Abstract

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.

中文翻译：

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通过靶向常见的鞭毛钩基因flgE，快速检测鞭毛和非鞭毛沙门氏菌

摘要

沙门氏菌属。会引起动物和人类沙门氏菌病。在这项研究中，我们建立了一种简单的方法，可通过扩增编码鞭毛钩蛋白的flgE基因内的特定区域来检测所有沙门氏菌。我们对沙门氏菌鞭毛相关基因进行的初步序列分析表明，尽管沙门氏菌Gallinarum和沙门氏菌Pullorum缺乏鞭毛，但它们确实具有鞭毛相关基因，包括flgE。为了详细研究，我们使用不同的细菌菌株（包括带鞭毛的和非带鞭毛的沙门氏菌以及非带菌的沙门氏菌）进行了比较性flgE序列分析。沙门氏菌菌株。发现在flgE开放阅读框内有两个独特的区域（参考序列的481–529 bp和参考序列的721–775 bp）是高度保守的，并且对所有沙门氏菌都具有特异性。接下来，我们设计了针对上述两个区域的一对PCR引物（flgE -UP和flgE -LO），并使用从总共76个细菌菌株（31个带鞭毛的沙门氏菌菌株，26个细菌菌株）中制备的模板DNA进行了flgE定制的PCR。非鞭毛沙门氏菌菌株和其他19种非沙门氏菌菌株）。结果表明，从所有样品中均获得了预期大小的特定正条带。沙门氏菌（包括带鞭毛的和非带鞭毛的沙门氏菌）菌株，而非沙门氏菌细菌菌株未产生特定产物。通过DNA测序证实来自阳性条带的PCR产物。每个PCR反应的基因组DNA和细菌细胞的最低检测量分别达到18.3 pg /μL和100个菌落形成单位（CFU）。使用flgE -PCR法检测沙门氏菌人工污染牛奶样品中，低至1 CFU / mL的沙门氏菌的8小时的预培养后，可检测的。同时，采用flgE -PCR技术对247株沙门氏菌感染的临床样本进行评估。来自不同的养鸡场。检测结果表明，与传统的基于细菌培养的检测方法相一致，flgE -PCR可用于特异性检测沙门氏菌。值得注意的是，使用flgE量身定制的PCR进行鉴定的结果应在不到1天的时间内完成，扩展结果的速度要比标准方法（耗时5天以上）快得多。总体而言，基于flgE的PCR方法可以特异性检测鞭毛和非鞭毛沙门氏菌，并且可以作为快速，简单和灵敏地检测沙门氏菌种类的强大工具。

关键点

•针对所有沙门氏菌属物种的flgE基因。找到了。

•已建立的PCR检测方法可用于特异性检测所有沙门氏菌。

•PCR方法用于检测临床沙门氏菌。不到1天的样品。