A nested reverse transcriptase PCR assay for detection of small round-structured viruses in environmentally...

Area of Science:

• Virology
• Food Safety
• Molecular Biology

Background:

• Small round-structured viruses (SRSVs) are a significant cause of gastroenteritis outbreaks linked to shellfish consumption.
• Current detection methods, like single-round RT-PCR, may lack the sensitivity to identify all relevant SRSV strains in molluscan shellfish.

Purpose of the Study:

• To evaluate a nested reverse transcriptase PCR (RT-PCR) procedure for detecting SRSVs in shellfish.
• To assess the assay's sensitivity and strain coverage for SRSV detection.
• To apply the nested RT-PCR assay in real-world scenarios, including commercial shellfish and outbreak investigations.

Main Methods:

• Development and evaluation of a nested RT-PCR assay.
• Testing the assay against a panel of 21 well-characterized SRSV strains.
• Seeding experiments to compare sensitivity with single-round RT-PCR.
• Application of the assay to shellfish from polluted harvesting areas and implicated in gastroenteritis outbreaks.

Main Results:

• The nested RT-PCR detected 15 out of 21 SRSV strains, covering both genogroup I and II.
• The nested RT-PCR was 10 to 1,000 times more sensitive than single-round RT-PCR in shellfish seeding experiments.
• The assay identified SRSV contamination in shellfish from polluted areas and in outbreak-associated shellfish that were negative by single-round RT-PCR.

Conclusions:

• The nested RT-PCR is a sensitive and broad-spectrum assay for SRSV detection in shellfish.
• This assay is effective for monitoring shellfish harvesting areas, investigating outbreaks, and assessing virus elimination during processing.
• The nested RT-PCR offers significant advantages over previous methods for ensuring shellfish safety.