| Validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples. | |
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MedLine Citation:
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PMID: 21602369 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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 primer-sharing controls. |
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Authors:
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Akihiko Hata; Hiroyuki Katayama; Masaaki Kitajima; Chettiyappan Visvanathan; Chea Nol; Hiroaki Furumai |
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Publication Detail:
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Type: Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't Date: 2011-05-20 |
Journal Detail:
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Title: Applied and environmental microbiology Volume: 77 ISSN: 1098-5336 ISO Abbreviation: Appl. Environ. Microbiol. Publication Date: 2011 Jul |
Date Detail:
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Created Date: 2011-06-24 Completed Date: 2011-10-12 Revised Date: 2012-01-02 |
Medline Journal Info:
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Nlm Unique ID: 7605801 Medline TA: Appl Environ Microbiol Country: United States |
Other Details:
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Languages: eng Pagination: 4336-43 Citation Subset: IM |
Affiliation:
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Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. hata@env.t.u-tokyo.ac.jp |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Adenoviridae
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genetics* DNA, Viral / genetics, isolation & purification* Norovirus / genetics* Polymerase Chain Reaction / methods, standards RNA, Viral / genetics, isolation & purification* Reference Standards Virology / methods*, standards* Water Microbiology* |
| Chemical | |
Reg. No./Substance:
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0/DNA, Viral; 0/RNA, Viral |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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