Williamson Charles H D, Vazquez Adam J, Hill Karen, Smith Theresa J, Nottingham Roxanne, Stone Nathan E, Sobek Colin J, Cocking Jill H, Fernández Rafael A, Caballero Patricia A, Leiser Owen P, Keim Paul, Sahl Jason W
Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, Arizona, USA.
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
Appl Environ Microbiol. 2017 Aug 31;83(18). doi: 10.1128/AEM.00806-17. Print 2017 Sep 15.
Diverse members of the genus produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of group I, , and two major subgroups within group II. Coding region sequences unique to each of the four species/subgroups were identified by analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present ( positive [] or ). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene () clusters. Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: group I, , and two major subgroups within group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the gene, which is a universally present component of gene clusters, and to provide information about the type ( or ) of gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.
该属的多种成员可产生肉毒杆菌神经毒素（BoNTs），这些毒素会导致一种称为肉毒中毒的弛缓性麻痹。虽然多种梭菌可产生BoNTs，但大多数人类肉毒中毒病例都归因于I群和II群。最近的比较基因组研究表明，这些产BoNT的物种具有基因组多样性。本报告介绍了一种多重PCR检测方法，用于区分I群、[具体物种名称未给出]以及II群中的两个主要亚群。通过对数千个基因组组装进行分析，确定了这四个物种/亚群各自独特的编码区序列，并设计了PCR引物来扩增每个标记。由此产生的多重PCR检测方法将41株受试菌株正确归类到相应的物种或亚群。还开发并验证了一种单独的PCR检测方法，用于确定[具体基因名称未给出]基因（一种与肉毒杆菌神经毒素基因簇相关的基因）的存在。该[具体基因名称未给出]基因PCR检测方法可提供有关肉毒杆菌神经毒素基因簇是否存在以及存在的基因簇类型（[具体基因名称未给出]阳性[]或[具体基因名称未给出]）的信息。全基因组序列数据和比较基因组工具的可用性增加，使得能够设计这些检测方法，它们为鉴定产BoNT的梭菌提供了有价值的信息。这些PCR检测方法是快速、廉价的检测手段，可应用于多种样本类型，以将分离株归类到物种/亚群，并检测带有肉毒杆菌神经毒素基因（[具体基因名称未给出]）簇的梭菌。多种梭菌可产生肉毒杆菌神经毒素，这是已知最有效的神经毒素之一。在本研究中，开发了一种多重PCR检测方法，以区分在人类肉毒中毒病例中最常分离出的梭菌：I群、[具体物种名称未给出]以及II群中的两个主要亚群。由于在每个物种中都能找到产BoNT和不产毒素的分离株，还开发了一种PCR检测方法，用于确定[具体基因名称未给出]基因的存在（该基因是[具体基因名称未给出]基因簇普遍存在的组成部分），并提供有关样本中存在的[具体基因名称未给出]基因簇类型（[具体基因名称未给出]或[具体基因名称未给出]）的信息。这些PCR检测方法为从临床或环境样本中筛选未鉴定的分离株提供了简单、快速且廉价的工具。这些检测方法提供的信息可用于流行病学研究，有助于识别培养物保藏中的分离株混合物和未知分离株，并确认感兴趣细菌的存在。
