An RNA extraction protocol for shellfish-borne viruses.

Area of Science:

• Food safety
• Virology
• Molecular biology

Background:

• Shellfish can be contaminated with human and animal viruses.
• Accurate viral RNA extraction is crucial for sensitive detection methods like RT-PCR.

Purpose of the Study:

• To evaluate the GPTT virus RNA extraction method for its efficacy with multiple picornaviruses and caliciviruses.
• To assess the method's performance in detecting viral RNA in oysters (Crassostrea virginica).

Main Methods:

• The GPTT method was tested for RNA extraction from Aichi virus (AiV), coxsackievirus strains A9 (CAV9) and B5 (CBV5), murine norovirus (MNV-1), and feline calicivirus (FCV).
• Extracted RNA was used for RT-PCR detection in seeded oyster extracts and oysters exposed to contaminated seawater.
• RT-PCR equivalent sensitivities were determined by comparing detection limits in oysters to virus stocks.

Main Results:

• The GPTT method demonstrated varying sensitivities for different viruses, with notable detection limits in seeded oysters and contaminated seawater.
• Equivalent sensitivities ranged from 0.68 to 26 RT-PCR(50) units in seeded oysters and 14.5 to 2600 RT-PCR(50) units in oysters exposed to contaminated seawater.
• The method showed effectiveness in extracting RNA from multiple target viruses within oyster matrices.

Conclusions:

• The GPTT RNA extraction method is a versatile tool for detecting a range of picornaviruses and caliciviruses in shellfish.
• This method supports the sensitive RT-PCR detection of viral contamination in oysters, contributing to food safety assessments.