Article pubs.acs.org/est
Characterization of Natural Organic Substances Potentially Hindering RT-PCR-Based Virus Detection in Large Volumes of Environmental Water Akihiko Hata,*,†,§ Manami Inaba,‡,§ Hiroyuki Katayama,§ and Hiroaki Furumai§ †
Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga 520-0811, Japan Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13, West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan § Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡
S Supporting Information *
ABSTRACT: Quantitative detection of pathogenic viruses in the environmental water is essential for the assessment of water safety. It is known that some of natural organic substances interfere with virus detection processes, i.e., nucleic acid extraction and reverse transcription-PCR. Such substances are carried over into a sample after virus concentration. In this study, inhibitory substances in coastal water samples were characterized in view of their effects on efficiency of virus detection and property as organic matters. Among 81 samples tested, 77 (95%) showed low recoveries (3 log10.8,12 © 2017 American Chemical Society
Received: Revised: Accepted: Published: 13568
January 17, 2017 November 13, 2017 November 14, 2017 November 22, 2017 DOI: 10.1021/acs.est.7b00306 Environ. Sci. Technol. 2017, 51, 13568−13579
Article
Environmental Science & Technology
and 950 μS/cm, respectively, throughout the study period. The Sumida River is a tidal river that flows into the Tokyo Bay. R2 was located at the mouth of the Sumida River, and the samples were characterized as having high salinity (up to 3.5%), indicating that the water at R2 is affected by seawater. The other sites (C1 and C2) were located on canals adjacent to the Tokyo Bay. The remaining three sites (S1−S3) were located at beach parks. The two-step virus concentration process using a cartridgetype electronegative membrane (Opticap XL2, 0.1 m2 filtration area, 0.5 μm pore size: Millipore) and a centrifugal UF device (Centricon plus-70, 30 kDa molecular weight cutoff: Millipore), which was described previously, was applied with slight modifications.20 The concentration method is based on the same principle to a method described by Katayama et al. (2002).13 For the R1 samples, 20 L of sample was collected in a 20 L polyethylene container presterilized by gamma irradiation, stored on ice, and transferred to the lab at the University of Tokyo. Subsequently, the sample was mixed with 200 mL of a 2.5 M MgCl2 solution and passed through a cartridge-type electronegative membrane. For the samples at other sites, 40 L of sample was directly passed through a cartridge-type electronegative membrane without adding MgCl2. The cartridge was kept cool and transported to the lab. After the sample filtration, 2.0 L of 0.5 mM H2SO4 (pH 3.0) (acid rinse step), 1.0 L of Milli-Q water (Milli-Q rinse step), and then 200 mL of 1.0 mM NaOH (pH 10.8) (alkaline elution step) were successively passed through the filter cartridge. The acid-rinse filtrate was recovered into a presterilized tube containing 40 mL of 100 mM NaOH (pH 11.8) for neutralization. Similarly, the final alkaline eluate was recovered as a primary concentrate into a presterilized tube containing 1 mL of 100 mM H2SO4. Then, 60 mL of the primary concentrate was concentrated using a Centricon Plus-70 centrifugal filter unit according to the manufacturer’s instructions. The resultant retentate (approximately 0.25 mL) was mixed with 2.5 μL of 100 × Tris-EDTA buffer (pH 8.0, Wako, Osaka, Japan) for stabilization and stored as a secondary concentrate. The concentration process was completed within 12 h after the sample collection. Performance of the primary concentration method tested using an artificial seawater sample spiked with viruses are shown in Table S1, in the Supporting Information. RNA Extraction and RT-qPCR. MNV was used as a molecular control to monitor the inhibitory effects on RNA extraction-RT-qPCR processes. Briefly, 140 μL of the secondary concentrate was spiked with 3.5 × 107 gene copies of MNV (5 μL) and subjected to RNA extraction to obtain 60 μL of an RNA extract using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The RNA extract was diluted 10- and 100-fold with nuclease-free water. Both 100-fold diluted and undiluted RNA extracts were subjected to RT-qPCR to quantify the spiked MNV as described below. As discussed previously,8 diluting the RNA extract, which reduces the effect of RT-qPCR inhibition, is effective to estimate the RNA extraction efficiency. Genes of genogroup I (GI-) and GII-NoVs, SaV, Aichi virus 1 (AiV), enterovirus (EV), GII- and GIII-F-specific RNA phages (FPHs), and spiked process controls, i.e., MNV and primer-sharing control (PSC)-RNAs,8,23 were quantified by a two-step RT-qPCR assay. For quantification of GI- and GIINoV and AiV, PSC-RNAs (GI- and GII-NoV- and AiV-PSCRNA, respectively) were used as RT-PCR controls to monitor the inhibitory effect on each RT-qPCR assay.8,23 In addition to
due to the lack of knowledge on the inhibitory substances. Evaluation of such inhibitory effects has been conducted by spiking and recovering viruses and/or nucleic acids, which serve as process controls.8,12,22−28 However, considering the effort required to measure the extent of inhibition, characterization of inhibitory substances has seldom been done. Rock et al.29 reported that excitation−emission matrix (EEM) spectroscopy, a method of fluorescence analysis used for the classification of natural organic substances, is useful for the estimation of the levels of PCR inhibitors. Our previous study has employed sizeexclusion chromatography and revealed that 10−100 kDa size fractions existing in commercially available humic acid powders were not excluded by the virus concentration process and could cause RT-PCR inhibition.21 We also found the similar size of organic fractions in virus concentrates obtained from 3 fresh water samples.21 These previous studies strongly suggest that an investigation is needed for the characterization of naturally occurring inhibitory substances. Components of the natural organic matter are variable depending on the sample matrix, geographical location, and season. Thus, it is possible that inhibitory substances are also variable depending on such environmental factors. Gibson et al.12 suggested that the impact of inhibitory substances is seasonal, although no relevant evidence was provided. As indicated by a previous study, virus concentration techniques selectively recover some inhibitory substances.21 Thus, it is important to determine the profiles of organic substances that are effectively recovered during virus concentration processes. Furthermore, to determine variations of such inhibitory organic substances along with environmental factors may help in the characterization of them and future establishment of an efficient sample purification technique. The Tokyo Bay coastal area has been a target of enteric virus monitoring.13,23,30,31 The area can present a challenge for studying the naturally occurring inhibitory substances, because it is surrounded by densely populated areas and is affected by a discharge from several wastewater treatment plants, either directly or via rivers and canals. During the summer season, the coastal area is often hit by red tides, which indicate that the coastal area is severely contaminated by organic matter. In this study, we aimed to determine variations of inhibitory organic substances along with environmental factors. River, canal, and seawater samples were collected monthly in the Tokyo Bay coastal area for a year. Concentrations of enteric viruses and indicators as well as properties of organic substances were investigated in terms of inhibitory effects on the virus molecular quantification.
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MATERIALS AND METHODS Viruses. Murine norovirus (MNV; strain S7-PP3) was replicated in RAW 264.7 cells (ATCC TIB-71; American Type Culture Collection, Manassas, VA, USA) and purified according to previous studies.8,32 The concentration of the purified MNV (7.0 × 106 gene copies/μL) was determined by RT-qPCR as described below. Collection and Concentration of Water Samples. In total, 81 water samples were collected from seven sampling sites (R1, R2, C1, C2, S1, S2, and S3), located in the coastal area of the Tokyo Bay from December 2010 to December 2011. Among the sampling sites, two sites were located on the Kanda River and Sumida River (R1 and R2, respectively). The Kanda River, which mainly consists of effluents from wastewater treatment plants, flows into the Sumida River. The salinity and electrical conductivity at R1 were below 0.5% 13569
DOI: 10.1021/acs.est.7b00306 Environ. Sci. Technol. 2017, 51, 13568−13579
Article
Environmental Science & Technology
Table 1. Efficiency of Spiked MNV Recovery and Positive Rates of Detection of Indigenous Viruses in Undiluted and 10-Fold Diluted RNA Extracts by RT-qPCRa,b station spiked MNV
GINoV GIINoV SaV
AiV
EV
GIIFPH GIIIFPH
virus
R1
R2
S1
S2
S3
C1
C2
total
