Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

Abstract

c Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loopmediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures. Microbially mediated reductive dechlorination plays a vital role in the bioremediation of the chlorinated ethenes tetrachloroethene (PCE) and trichloroethene (TCE). Under the appropriate conditions, PCE and TCE undergo sequential reductive dechlorination via hydrogenolysis to cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), respectively, finally forming the nontoxic end product ethene (ETH) (1). When reductive dechlorination is linked to growth, it is called organohalide respiration, a metabolism commonly associated with microbial taxa, like Dehalococcoides and Dehalobacter (2-9). Commercially available reductive dechlorinating mixed cultures (e.g., KB-1 and SDC-9) containing such strains are frequently used for the bioaugmentation of contaminated groundwater aquifers (10-12). In fact, in 2009, it was estimated that bioaugmentation with Dehalococcoides spp. had been used at several hundred sites in the United States (13). The growth of these strains in the field and in the laboratory is commonly monitored using real-time quantitative PCR (qPCR) targeting the genes vcrA, bvcA, and tceA, which code for distinct reductive dehalogenases (RDases) implicated in organohalide respiration (14). To date, a number of qPCR protocols with DNA binding dyes or TaqM...

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of Dreissena sp.

“…Direct amplification, i.e., amplification without DNA extraction or purification, satisfies both these attributes. Elimination of DNA extraction and purification steps simplifies the process and may avoid the need for sample transport [ 3 , 4 ]. For detection of invasive species at very low abundance, sample concentration is often useful and necessary.…”

Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater

“…The chip configuration consisted of eight reaction wells per sample lane, and four sample lanes per chip (i.e. 32 reaction wells per chip, each with 20 μL reaction volume), as previously described (Kanitkar et al, 2016). Primers targeting rdhA gene and 16S rRNA gene were each dispensed into three separate reaction wells per sample lane, and primers targeting the MIAC were dispensed into two reaction wells.…”

“…Previous studies demonstrated LAMP assays for quantification of Dehalococcoides spp. in groundwater (Kanitkar et al, 2016). Our studies also examined a field-able means to concentrate bio-mass from groundwater via Sterivex cartridges and direct amplification from filtrate elution on a field-ready Gene-Z device (Stedtfeld et al, 2014).…”

Microbiological Sensing Technologies: A Review

“…Microbial genomic studies are currently the most popular molecular identification and classification method of microorganisms in different types of samples based on the selection of one or more specific molecular target(s), such as DNA, RNA and protein, without the requirement to grow in their culture media [ 107 ]. Genetic microbial detection assays can be classified into genome GC% (Guanine + Cytosine) contents, pattern amplification (polymerase chain reaction; PCR, and loop mediated isothermal amplification; LAMP), DNA/RNA hybridization, genome polymorphism (such as RFLP: restriction fragment length polymorphism and AFLP: amplified fragment length polymorphisms) and gene sequencing [ 108 , 109 , 110 ]. In addition to the sample preparation and genome extraction, the majority of these methods need a nucleic acid amplification and an amplicon analysis step [ 110 ].…”

“…The sample preparation and DNA extraction steps are very challenging steps due to the removal of inhibitors from genomic samples and accurate measurement of the initial genomic contents per specific amounts of sample, which is used in quantitative methods [ 110 ]. These samples are always passed through an enrichment step, which can be performed by overnight incubation of bacteria in a specific sample (only limited to cultivable bacteria), centrifugation, and immunomagnetic separation [ 108 , 109 , 110 ]. These two latest samples can be applied for the removal of sample matrix from the microorganisms, which indeed is useful for removal of inhibitors from the genome [ 108 , 109 , 110 ].…”