Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics

Molenkamp, Richard; Ham, Alwin J. van der; Schinkel, Janke; Beld, Marcel

doi:10.1016/j.jviromet.2006.12.007

Cited by 57 publications

(40 citation statements)

References 25 publications

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“…Therefore, developing methods to evaluate the magnitude of inhibition is essential (14,42). Exogenously added control nucleic acids have been used for this purpose (14,27,30,32,37,42). Although the inhibition of polymerase activity depends on the target nucleotide sequences (22,40), most previous studies have not employed sequence-matched controls (27,30,32,37,42).…”

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confidence: 99%

Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

Hata

Katayama

Kitajima

et al. 2011

Appl Environ Microbiol

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Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primersharing controls.

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mentioning

confidence: 99%

Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

Hata

Katayama

Kitajima

et al. 2011

Appl Environ Microbiol

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“…Our study describes only the second reported use of a pentaplexed real-time PCR assay, following a recent description of a pentaplexed assay for the simultaneous quantification of respiratory RNA viruses (25). While setting up our pentaplexed assay, we empirically learned that mixing of MGB-and LNA-containing oligoprobes rendered a superior PCR performance, compared to a multiplex assay with LNA probes only.…”

Section: Discussionmentioning

confidence: 99%

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Kirchner

Kramer

Schulze

et al. 2010

Appl Environ Microbiol

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Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder-and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10 3 and 10 5 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.

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“…The IAC is added at a low level to maximize its responsiveness to a loss of DNA during extraction and to minimize potential interference with the detection of the target DNAs (18); we found that the addition of IAC to a level such that 100 copies are present in the qPCR assay was consistently detected with a low Cp variability and a low rate of IAC-negative samples (Table 3). We designed this assay for the LightCycler 480 system, which is a platform that performs very well for viral diagnostics, particularly for multiplex applications (31). The dye configuration for this platform featured detection of BKPyV on the FAM channel, the IAC on the VIC/HEX channel, and JCPyV on the Cy5 channel.…”

Section: Vol 46 2008 Triplex Qpcr Assay For Jc and Bk Polyomavirusementioning

confidence: 99%

Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK

2008

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We have developed a triplex TaqMan-based quantitative PCR assay for the human polyomaviruses JC (JCPyV) and BK (BKPyV). The assay simultaneously detects and quantifies both JCPyV and BKPyV in human clinical samples, and it includes an internal amplification control consisting of murine polyomavirus (MuPyV) plasmid DNA. We developed the assay for the Roche LightCycler 480 platform with the reporter dyes VIC, 6-FAM, and Cy5 for MuPyV, BKPyV, and JCPyV, respectively. The assay had a high specificity for BKPyV and JCPyV when either viral genome was present alone or in mixed samples over a range of 10 1 to 10 7 copy numbers per reaction. The analytical sensitivity was 50 copies for BKPyV and 10 copies for JCPyV. The use of the MuPyV internal control ensured monitoring of the quality of the extraction and of PCR inhibition, even in samples such as cerebrospinal fluid and plasma in which controls based on host genes cannot be effectively used. In addition, we developed a similar assay using a different dye configuration (6-FAM, VIC, and NED) that could be used on an ABI 7500 Fast platform. This assay had sensitivities similar to those of the LightCycler 480 configuration for BKPyV and JCPyV when either viral genome was present alone, but the sensitivity of detection of BKPyV was greatly decreased when an excess of JCPyV (>100-fold) was present in the sample. This internally controlled combined assay offers greater convenience and cost-effectiveness compared to separate assays for each virus and can also detect unexpected PyV activations by testing for both viruses in all samples.

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Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics

Cited by 57 publications

References 25 publications

Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK

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