Development of an integrated cell culture--real-time RT-PCR assay for detection of reovirus in biosolids.

The current method for viral detection in biosolids is a plaque assay, as specified by the EPA in the 40 CFR Part 503 rule. Development of an integrated cell culture-polymerase chain reaction (ICC-PCR) assay has allowed detection of viruses that are under-detected and undetected by the plaque assay. This study examined the efficiency of the ICC-PCR method to detect mammalian orthoreovirus, a virus typically under-detected in biosolids. Biosolid samples seeded with mammalian orthoreovirus type 1 (Lang) detected to 3 x 10(5) plaque forming units (pfu) with a plaque assay, 10(2)pfu equivalents with real-time RT-PCR and no incubation, and 10(8)pfu equivalents with real-time RT-PCR after 7 days incubation. More infectious virus was detected using ICC-real-time RT-PCR than a plaque assay. Twenty-four environmental samples from three locations around the United States did not plaque with the EPA method; however the ICC-PCR detected infectious reovirus in 13 of the samples. Raw biosolids samples accounted for 12 of the positive samples, and 1 positive was from an aerobically digested sample.