Zhai Ligong, Li Junjie, Tao Tingting, Lu Zhaoxin, Lv Fengxia, Bie Xiaomei
a College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing, Jiangsu Province, China.
b College of Food Engineering, Anhui Science and Technology University Chuzhou, Anhui Province, China.
Can J Microbiol. 2019 Jul;65(7):477-485. doi: 10.1139/cjm-2018-0547. Epub 2019 Mar 13.
serovar Heidelberg causes foodborne infections and is a major threat to the food chain and public health. In this study, we aimed to develop a rapid molecular typing approach to identify serovar Heidelberg. Using comparative genomics, four serovar-specific gene fragments were identified, and a real-time polymerase chain reaction (PCR) combined with a propidium monoazide (PMA) pretreatment method was developed for simultaneous detection of viable sp. () and Heidelberg (). The assay showed 100% specificity for all strains tested. The assay was able to distinguish effectively viable or dead cells with the PMA. The detection limit was 2.4 CFU/mL following 6 h of incubation in enrichment Luria-Bertani medium, and the assay could detect 1.7 × 10 CFU/mL in the presence of pork background flora. In artificially contaminated pork, real-time PCR detected inoculum levels of 1.15 CFU/25 g of pork after a 6 h enrichment. Thus, our findings indicated that this comparative genomics approach could be used to screen for serovar-specific fragments and that real-time PCR with PMA was a simple and reliable method for detecting viability of species and Heidelberg.
海德堡血清型可导致食源性感染，对食物链和公众健康构成重大威胁。在本研究中，我们旨在开发一种快速分子分型方法来鉴定海德堡血清型。通过比较基因组学，鉴定出四个血清型特异性基因片段，并开发了一种实时聚合酶链反应（PCR）结合单叠氮化丙锭（PMA）预处理方法，用于同时检测活的sp.（）和海德堡（）。该检测方法对所有测试菌株显示出100%的特异性。该检测方法能够通过PMA有效区分活细胞和死细胞。在富集的Luria-Bertani培养基中孵育6小时后，检测限为2.4 CFU/mL，在存在猪肉背景菌群的情况下，该检测方法能够检测到1.7×10 CFU/mL。在人工污染的猪肉中，实时PCR在富集6小时后检测到接种量为1.15 CFU/25 g猪肉。因此，我们的研究结果表明，这种比较基因组学方法可用于筛选血清型特异性片段，并且实时PCR结合PMA是一种检测sp.物种和海德堡血清型活力的简单可靠方法。
