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.