Abstract
Listeria spp. is an important foodborne disease agent, often found in the fresh mushroom (Flammulina velutipes) and its production environment. The aim of this study was to develop multiplex PCR for rapid identification of Listeria monocytogenes and Listeria ivanovii, and nonpathogenic Listeria in F. velutipes plants. Pan-genome analysis was first used to identify five novel Listeria-specific targets: one for the Listeria genus, one for L. monocytogenes, and three for L. ivanovii. Primers for the novel targets were highly specific in individual reactions. The detection limits were 103–104 CFU/mL, meeting the requirements of molecular detection. A mPCR assay for the identification of pathogenic Listeria, with primers targeting the novel genes specific for Listeria genus (LMOSLCC2755_0944), L. monocytogenes (LMOSLCC2755_0090), and L. ivanovii (queT_1) was then designed. The assay specificity was robustly verified by analyzing nonpathogenic Listeria and non-Listeria spp. strains. The determined detection limits were 2.0 × 103 CFU/mL for L. monocytogenes and 3.4 × 103 CFU/mL for L. ivanovii, for pure culture analysis. Further, the assay detected 7.6 × 104 to 7.6 × 100 CFU/10 g of pathogenic Listeria spiked into F. velutipes 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 F. velutipes plants. The prevalence of Listeria spp. and L. monocytogenes was 58.1% and 41.1%, respectively. The calculated κ factors for Listeria spp., L. monocytogenes, and L. ivanovii 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 Listeria in food and its production environment.
Highlights
Listeria is a genus of gram-positive facultative anaerobic bacilli that is widely distributed in food and natural environments
The specific targets reported in the previous studies [including prs specific for Listeria genus (Doumith et al, 2004), actA (Gouin et al, 1995; Zhou and Jiao, 2005), hly (Paziak-Domanska et al, 1999), and inlA (Jung et al, 2003) specific for L. monocytogenes, and iactA specific for L. ivanovii (Gouin et al, 1995)] were analyzed in the 0/1 matrix to evaluate their absence/existence profile
The detection limits of the corresponding primer pairs (103–104 CFU/mL) of these novel target genes were comparable with those of existing molecular detection targets (Chiang et al, 2012; Tao et al, 2017). These Listeria-specific targets obtained by this approach have better specificity, and their sensitivity can meet the needs of existing molecular detection methods, so these novel targets can represent unique detection targets for monitoring of pathogenic Listeria in food and its production environment
Summary
Listeria is a genus of gram-positive facultative anaerobic bacilli that is widely distributed in food and natural environments. L. monocytogenes and L. ivanovii are human and animal pathogens (Snapir et al, 2006; Jagadeesan et al, 2019; Lee et al, 2020). These bacteria can cause severe listeriosis, leading to spontaneous abortion, neonatal sepsis, and meningoencephalitis, and the post-infection mortality rate ranges from 20 to 30% (Radoshevich and Cossart, 2018; Ranjbar and Halaji, 2018), which places this pathogen among the most frequent causes of death from foodborne illnesses (Tamburro et al, 2015b). It is important to develop rapid and accurate diagnostic techniques or tools for the detection and distinguishing of pathogenic Listeria in F. velutipes, to facilitate introduction of appropriate intervention measures in the F. velutipes production chain to reduce the risk of Listeria contamination
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