Characterization of Natural Organic Substances Potentially Hindering RT-PCR-Based Virus Detection in Large Volumes of Environmental Water.

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 (<10%) of spiked murine norovirus. These recovery rates were correlated with the levels of organic matter present in virus concentrates as measured by ultraviolet absorbance at 254 nm (r = -0.70 - -0.71, p < 0.01). High-performance gel chromatography and fluorescence excitation-emission matrix spectroscopy revealed that organic fractions in the 10-100 kDa size range, which were not dominant in the original samples, and those possessing humic acid-like fluorescence properties were dominant in virus concentrates. The inhibitory effect was more pronounced during summer. Substances originating from seawater seemed to cause a more pronounced effect than those originating from wastewater. Our data highlight the previously unknown characteristics of natural inhibitory substances and are helpful in establishing an effective sample purification technique.

[1]  Kwang Il Kim,et al.  Surveillance of aquatic animal viruses in seawater and shellfish in Korea , 2016 .

[2]  M. Sobsey,et al.  Evaluation of FRNA coliphages as indicators of human enteric viruses in a tropical urban freshwater catchment. , 2015, Water research.

[3]  M. Hamdi,et al.  Removal of Rotavirus and Bacteriophages by Membrane Bioreactor Technology from Sewage , 2015, Current Microbiology.

[4]  F. Lucas,et al.  Large scale survey of enteric viruses in river and waste water underlines the health status of the local population. , 2015, Environment international.

[5]  S. Delannoy,et al.  A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples. , 2015, International journal of food microbiology.

[6]  D. Sano,et al.  Removal properties of human enteric viruses in a pilot-scale membrane bioreactor (MBR) process. , 2015, Water research.

[7]  M. Kitajima,et al.  Concentration of Enteric Viruses in Large Volumes of Water Using a Cartridge-Type Mixed Cellulose Ester Membrane , 2015, Food and Environmental Virology.

[8]  S. Takizawa,et al.  Pepper mild mottle virus as an indicator and a tracer of fecal pollution in water environments: comparative evaluation with wastewater-tracer pharmaceuticals in Hanoi, Vietnam. , 2015, The Science of the total environment.

[9]  V. Harwood,et al.  Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters , 2015, Applied and Environmental Microbiology.

[10]  H. Furumai,et al.  Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples , 2014, Applied and Environmental Microbiology.

[11]  Maria Hellmér,et al.  Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks , 2014, Applied and Environmental Microbiology.

[12]  C. Gerba,et al.  Relative abundance and treatment reduction of viruses during wastewater treatment processes--identification of potential viral indicators. , 2014, The Science of the total environment.

[13]  Wenbo Xu,et al.  Environmental Surveillance of Human Enteroviruses in Shandong Province, China, 2008 to 2012: Serotypes, Temporal Fluctuation, and Molecular Epidemiology , 2014, Applied and Environmental Microbiology.

[14]  Keisuke Kojima,et al.  Effects of rainfall events on the occurrence and detection efficiency of viruses in river water impacted by combined sewer overflows. , 2014, The Science of the total environment.

[15]  Maja Ravnikar,et al.  One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses , 2013, Analytical and Bioanalytical Chemistry.

[16]  L. Guillier,et al.  Comparison of two extraction methods for the detection of hepatitis A virus in lettuces using the murine norovirus as a process control. , 2013, Journal of virological methods.

[17]  J. Flannery,et al.  Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters. , 2013, Water research.

[18]  J L Cashdollar,et al.  Methods for primary concentration of viruses from water samples: a review and meta‐analysis of recent studies , 2013, Journal of applied microbiology.

[19]  T. Yamashita,et al.  Development of a Reverse Transcription-Quantitative PCR System for Detection and Genotyping of Aichi Viruses in Clinical and Environmental Samples , 2013, Applied and Environmental Microbiology.

[20]  M. Kitajima,et al.  Occurrence and reduction of human viruses, F‐specific RNA coliphage genogroups and microbial indicators at a full‐scale wastewater treatment plant in Japan , 2013, Journal of applied microbiology.

[21]  C. Schrader,et al.  PCR inhibitors – occurrence, properties and removal , 2012, Journal of applied microbiology.

[22]  K. Gibson,et al.  Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples. , 2012, Water research.

[23]  Frank J. Loge,et al.  Viruses in Nondisinfected Drinking Water from Municipal Wells and Community Incidence of Acute Gastrointestinal Illness , 2012, Environmental health perspectives.

[24]  K. Schwab,et al.  Challenges in environmental detection of human viral pathogens. , 2012, Current opinion in virology.

[25]  A. Imai,et al.  Fast and precise method for HPLC-size exclusion chromatography with UV and TOC (NDIR) detection: importance of multiple detectors to evaluate the characteristics of dissolved organic matter. , 2011, Water research.

[26]  C. Visvanathan,et al.  Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples , 2011, Applied and Environmental Microbiology.

[27]  L. Ikner,et al.  New Method Using a Positively Charged Microporous Filter and Ultrafiltration for Concentration of Viruses from Tap Water , 2011, Applied and Environmental Microbiology.

[28]  C. Rock,et al.  PCR Inhibitor Levels in Concentrates of Biosolid Samples Predicted by a New Method Based on Excitation-Emission Matrix Spectroscopy , 2010, Applied and Environmental Microbiology.

[29]  M. Sobsey,et al.  Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses, adenoviruses and male‐specific coliphages from seawater , 2010, Journal of applied microbiology.

[30]  Jesus Rodriguez-Manzano,et al.  Molecular detection of pathogens in water--the pros and cons of molecular techniques. , 2010, Water research.

[31]  J. Rose,et al.  Quantitative Detection of Human Adenoviruses in Wastewater and Combined Sewer Overflows Influencing a Michigan River , 2009, Applied and Environmental Microbiology.

[32]  J. Vinjé,et al.  Norovirus Distribution within an Estuarine Environment , 2009, Applied and Environmental Microbiology.

[33]  Y. Tohya,et al.  Use of Murine Norovirus as a Novel Surrogate to Evaluate Resistance of Human Norovirus to Free Chlorine Disinfection in Drinking Water Supply System , 2008 .

[34]  G. Greening,et al.  Detection and characterization of F+ RNA bacteriophages in water and shellfish: application of a multiplex real-time reverse transcription PCR. , 2008, Journal of virological methods.

[35]  K. Oguma,et al.  Quantitative analysis of human enteric adenoviruses in aquatic environments , 2007, Journal of applied microbiology.

[36]  M. Elimelech,et al.  Evaluation of Removal of Noroviruses during Wastewater Treatment, Using Real-Time Reverse Transcription-PCR: Different Behaviors of Genogroups I and II , 2007, Applied and Environmental Microbiology.

[37]  T. Oka,et al.  Detection of human sapovirus by real‐time reverse transcription‐polymerase chain reaction , 2006, Journal of medical virology.

[38]  K. Oguma,et al.  Effects of rainfall on the occurrence of human adenoviruses, total coliforms, and Escherichia coli in seawater. , 2006, Water science and technology : a journal of the International Association on Water Pollution Research.

[39]  R. Gersberg,et al.  Quantitation of hepatitis A virus and enterovirus levels in the lagoon canals and Lido beach of Venice, Italy, using real-time RT-PCR. , 2006, Water research.

[40]  A. Bosch,et al.  Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples , 2006, Applied and Environmental Microbiology.

[41]  Larissa B. Thackray,et al.  Replication of Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and Macrophages , 2004, PLoS biology.

[42]  M. Mckendrick,et al.  A review of viral gastroenteritis , 2004, Current opinion in infectious diseases.

[43]  影山 努.  Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real Time Quantitative Reverse Transcription-PCR , 2004 .

[44]  K. Booksh,et al.  Fluorescence excitation-emission matrix regional integration to quantify spectra for dissolved organic matter. , 2003, Environmental science & technology.

[45]  S. Spencer,et al.  Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin , 2003, Applied and Environmental Microbiology.

[46]  Hiroyuki Katayama,et al.  Development of a Virus Concentration Method and Its Application to Detection of Enterovirus and Norwalk Virus from Coastal Seawater , 2002, Applied and Environmental Microbiology.

[47]  S. Farrah,et al.  Influence of Salts on Virus Adsorption to Microporous Filters , 2000, Applied and Environmental Microbiology.

[48]  H. Tachibana,et al.  CHARACTERIZATION OF DISSOLVED ORGANIC MATTER BY THREE DIMENSIONAL EXCITATION EMISSION MATRIX SPECTROSCOPY , 1999 .

[49]  M. Sobsey,et al.  Methods to remove inhibitors in sewage and other fecal wastes for enterovirus detection by the polymerase chain reaction. , 1995, Journal of virological methods.

[50]  E. O’Loughlin,et al.  Molecular weight, polydispersity, and spectroscopic properties of aquatic humic substances. , 1994, Environmental science & technology.

[51]  C. Gerba,et al.  Detection of enteroviruses in groundwater with the polymerase chain reaction , 1993, Applied and environmental microbiology.

[52]  B H Olson,et al.  Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction , 1992, Applied and environmental microbiology.

[53]  N. Granboulan,et al.  Molecular Weight of Poliovirus Ribonucleic Acid , 1969, Journal of virology.

[54]  H. Furumai,et al.  Quantification of enteric viruses and microbial indicators in Ishinomaki coastal area , 2012 .

[55]  A. Imai,et al.  Characterization of Dissolved Organic Matter (DOM) in Lake Kasumigaura and Several DOM Sources Using Method of Three-Dimensional Excitation-Emission Matrix Fluorescence Spectra , 2008 .