Quantitative real-time PCR (qPCR) detection chemistries affect enumeration of the Dehalococcoides 16S rRNA gene in groundwater

Hatt, Janet K.; Löffler, Frank E.

doi:10.1016/j.mimet.2011.12.005

Cited by 34 publications

(26 citation statements)

References 37 publications

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“…IDT DNA Primer Quest software (http: //scitools.idtdna.com/Primerquest/) was used to design qPCR primers dcpA-1257F and dcpA-1449R and TaqMan probe dcpA-1426 to enumerate dcpA gene copies (Table 2). First, the primers were used with SYBR green chemistry to recognize nonspecific amplification and/or primer dimer formation (19). Standard curves were generated with 10-fold serial dilutions (10 8 to 10 0 copies) of the partial D. mccartyi strain KS dcpAB gene fragment cloned into the TOPO TA (Invitrogen) pCRII plasmid.…”

Section: 2-d-dechlorinating Cultures the 12-d-dechlorinating Mixementioning

confidence: 99%

Identification and Environmental Distribution ofdcpA, Which Encodes the Reductive Dehalogenase Catalyzing the Dichloroelimination of 1,2-Dichloropropane to Propene in Organohalide-Respiring Chloroflexi

Padilla-Crespo

Yan

Swift

et al. 2014

Appl Environ Microbiol

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Section: 2-d-dechlorinating Cultures the 12-d-dechlorinating Mixementioning

confidence: 99%

Identification and Environmental Distribution ofdcpA, Which Encodes the Reductive Dehalogenase Catalyzing the Dichloroelimination of 1,2-Dichloropropane to Propene in Organohalide-Respiring Chloroflexi

Padilla-Crespo

Yan

Swift

et al. 2014

Appl Environ Microbiol

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“…All TaqMan assays were set up as 20-l reaction mixtures. Each 20-l reaction mixture contained 10 l of iTaq Universal super mix supplied by Bio-Rad, 1.2 l of TaqMan probe, described previously (15,17,26,27), and balance water to make up 18 l. PCR amplifications were performed using cycling conditions of 95°C for 15 s, 60°C for 1 min, a slow ramp of 1% to 95°C for 15 s, and 60°C for 15 s. DNA templates and plasmid standards were added to each LAMP and qPCR mixtures as 2-l aliquots. All qPCR primers and probes used in this study are listed in Table 2.…”

Section: Methodsmentioning

confidence: 99%

“…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 TaqMan probes to quantify vcrA, bvcA, and tceA genes have been developed (2,(15)(16)(17). Although qPCR has been successful for monitoring reductive dechlorination, alternative methods would be advantageous for laboratories or practitioners without access to a real-time thermal cycler.…”

mentioning

confidence: 99%

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

Kanitkar

Stedtfeld

Steffan³

et al. 2016

Appl Environ Microbiol

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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...

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“…The rPCR technology allows the detection of PCR amplification and the measurement of the reaction kinetics during the early phases of the reaction, thus providing a distinct advantage over cPCR detection [33][34][35]. Because rPCR detects the accumulation of amplicons as the reaction progresses, the data are acquired during the exponential phase of the PCR reaction.…”

Section: Introductionmentioning

confidence: 99%

“…The exponential phase, or real-time, is commonly considered to be the optimal point for data analysis, and rPCR is considered to be the easiest method for quantifying detected DNA and RNA. Theoretically, there is a quantitative relationship between the amount of DNA in the starting target sample and the amount of PCR product at any given cycle number [34,35].…”

Section: Introductionmentioning

confidence: 99%

Next-Generation Sequencing Confirmation of Real-Time RT-PCR False Positive Influenza-A Virus Detection in Waterfowl and Swine Swab Samples

Lu¹,

Tang²,

Lin³

2016

Next Generat Sequenc & Applic

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Surveillance for the Influenza A virus is the most important method used to monitor poultry and other animal species for the presence of the Influenza A viruses. Waterfowl and swine swab samples that tested positive for the Influenza A virus by real-time RT-PCR (rRT-PCR) but tested negative for the virus by virus isolation were further investigated in our recent studies using next-generation sequencing (NGS). A total of seven such pooled swab samples (four swine oral-pharyngeal (OPH) swab pools and three wild duck OPH and cloacal swab pools) were tested for influenza A virus by whole genome sequencing using NGS with the Illumina MiSeq system. Our sequencing results confirmed that none of these rRT-PCR positive samples (Ct-values of 22 to 28) contained Influenza A virus contigs; instead, all the samples contained multiple non-target contig sequences mismatched to the rRT-PCR primer and probe sequences, which led to the positive rRT-PCR results. These genome sequence findings provide scientific evidence that the rRT-PCR false positive results were caused by non-target contigs and not by target viral RNA. Additionally, these samples tested negative for the influenza A virus by conventional RT-PCR (cRT-PCR), which suggests cRT-PCR can serve as an alternative approach for identifying rRT-PCR false positive results.

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Quantitative real-time PCR (qPCR) detection chemistries affect enumeration of the Dehalococcoides 16S rRNA gene in groundwater

Cited by 34 publications

References 37 publications

Identification and Environmental Distribution ofdcpA, Which Encodes the Reductive Dehalogenase Catalyzing the Dichloroelimination of 1,2-Dichloropropane to Propene in Organohalide-Respiring Chloroflexi

Identification and Environmental Distribution ofdcpA, Which Encodes the Reductive Dehalogenase Catalyzing the Dichloroelimination of 1,2-Dichloropropane to Propene in Organohalide-Respiring Chloroflexi

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

Next-Generation Sequencing Confirmation of Real-Time RT-PCR False Positive Influenza-A Virus Detection in Waterfowl and Swine Swab Samples

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