A Nested Reverse Transcriptase PCR Assay for Detection of Small Round-Structured Viruses in Environmentally Contaminated Molluscan Shellfish

We describe the evaluation of a nested reverse transcriptase PCR (RT-PCR) procedure for the detection of small round-structured viruses (SRSVs) in molluscan shellfish and the application of this assay for the detection of SRSVs in commercially produced shellfish and in shellfish implicated in outbreaks of gastroenteritis. The range of virus strains detected and the sensitivity of detection were evaluated by using a representative panel of 21 well-characterized SRSV strains. The nested RT-PCR detected 15 of 21 SRSVs, demonstrating that the assay detects a broad range of SRSVs including strains from both genogroup I and genogroup II. Seeding experiments showed the nested RT-PCR assay to be 10 to 1,000 times more sensitive than the single-round RT-PCR assay for the detection of SRSV in shellfish. SRSV-contaminated samples were identified by nested RT-PCR for shellfish grown in polluted harvesting areas and for shellfish associated with outbreaks of gastroenteritis which were negative by a previously described single-round RT-PCR. The assay was shown to be effective for investigation of virus elimination during commercial shellfish processing procedures such as depuration and relaying and has potential applications for monitoring at-risk shellfish harvesting areas, for investigation of SRSV contamination in shellfish from producers linked to gastroenteritis outbreaks, and for the direct detection of virus in shellfish implicated in outbreaks.