title: Nanoliter qPCR platform for highly parallel, quantitative assessment of reductive dehalogenase genes and populations of dehalogenating microorganisms in complex environments.
abstract: 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.
authors.affiliation: Civil and Environmental Engineering, §Geological and Environmental Sciences, and ⊥Chemical Engineering, Stanford University , Stanford, California 94305, United States.
authors.forename: Koshlan
authors.initials: K
authors.lastname: Mayer-Blackwell
authors.forename: Mohammad F
authors.initials: MF
authors.lastname: Azizian
authors.forename: Christina
authors.initials: C
authors.lastname: Machak
authors.forename: Elena
authors.initials: E
authors.lastname: Vitale
authors.forename: Giovanna
authors.initials: G
authors.lastname: Carpani
authors.forename: Francesca
authors.initials: F
authors.lastname: de Ferra
authors.forename: Lewis
authors.initials: L
authors.lastname: Semprini
authors.forename: Alfred M
authors.initials: AM
authors.lastname: Spormann
mesh_terms: D001419:Bacteria / Q000235:genetics*; D001673:Biodegradation, Environmental; D019149:Bioreactors; D054879:Halogenation / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D010088:Oxidoreductases; C112990:tetrachloroethene dehalogenase