College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, 225009, China.
Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China.
Appl Microbiol Biotechnol. 2020 Nov;104(22):9719-9732. doi: 10.1007/s00253-020-10925-0. Epub 2020 Oct 3.
Salmonella spp. can cause animal and human salmonellosis. In this study, we established a simple method to detect all Salmonella species by amplifying a specific region within the flgE gene encoding the flagellar hook protein. Our preliminary sequence analysis among flagella-associated genes of Salmonella revealed that although Salmonella Gallinarum and Salmonella Pullorum are lacking flagella, they did have flagella-associated genes, including flgE. To investigate in detail, a comparative flgE sequence analysis was conducted using different bacterial strains including flagellated and non-flagellated Salmonella as well as non-Salmonella strains. Two unique regions (481-529 bp and 721-775 bp of the reference sequence) within the flgE open reading frame were found to be highly conserved and specific to all Salmonella species. Next, we designed a pair of PCR primers (flgE-UP and flgE-LO) targeting the above two regions, and performed a flgE-tailored PCR using as template DNA prepared from a total of 76 bacterial strains (31 flagellated Salmonella strains, 26 non-flagellated Salmonella strains, and 19 other non-Salmonella bacteria strains). Results showed that specific positive bands with expected size were obtained from all Salmonella (including flagellated and non-flagellated Salmonella) strains, while no specific product was generated from non-Salmonella bacterial strains. PCR products from the positive bands were confirmed by DNA sequencing. The minimum detection amount for genomic DNA and bacteria cells reached 18.3 pg/μL and 100 colony-forming unit (CFU) per PCR reaction, respectively. Using the flgE-PCR method to detect Salmonella in artificially contaminated milk samples, as low as 1 CFU/mL Salmonella was detectable after an 8-h pre-culture. Meanwhile, the flgE-tailored PCR method was applied to evaluate 247 clinical samples infected with Salmonella from different chicken breeding farms. The detection results indicated that flgE-PCR could be used to specifically detect Salmonella in concordance with the traditional bacterial culture-based detection method. It is worthwhile noticed that identification results using flgE-tailored PCR should be completed within less than 1 day, expanding the result of much faster than the standard method, which took more than 5 days. Overall, the flgE-tailored PCR method can specifically detect flagellated and non-flagellated Salmonella and can serve as a powerful tool for rapid, simple, and sensitive detection of Salmonella species. KEY POINTS : • Targeting flgE gene for all Salmonella spp. found. • The established PCR assay is used to specifically detect all Salmonella spp. • The PCR method is applied to detect clinical Salmonella spp. samples within less than 1 day.
肠沙门氏菌可引起动物和人类沙门氏菌病。在这项研究中，我们建立了一种通过扩增编码鞭毛钩蛋白的 flgE 基因内特定区域来检测所有沙门氏菌物种的简单方法。我们对沙门氏菌鞭毛相关基因的初步序列分析表明，尽管鸡沙门氏菌和鸡白痢沙门氏菌缺乏鞭毛，但它们确实有鞭毛相关基因，包括 flgE。为了详细研究，使用不同的细菌菌株（包括有鞭毛和无鞭毛的沙门氏菌以及非沙门氏菌菌株）进行了 flgE 序列比较分析。在 flgE 开放阅读框内发现两个独特的区域（参考序列的 481-529 bp 和 721-775 bp）高度保守且特异性针对所有沙门氏菌物种。接下来，我们设计了一对针对上述两个区域的 PCR 引物（flgE-UP 和 flgE-LO），并使用总共 76 株细菌菌株（31 株有鞭毛的沙门氏菌菌株、26 株无鞭毛的沙门氏菌菌株和 19 株其他非沙门氏菌菌株）制备的模板 DNA 进行 flgE 定制 PCR。结果表明，从所有沙门氏菌（包括有鞭毛和无鞭毛的沙门氏菌）菌株中均获得了预期大小的特异性阳性条带，而非沙门氏菌菌株则未产生特异性产物。通过 DNA 测序确认阳性条带的 PCR 产物。基因组 DNA 和细菌细胞的最低检测量分别达到 18.3 pg/μL 和每 PCR 反应 100 个菌落形成单位 (CFU)。使用 flgE-PCR 方法检测人工污染牛奶样品中的沙门氏菌，经过 8 小时的预培养后，可检测到低至 1 CFU/mL 的沙门氏菌。同时，将 flgE 定制 PCR 方法应用于评估来自不同鸡养殖场的 247 份临床感染沙门氏菌的样本。检测结果表明，flgE-PCR 可与传统的基于细菌培养的检测方法相结合，特异性检测沙门氏菌。值得注意的是，flgE 定制 PCR 的鉴定结果应在 1 天内完成，比标准方法（超过 5 天）的结果快得多。总的来说，flgE 定制 PCR 方法可以特异性检测有鞭毛和无鞭毛的沙门氏菌，可作为快速、简单、灵敏检测沙门氏菌的有力工具。要点：• 针对 flgE 基因检测所有沙门氏菌属。• 建立的 PCR 检测法用于特异性检测所有沙门氏菌属。• 该方法用于在 1 天内检测临床沙门氏菌属样本。
