AUTHOR=Li Fan , Ye Qinghua , Chen Moutong , Zhang Jumei , Xue Liang , Wang Juan , Wu Shi , Zeng Haiyan , Gu Qihui , Zhang Youxiong , Wei Xianhu , Ding Yu , Wu Qingping 

TITLE=Multiplex PCR for the Identification of Pathogenic Listeria in Flammulina velutipes Plant Based on Novel Specific Targets Revealed by Pan-Genome Analysis

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.634255

DOI=10.3389/fmicb.2020.634255

ISSN=1664-302X

ABSTRACT=<p><italic>Listeria</italic> spp. is an important foodborne disease agent, often found in the fresh mushroom (<italic>Flammulina velutipes</italic>) and its production environment. The aim of this study was to develop multiplex PCR for rapid identification of <italic>Listeria monocytogenes</italic> and <italic>Listeria ivanovii</italic>, and nonpathogenic <italic>Listeria</italic> in <italic>F. velutipes</italic> plants. Pan-genome analysis was first used to identify five novel <italic>Listeria</italic>-specific targets: one for the <italic>Listeria</italic> genus, one for <italic>L. monocytogenes</italic>, and three for <italic>L. ivanovii</italic>. Primers for the novel targets were highly specific in individual reactions. The detection limits were 10<sup>3</sup>–10<sup>4</sup> CFU/mL, meeting the requirements of molecular detection. A mPCR assay for the identification of pathogenic <italic>Listeria</italic>, with primers targeting the novel genes specific for <italic>Listeria</italic> genus (<italic>LMOSLCC2755_0944</italic>), <italic>L. monocytogenes</italic> (<italic>LMOSLCC2755_0090</italic>), and <italic>L. ivanovii</italic> (<italic>queT_1</italic>) was then designed. The assay specificity was robustly verified by analyzing nonpathogenic <italic>Listeria</italic> and non-<italic>Listeria</italic> spp. strains. The determined detection limits were 2.0 × 10<sup>3</sup> CFU/mL for <italic>L. monocytogene</italic>s and 3.4 × 10<sup>3</sup> CFU/mL for <italic>L. ivanovii</italic>, for pure culture analysis. Further, the assay detected 7.6 × 10<sup>4</sup> to 7.6 × 10<sup>0</sup> CFU/10 g of pathogenic <italic>Listeria</italic> spiked into <italic>F. velutipes</italic> samples following 4–12 h enrichment. The assay feasibility was evaluated by comparing with a traditional culture-based method, by analyzing 129 samples collected from different <italic>F. velutipes</italic> plants. The prevalence of <italic>Listeria</italic> spp. and <italic>L. monocytogenes</italic> was 58.1% and 41.1%, respectively. The calculated κ factors for <italic>Listeria</italic> spp., <italic>L. monocytogenes</italic>, and <italic>L. ivanovii</italic> were 0.97, 0.97, and 1, respectively. The results of the novel mPCR assay were highly consistent with those of the culture-based method. The new assay thus will allow rapid, specific, and accurate detection and monitoring of pathogenic <italic>Listeria</italic> in food and its production environment.</p>