Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real‐time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis‐specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis‐specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis‐specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis‐spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die‐off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis‐specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent.

中文翻译：

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Pentaplex 实时 PCR 用于鼠疫耶尔森氏菌和假结核耶尔森氏菌的差异检测，并应用于检测鼠疫流行期间收集的跳蚤。


在从鼠疫病原体Y假结核耶尔森氏菌进化过程中获得两个独特的质粒（pMT1 和 pPCP1）和基因组重排后。鼠疫菌与Y密切相关。假结核病具有遗传性，但毒性很强。我们开发了一种五重实时 PCR 检测方法，不仅可以检测两种耶尔森氏菌，还可以区分耶尔森氏菌。鼠疫菌株的质粒谱。使用的五个目标是Y 。鼠疫菌特异性ypo2088 、 caf1和pst分别位于染色体、质粒 pMT1 和 pPCP1 上；是的。假结核病特异性染色体基因opgG ； 18S核糖体RNA基因作为跳蚤DNA的内部对照。所有目标均显示 100% 特异性和高灵敏度，检测限范围为 1 fg 至 100 fg， Y 。鼠疫菌特异性pst作为最敏感的目标。使用该测定， Y 。鼠疫菌株通过其已知的质粒谱进行 100% 区分。测试Y 。鼠疫菌和耶氏菌。假结核- 掺入跳蚤 DNA 表明跳蚤 DNA 对目标基因的扩增没有干扰。最后，我们应用该测定法测试了从草原土拨鼠洞穴中收集的 102 只跳蚤，这些地方在跳蚤收集前几个月就报告了草原土拨鼠死亡的情况。所有跳蚤DNA均通过18S rRNA扩增；没有Y 。检出假结核病；一只跳蚤对所有Y呈阳性。鼠疫菌特定目标，确认本地Y 。鼠疫传播。我们的结果表明该测定对于Y的检测和分化是敏感且特异的。鼠疫菌和耶氏菌。假结核病。该测定可用于现场调查，以快速识别鼠疫病原体。