AUTHOR=Bouju-Albert Agnès , Saltaji Sabrina , Dousset Xavier , Prévost Hervé , Jaffrès Emmanuel 

TITLE=Quantification of Viable Brochothrix thermosphacta in Cold-Smoked Salmon Using PMA/PMAxx-qPCR

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.654178

DOI=10.3389/fmicb.2021.654178

ISSN=1664-302X

ABSTRACT=<p>The aim of this study was to develop a rapid and accurate PMA-qPCR method to quantify viable <italic>Brochothrix thermosphacta</italic> in cold-smoked salmon. <italic>B. thermosphacta</italic> is one of the main food spoilage bacteria. Among seafood products, cold-smoked salmon is particularly impacted by <italic>B. thermosphacta</italic> spoilage. Specific and sensitive tools that detect and quantify this bacterium in food products are very useful. The culture method commonly used to quantify <italic>B. thermosphacta</italic> is time-consuming and can underestimate cells in a viable but not immediately culturable state. We designed a new PCR primer set from the single-copy <italic>rpoC</italic> gene. QPCR efficiency and specificity were compared with two other published primer sets targeting the <italic>rpoC</italic> and <italic>rpoB</italic> genes. The viability dyes PMA or PMAxx were combined with qPCR and compared with these primer sets on viable and dead <italic>B. thermosphacta</italic> cells in BHI broth and smoked salmon tissue homogenate (SSTH). The three primer sets displayed similar specificity and efficiency. The efficiency of new designed <italic>rpoC</italic> qPCR on viable <italic>B. thermosphacta</italic> cells in SSTH was 103.50%, with a linear determination coefficient (r<sup>2</sup>) of 0.998 and a limit of detection of 4.04 log CFU/g. Using the three primer sets on viable cells, no significant difference was observed between cells treated or untreated with PMA or PMAxx. When dead cells were used, both viability dyes suppressed DNA amplification. Nevertheless, our results did not highlight any difference between PMAxx and PMA in their efficiency to discriminate viable from unviable <italic>B. thermosphacta</italic> cells in cold-smoked salmon. Thus, this study presents a rapid, specific and efficient <italic>rpoC</italic>-PMA-qPCR method validated in cold-smoked salmon to quantify viable <italic>B. thermosphacta</italic> in foods.</p>