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
Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China.
School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Front Microbiol. 2021 Jan 15;11:634255. doi: 10.3389/fmicb.2020.634255. eCollection 2020.
spp. is an important foodborne disease agent, often found in the fresh mushroom () and its production environment. The aim of this study was to develop multiplex PCR for rapid identification of and , and nonpathogenic in plants. Pan-genome analysis was first used to identify five novel -specific targets: one for the genus, one for , and three for . Primers for the novel targets were highly specific in individual reactions. The detection limits were 10-10 CFU/mL, meeting the requirements of molecular detection. A mPCR assay for the identification of pathogenic , with primers targeting the novel genes specific for genus (), (), and () was then designed. The assay specificity was robustly verified by analyzing nonpathogenic and non- spp. strains. The determined detection limits were 2.0 × 10 CFU/mL for s and 3.4 × 10 CFU/mL for , for pure culture analysis. Further, the assay detected 7.6 × 10 to 7.6 × 10 CFU/10 g of pathogenic spiked into 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 plants. The prevalence of spp. and was 58.1% and 41.1%, respectively. The calculated κ factors for spp., , and 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 in food and its production environment.
某菌属是一种重要的食源性疾病病原体，常见于新鲜蘑菇及其生产环境中。本研究旨在开发多重聚合酶链反应（mPCR）以快速鉴定某菌属、某菌及某植物中的非致病性某菌。首先通过泛基因组分析确定了五个新的某菌属特异性靶点：一个针对某菌属，一个针对某菌，三个针对某菌。针对新靶点设计的引物在单个反应中具有高度特异性。检测限为10⁻¹⁰CFU/mL，满足分子检测要求。随后设计了一种用于鉴定致病性某菌的mPCR检测方法，其引物靶向某菌属（某基因）、某菌（某基因）和某菌（某基因）的新基因。通过分析非致病性某菌和非某菌属菌株，有力地验证了该检测方法的特异性。对于纯培养分析，确定的检测限为某菌2.0×10 CFU/mL，某菌3.4×10 CFU/mL。此外，该检测方法在4至12小时富集后，能够检测出添加到某样品中的7.6×10至7.6×10 CFU/10 g致病性某菌。通过与传统的基于培养的方法比较，分析从不同某植物采集的129个样品，评估了该检测方法的可行性。某菌属和某菌的流行率分别为58.1%和41.1%。某菌属、某菌和某菌计算得到的κ系数分别为0.97、0.97和1。新的mPCR检测结果与基于培养的方法高度一致。因此，这种新检测方法将能够快速、特异性和准确地检测和监测食品及其生产环境中的致病性某菌。
