Discrimination of viable and dead Vibrio parahaemolyticus subjected to low temperatures using Propidium Monoazide – Quantitative loop mediated isothermal amplification (PMA-qLAMP) and PMA-qPCR

Abstract

The aim of this study was to determine the effect of cold (4 °C) and subzero (−18 °C, −45 °C) temperatures on the occurrence time of membrane damage to provide Propidium Monoazide (PMA) penetration of Vibrio parahaemolyticus inoculated to the sea bass. Direct plate counting (DPC) and PMA-based quantitative loop-mediated isothermal amplification (qLAMP) and qPCR was utilized for discrimination of dead and live bacteria on the designated storage days (1, 3, 7, and 14).The optimum amount of PMA was 50 μM for inhibition of amplification derived from dead cells in spiked samples. The number of live V. parahaemolyticus was detectable at the end of the 14. day using PMA-qLAMP and PMA-qPCR at all the temperatures. On the 7th day, culturability has lost at any of the storage temperatures and DPCs at −18 °C and −45 °C revealed a difference of about 1 log10 CFU/ml between 1st and 3rd days. The same difference was also observed in PMA–qLAMP and PMA–qPCR on the same days (0.59–0.95 log10 CFU/ml). Subzero temperatures have the highest rate of viability while causing the fastest decrease in culturability in sample groups as a result of the higher level of transition to VBNC state. qLAMP and qPCR methods in the PMA-treated and nontreated groups on the storage days at all temperatures gave similar results (p > 0.05).