 gene of Vibrio spp. and toxR gene of V. parahaemolyticus. Virulence property of the isolates were determined by tdh based qLAMP. The detection limit of the qLAMP was 1.2×104 CFU/g in artificially contaminated seafoods. DPC, qLAMP and PMA-qLAMP detected V. parahaemolyticus in 8 (4%), 12 (6%) and 12 (6%) samples, respectively. The CFUs of V. parahaemolyticus detected in qLAMP (5.96±0.10 log10 CFU/ml) and PMA-qLAMP (4.71±0.13 log10 CFU/ml) were higher than those of DPC (1.99±0.44 log10 CFU/ml) (P<0.05). The mean tt reduction in PMA treated samples was 1.25±0.38 log10 CFU/sample. The tdh gene was not detected in any of the isolates. In conclusion, the toxR-based PMA-qLAMP method could be an alternative to be used more widely and effective assay for the quantification of live V. parahaemolyticus in seafoods.

Keywords

• DPC,propidium monoazide,qLAMP,VBNC,V. parahaemolyticus

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