Nanoliter qPCR Platform for Highly Parallel, Quantitative Assessment of Reductive Dehalogenase Genes and Populations of Dehalogenating Microorganisms in Complex Environments

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• 作者：Koshlan Mayer-Blackwell ; Mohammad F. Azizian ; Christina Machak ; Elena Vitale ; Giovanna Carpani ; Francesca de Ferra ; Lewis Semprini ; Alfred M. Spormann
• 刊名：Environmental Science & Technology
• 出版年：2014
• 出版时间：August 19, 2014
• 年：2014
• 卷：48
• 期：16
• 页码：9659-9667
• 全文大小：442K
• ISSN：1520-5851

文摘

Idiosyncratic combinations of reductive dehalogenase (rdh) genes are a distinguishing genomic feature of closely related organohalogen-respiring bacteria. This feature can be used to deconvolute the population structure of organohalogen-respiring bacteria in complex environments and to identify relevant subpopulations, which is important for tracking interspecies dynamics needed for successful site remediation. Here we report the development of a nanoliter qPCR platform to identify organohalogen-respiring bacteria and populations by quantifying major orthologous reductive dehalogenase gene groups. The qPCR assays can be operated in parallel within a 5184-well nanoliter qPCR (nL-qPCR) chip at a single annealing temperature and buffer condition. We developed a robust bioinformatics approach to select from thousands of computationally proposed primer pairs those that are specific to individual rdh gene groups and compatible with a single amplification condition. We validated hundreds of the most selective qPCR assays and examined their performance in a trichloroethene-degrading bioreactor, revealing population structures as well as their unexpected shifts in abundance and community dynamics.