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. 2011 Jul;77(13):4336-43.
doi: 10.1128/AEM.00077-11. Epub 2011 May 20.

Validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples

Akihiko Hata  1 , Hiroyuki Katayama, Masaaki Kitajima, Chettiyappan Visvanathan, Chea Nol, Hiroaki Furumai
Affiliations

Validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples

Akihiko Hata et al. Appl Environ Microbiol. 2011 Jul.

Abstract

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 primer-sharing controls.

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Figures

Fig. 1.
Fig. 1.
Schematic diagram of PSC DNA amplified by the same primer set as target genome DNA, which was of the same amplicon size but was recognized by a different TaqMan probe.
Fig. 2.
Fig. 2.
Input levels and detection of serially diluted Ad40 DNA and the indicated amount (1.0 × 102 copies per reaction) of EAdV PSC DNA by duplex qPCR. Horizontal and vertical axes indicate the added and observed concentrations of Ad40 DNA and EAdV PSC DNA, respectively. The dotted line indicates the added concentration of EAdV PSC DNA. Error bars indicate the standard deviations (n = 3).
Fig. 3.
Fig. 3.
Inhibition of target viral genome and control amplification in the presence of humic acid. (A) Underestimation of Ad40 DNA and EAdV PSC DNA and the underestimation of Ad40 DNA and MNV cDNA. The vertical axis indicates the observed recovery of EAdV PSC DNA or MNV cDNA. The horizontal axis indicates the observed recovery of Ad40 DNA. (B) Underestimation of GII NoV RNA and GII NoV PSC RNA. A plot bracket on (x, y) of (−2.1, −3.3) indicates the possible outlier. The underestimation of GII NoVRNA and MNV-RNA is indicated. The vertical axis indicates the observed recoveries of GII NoV PSC RNA or MNV RNA. The horizontal axis indicates the observed recovery of GII NoV RNA. In both panels, ND on the vertical and horizontal axes indicates not detected. Open circles and triangles indicate the lower limits of detection.
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