Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology
Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology
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DOI：
10.1016/j.mimet.2012.05.012
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发表时间：
2012-09-01
影响因子：
2.2
通讯作者：
Goji, Noriko
中科院分区：
文献类型：
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作者：
Amoako, Kingsley K.;Thomas, Matthew C.;Goji, Noriko
当生物恐怖主义袭击企图或实施时，会对公共健康造成相当大的风险，并造成大规模的社会经济后果。我们必须拥有高灵敏度和特异性的快速识别生物威胁因子的既定检测方法，以确保能够适当部署应急响应措施。需要在相关时间范围内获得高度可信的信息才能做出快速、明智的决策。与假定的阳性 PCR 扩增子相比，从可疑物质获取 DNA 序列数据可提供额外的置信度。基于测序的技术，例如焦磷酸测序，具有足够的区分潜力，可用于微生物鉴定，也可用于鉴定抗菌素耐药性 (AMR) 基因。我们在这项研究中展示了焦磷酸测序在基于毒力基因明确检测和鉴定九种鼠疫耶尔森氏菌菌株方面的力量。此外，我们开发了测定法来表征其 AMR 基因谱。初始 PCR 扩增后约 60 分钟即可生成 40 至 84 bp 范围的序列结果，与需要数天的传统平板方法相比，提供了一种快速确定 AMR 谱的方法。观察到的高序列同一性 (95-100%) 和特异性表明焦磷酸测序技术的准确性很高。此外，本研究中观察到的高达 84 bp 的读长对于使用 Pyromark Q24 进行焦磷酸测序来说是前所未有的。我们提出这种方法作为一种新颖、快速、基于序列的鼠疫杆菌检测和识别工具，在生物防御方面具有潜在的应用前景。 Crown 版权所有 (C) 2012 由 Elsevier B.V. 出版。保留所有权利。
When a bioterrorism attack is attempted or perpetrated there is considerable risk for public health and large scale socioeconomic consequences. It is imperative that we possess established assays for the rapid identification of biothreat agents with high sensitivity and specificity to ensure emergency response measures can be deployed appropriately. Highly trustworthy information within a relevant timeframe is required to make a rapid and informed decision. Obtaining DNA sequence data from a suspected agent provides an added layer of confidence compared to a presumptive positive PCR amplicon. Sequencing based technologies, such as pyrosequencing, have sufficient discrimination potential to be used for microbial identification and can also be used to identify antimicrobial resistance (AMR) genes. We have shown in this study the power of pyrosequencing in the unambiguous detection and identification of nine Yersinia pestis strains based on virulence genes. Furthermore, we developed assays to characterize their AMR gene profiles. Sequence results ranging from 40 to 84 bp were generated in about 60 min following initial PCR amplification and provide a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The high sequence identities (95-100%) and specificity observed indicate the high level of accuracy of pyrosequencing technology. In addition, the read lengths of up to 84 bp observed in this study are unprecedented for pyrosequencing using the Pyromark Q24. We propose this method as a novel, rapid, sequence based detection and identification tool for Y. pestis with a potential application in biodefence. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.