Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets

Volkmann, Holger; Schwartz, Thomas; Kirchen, Silke; Stofer, Carmen; Obst, Ursula

doi:10.1016/j.mcp.2006.08.009

Cited by 67 publications

(36 citation statements)

References 28 publications

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“…DNA was eluted in 200 μl. Extracted DNA was tested using qPCR with P. aeruginosa specific primers (Pa23F-TCC AAG TTT AAG GTG GTA GGC TG and Pa23R-ACC ACT TCG TCA TCT AAA AGA CGA C) and Taqman probe (Pa23P-6-FAM-AGG TAA ATC CGG GGT TTC AAG GCC-TAMRA) as previously described [11]. The qPCR program consisted of 50°C for 2 min, 95°C for 15 min and 40 cycles of 95°C for 15 s and 60°C for 1 min with a plate read after every cycle.…”

Section: Methodsmentioning

confidence: 99%

Improved early diagnosis of Pseudomonas aeruginosa by real-time PCR to prevent chronic colonisation in a paediatric cystic fibrosis population

McCulloch

Lucas

Ramage

et al. 2011

Journal of Cystic Fibrosis

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Early detection of Pseudomonas aeruginosa in children with cystic fibrosis is hampered by the need to process a sub-optimal specimen type, namely cough swabs, which are known to have a lower positive yield than sputa or more invasive samples. This delay in the detection of low levels of P. aeruginosa could potentially result in the loss of an opportunity to initiate early aggressive antibiotic therapy and result in chronic colonisation, with a poorer overall prognosis. Quantitative real-time PCR (qPCR) offers an opportunity to increase the detection rate of P. aeruginosa compared to traditional culture techniques. This study examined 500 cough swabs and 42 sputum samples from paediatric patients and showed that detection of P. aeruginosa could be increased in both sample types by 100% and 45% respectively. Overall the sensitivity was 100% and specificity of 58% when compared to culture as a gold standard. These results although initially promising require careful consideration both from a treatment and infection control standpoint as the significance of detection of very low levels of P. aeruginosa is unclear.

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Section: Methodsmentioning

confidence: 99%

Improved early diagnosis of Pseudomonas aeruginosa by real-time PCR to prevent chronic colonisation in a paediatric cystic fibrosis population

McCulloch

Lucas

Ramage

et al. 2011

Journal of Cystic Fibrosis

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“…Singleplex qPCR using SYBR green detection has previously been used to detect certain antibiotic resistance genes in environmental samples, most frequently identified in water or waste water samples (Volkmann et al, 2004(Volkmann et al, , 2007Borjesson et al, 2009;Knapp et al, 2008;Koike et al, 2007;Pruden et al, 2006;Smith et al, 2004). The SYBR Green RT-PCR chemistry may generate false positive results as it binds to any double-stranded DNA, and as such can also bind to non-specific double-stranded DNA sequences.…”

Section: Discussionmentioning

confidence: 99%

Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

Walsh

Ingenfeld

Zampicolli

et al. 2011

Journal of Microbiological Methods

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Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora in pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we developed and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin (strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in plant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly from bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry treatment. The genes most frequently identified in soils pre-and post-slurry treatment were strB, aadA, tetB and tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative concentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry treatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification of antibiotic resistance genes in complex, environmental sample matrices.

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“…In this assay, fluorescent radicals are added into PCR reaction system to make use of the accumulation of the fluorescent signals to monitor the whole process of PCR at any time. Compared with conventional PCR (Volkmann et al 2007), fluorescent qPCR not only realizes the leap that converts from the qualitative analysis into quantitative one, but also has some clear advantages such as high sensitivity and strong specificity. In this study, when DNA probe concentration ranged from 10 5 to 10 9 copies ll -1 , Ct values and DNA copies showed linear correlation (R 2 = 0.999).…”

Section: Discussionmentioning

confidence: 99%

Developing a qPCR method to quantify AhR–PCP–DNA complex for detection of environmental trace-level PCP

2011

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Pentachlorophenol (PCP), a widely-used aseptic or biocide, is known as an environmental toxicant involved in endocrine disruption even at a trace level. In order to reliably and efficiently quantify environmental trace-quantity PCP, this study developed a novel PCP detection method using the aryl hydrocarbon receptor (AhR) and fluorescence quantitative PCR (qPCR). DNA probe with AhR binding sites was synthesized by PCR before added into AhR-PCP complex. After AhR-PCP-DNA complex was digested with exonuclease, copy number of DNA probe was determined using fluorescence qPCR. To calculate PCP concentration in samples, a standard curve (PCP concentration versus Ct value) was constructed and the detection range was 10(-13) to 10(-9) M. PCP detection limit was 0.0089 ppt for the AhR-PCP-DNA complex assay and 8.8780 ppm for high performance liquid chromatography, demonstrating that the method developed in this study is more sensitive. These results suggest that AhR-PCP-DNA complex method may be successfully applicable in detection and quantification of environmental trace-level PCP.

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Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets

Cited by 67 publications

References 28 publications

Improved early diagnosis of Pseudomonas aeruginosa by real-time PCR to prevent chronic colonisation in a paediatric cystic fibrosis population

Improved early diagnosis of Pseudomonas aeruginosa by real-time PCR to prevent chronic colonisation in a paediatric cystic fibrosis population

Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

Developing a qPCR method to quantify AhR–PCP–DNA complex for detection of environmental trace-level PCP

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