利用非标准核酸进行高灵敏度二十联体(20-Plex)微生物威胁检测用于环境监测

IF 3.9 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Hinako Kawabe, Luran Manfio, Sebastian Magana Pena, Nicolette A. Zhou, Kevin M. Bradley, Cen Chen, Chris McLendon, Steven A. Benner, Karen Levy, Zunyi Yang*, Jorge A. Marchand* and Erica R. Fuhrmeister*, 
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引用次数: 0

摘要

环境监测和临床诊断在很大程度上依赖于聚合酶链反应(PCR)的目标检测。越来越多的微生物威胁需要新的基于pcr的检测方法,这些方法具有高度的敏感性、特异性和可复用性。在这里,我们介绍了一种基于pcr的二十plex (20-plex)检测方法,用于检测18种肠病原体和2种抗微生物药物耐药性基因。该多重PCR检测利用自回避分子识别系统(SAMRS)来避免引物二聚体的形成,人工扩展遗传信息系统(AEGIS)来扩增特异性,以及下一代测序来扩增子鉴定。使用并行多目标TaqMan阵列卡(TAC)对废水、土壤和人类粪便样本的20-plex检测进行基准性能测试,我们发现90%的阳性呼叫一致性和89%的阴性呼叫一致性。此外,我们展示了来自20-plex的长读和短读测序信息如何用于进一步分类基因的等位基因变异和区分亚种。所提出的战略提供了敏感、负担得起和可靠的多重检测,可用于支持基于废水的流行病学、环境监测和人/动物诊断方面的工作。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance

Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

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来源期刊
CiteScore
8.00
自引率
10.60%
发文量
380
审稿时长
6-12 weeks
期刊介绍: The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.
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