Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters

Abstract

Quantitative real-time PCR (qPCR) is used commonly to detect adenovirus (Ads) and norovirus (Nvs) in recreational waters. However, qPCR detection may be limited by interference from inhibitory substances found in recreational waters. In previous work, viruses in Avalon and Doheny Beach water samples were concentrated by electropositive cartridge filtration and PEG precipitation, and high inhibition was found in the samples when using qPCR for detection of Ads and Nvs. Therefore, different approaches were evaluated for removal or blocking of inhibitory compounds that affect qPCR. Avalon and Doheny concentrates were spiked with known amounts of Ads 41 and Nvs GII, and spiked deionized water was used as a positive control. Modifications included gel chromatography with columns of Sephadex G-200/Chelex 100, different sample volumes for nucleic acid extraction, organic solvent extraction, and nucleic acid precipitation. The efficiency of each treatment varied according to sampling location and virus type. The best option for improved Nvs detection by reverse transcription-qPCR was to reduce the sample volume for nucleic acid purification. The best option for improving Ads detection in both beach samples was Sephadex/Chelex spin column chromatography. Chloroform extraction only improved virus detection in Doheny Beach samples but not in Avalon Beach samples. Observed differences in effective treatments between viruses may be related to the different PCR targets, amplification conditions, and enzymes used in each assay, and differences between beaches may be related to differences in PCR inhibitory environmental compounds at each location. The results suggest that methods for detecting viruses from marine beaches, including treatments for the removal of PCR inhibitory compounds, should be optimized for each sampling site and probably for each virus of interest.