Using unique ORFan genes as strain-specific identifiers for Escherichia coli.
Using unique ORFan genes as strain-specific identifiers for Escherichia coli.
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DOI：
10.1186/s12866-022-02508-y
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发表时间：
2022-05-18
期刊：
影响因子：
4.2
通讯作者：
中科院分区：
文献类型：
--
作者：
菌株水平的细菌鉴定是一项迫切需要但艰巨且具有挑战性的任务。本研究的目的是建立一种方法来鉴定和区分同一种细菌的多个菌株之间的单个菌株。用于检测该方法的组包括从德国、西班牙、英国和越南从人、牛、猪、野猪和鸡中分离的菌株集合中挑选的17种大肠杆菌菌株。我们针对独特或罕见的ORFan基因来解决选择性和特异性菌株鉴定的问题。这些ORFan基因是每个菌株独有的，可作为开发菌株特异性引物的模板。大多数实验菌株（17个中的14个）具有用于开发菌株特异性引物的独特ORFan基因。通过将用于稀有基因的PCR与用于分离实验菌株的选择步骤相结合来鉴定剩余的三种菌株。多重PCR允许在体外加标粪便材料中以及在猪实验性感染和从粪便材料中回收细菌后的体内成功鉴定菌株。此外，还开发了qPCR的引物，实验感染后粪便样品的定量读数也是可能的。该手稿中描述的方法使用菌株特异性的独特基因来鉴定菌株混合物中的单个菌株，证明了其在体外和体内检测和跟踪单个菌株方面的有效性和可靠性，代表了更昂贵方法的快速且廉价的替代方法。在线版本包含补充材料，可通过10.1186/s12866-022-02508-y获得。
Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods. The online version contains supplementary material available at 10.1186/s12866-022-02508-y.
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影响因子：
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