title: Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction.
abstract: We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.
authors.affiliation: Area on Water Management, Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351-0197, Japan E-mail: kishida@niph.go.jp.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kishida
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
authors.affiliation: Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Haramoto
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Kawaharasaki
authors.affiliation: Area on Water Management, Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351-0197, Japan E-mail: kishida@niph.go.jp.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Akiba
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
mesh_terms: D000260:Adenoviruses, Human / Q000302:isolation & purification*; D005618:Fresh Water / Q000382:microbiology*; D007422:Intestines / Q000821:virology; D044085:Microfluidics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D045483:Rivers; D014871:Water Microbiology