Development of a novel internal positive control for Taqman® based assays

Hartman, Laurie J.; Coyne, S. R.; Norwood, David

doi:10.1016/j.mcp.2004.07.006

Cited by 152 publications

(154 citation statements)

References 17 publications

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“…Surprisingly, and likely a result of the low bacterial burden in the blood, our real-time PCR assays were not capable of detecting B. mallei in the blood of infected mice (Table 4). The presence of PCR inhibitors in all sample eluates was tested by using an inhibition assay previously described (Hartman et al, 2005). The extracted DNA material from whole blood required a dilution of 1 : 8 to overcome the effects of PCR inhibitors present in the sample (Table 4).…”

Section: In Vivo Analysismentioning

confidence: 99%

Using real-time PCR to specifically detect Burkholderia mallei

Ulrich¹,

Norwood²,

Christensen³

et al. 2006

Journal of Medical Microbiology

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Section: In Vivo Analysismentioning

confidence: 99%

Using real-time PCR to specifically detect Burkholderia mallei

Ulrich¹,

Norwood²,

Christensen³

et al. 2006

Journal of Medical Microbiology

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“…The amplification efficiencies were evaluated using a TaqMan® Exogenous Internal Positive Control (IPC) (Applied Biosystems) (Hartman et al, 2005). Each sludge sample was assayed together with their 10-fold dilution in triplo.…”

Section: Qpcr Reactionmentioning

confidence: 99%

“…PCR inhibition was determined by comparing the amplification plot of the IPC of the samples with the amplification plot of the IPC of the positive control DNA. As previously mentioned, IPC amplifies under the same PCR conditions as the target DNA but by using its own primer and probe sets (Hartman et al, 2005), meaning that the target DNA amplification efficiency will never be affected by competition of both IPC and target DNA for the same primer and probe sets. As PCR inhibition mostly concerns inhibition of the DNA polymerase (Stark et al, 2000), the IPC and the target DNA are exposed to the same degree of amplification inhibition.…”

Section: Qpcr Reactionmentioning

confidence: 99%

“…In addition the use of different fluorophores enables the development of multiplex qPCR protocols whereby different targets can be co-amplified and quantified within one single reaction (Baldwin et al, 2003(Baldwin et al, , 2008Neretin et al, 2003). An example of the latter is the use of an exogenous internal positive control (IPC) (Hartman et al, 2005). In contrast to an internal inhibitor control, an IPC amplifies under the same PCR conditions as the target DNA, but using its own primer and probe sets (Hartman et al, 2005;Welti et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

“…An example of the latter is the use of an exogenous internal positive control (IPC) (Hartman et al, 2005). In contrast to an internal inhibitor control, an IPC amplifies under the same PCR conditions as the target DNA, but using its own primer and probe sets (Hartman et al, 2005;Welti et al, 2003). PCR inhibitors have been a persistent problem when working with DNA from environmental samples.…”

Section: Introductionmentioning

confidence: 99%

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Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Vanysacker

Denis

Roels

et al. 2014

Journal of Microbiological Methods

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Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93 × 10 9 to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R 2 = 0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events.

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Detection of Microbial Water Quality Indicators and Fecal Waterborne Pathogens in Environmental Waters: A Review of Methods, Applications, and Limitations

Domingo

Rice

2009

Encyclopedia of Analytical Chemistry

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Environmental waters are important reservoirs of pathogenic microorganisms, many of which are of fecal origin. In most cases, the presence of pathogens is determined using surrogate bacterial indicators. In other cases, direct detection of the pathogen in question is required. Microbiological methods to determine the presence of indicator organisms or pathogens in environmental samples vary depending on the target organism, sample type, need for quantification, speed of detection, sensitivity, and detection limits. Classical methods depend on cultivation steps while emerging techniques rely on detecting genetic signals from nucleic acid extracted from water samples. Regardless of the method, accurate detection is important to public health officials and monitoring agencies. In this article, we review some of the methods used to detect and quantify indicators of microbial water quality and human fecal pathogens as well as other issues relevant to water fecal pollution.

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Development of a novel internal positive control for Taqman® based assays

Cited by 152 publications

References 17 publications

Using real-time PCR to specifically detect Burkholderia mallei

Using real-time PCR to specifically detect Burkholderia mallei

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Detection of Microbial Water Quality Indicators and Fecal Waterborne Pathogens in Environmental Waters: A Review of Methods, Applications, and Limitations

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