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.

High-throughput multi-parallel enteropathogen quantification via nano-liter qPCR

“…We selected bacterial, protozoan, and helminthic enteropathogens identified as contributing to diarrheal disease in children across twelve countries (33, 34). We computationally designed and screened 175,000 candidate primer pairs to target 16 virulence genes using methods described previously (32). Briefly, amino acid sequences corresponding to all non-redundant members of each target gene’s protein family (Pfam v 27.0) were clustered based on percent pairwise identity using BLASTp all-vs-all search (35).…”

“…In more recent studies, a commercial nL-qPCR technology (SmartChip™ Real-Time PCR, TakaraBio Inc.) was used to design multi-target diagnostics to detect the presence of antibiotic resistance genes in urban wastewater treatment plant effluent, reclaimed water, and environmental samples (29–31) and to evaluate a suite of related dehalogenase genes in complex microbial communities (32). This technology uses 100 nL reaction volumes and allows for flexible configuration of a 5184-well chip that can analyze up to 384 samples (depending on the number of assays included).…”

Isothermal assay targeting class 1 integrase gene for environmental surveillance of antibiotic resistance markers

“…ARG primers used on the SmartChip included: 35 primers targeting mobile genetic elements (MGEs, 4 different primers for the intI1 gene (Barraud et al, 2010; Gillings et al, 2015; Power et al, 2013; F. Wang et al, 2016), 2 primers targeting the universal bacteria 16S rRNA gene (Lane, 1991; Mayer-blackwell et al, 2014) for normalization, 4 primers specific to E. coli functional genes, multiple drug resistance genes (n = 60 primers), amphenicol resistance (n = 4 primers), beta-lactam resistance (n = 66 primers), non-classifiable resistance (n = 16 primers), macrolide-lincosamide-streptogramin B resistance (n = 53 primers), tetracycline resistance (n = 47 primers), aminoglycoside resistance (n=35 primers), vancomycin resistance g (n = 36 primers), and sulfonamide resistance (n = 9) as previously described (F. Wang et al, 2016; Wang et al, 2014).…”

Ecophysiology and environmental distribution of organohalide-respiring bacteria

“…Additionally, primer-independent metagenomic surveys are expected to further broaden the diversity of currently known rdh genes, as has already been demonstrated in marine subsurface sediments [216]. Combined application of amplicon-based or metagenomic surveys with high-throughput quantitative analysis methods [288] should be instrumental in obtaining a comprehensive understanding of OHRB and their catabolic reductive dehalogenation gene pools. …”

“…The method was helpful in quantitative analysis of rdhA repertoires and identification of closely related populations of OHRB [287,288]. It should be noted that to date all known RDase and 16S rRNA genes identified in the genomes of known D. mccartyi strains occur as single copies (unlike Desulfitobacterium spp.…”