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Development of a method for detection of enteroviruses in shellfish by PCR with poliovirus as a model
D N Lees1, K Henshilwood, W J Doré
1Fish Diseases Laboratory, Ministry of Agriculture, Fisheries and Food, Weymouth, Dorset, England.
Abstract:
The application of the PCR to complex samples is hindered by amplification inhibitors. We describe a reverse transcription-PCR-based method capable of inhibitor removal for the detection of enteroviruses in shellfish. Initial virus extraction stages based on a modified polyethylene glycol precipitation technique (G.D. Lewis and T.G. Metcalf, Appl. Environ. Microbiol. 54:1983-1988, 1988) were followed by virus purification with 1,1,2-trichloro,2,2,1-trifluoroethane and concentration by ultrafiltration. A guanidine isothiocyanate-glass powder extraction system was utilized for sample lysis, RNase protection, and nucleic acid purification. Removal of PCR inhibitors and method sensitivity were quantified in shellfish (oysters and mussels) seeded with poliovirus. PCR sample tolerance exceeded 4 g for depurated shellfish; however, polluted field samples were more inhibitory. Virus recoveries of 31% for oyster extracts and 17% for mussel extracts and nucleic acid extraction reverse transcription-PCR detection limits down to 1 PFU yielded an overall sensitivity limit of < 10 PFU of poliovirus in up to 5 g of shellfish. PCR-positive results were obtained from a variety of polluted field samples naturally contaminated with human enteroviruses. The methods developed for virus recovery and PCR inhibitor removal should be equally applicable to detection of other RNA viruses such as hepatitis A virus, Norwalk virus, and other small round-structured viruses in shellfish.
Insights
This study presents a new reverse transcription-polymerase chain reaction (RT-PCR) method to detect enteroviruses in shellfish by removing amplification inhibitors. The method achieves high sensitivity for detecting viruses in oysters and mussels.
Area of Science:
- Environmental Microbiology
- Molecular Virology
- Food Safety
Background:
- Polymerase chain reaction (PCR) is crucial for detecting viruses but is often inhibited by complex sample matrices like shellfish.
- Existing methods struggle with inhibitor removal, limiting sensitive viral detection in food sources.
Purpose of the Study:
- To develop and validate a reverse transcription-PCR (RT-PCR) based method for efficient enterovirus detection in shellfish.
- To address the challenge of amplification inhibitors present in shellfish samples.
Main Methods:
- A multi-step approach involving modified polyethylene glycol precipitation, solvent purification, ultrafiltration, and guanidine isothiocyanate-glass powder extraction.
- Quantification of PCR inhibitor removal and method sensitivity using poliovirus-spiked oysters and mussels.
Main Results:
- The RT-PCR method demonstrated effective inhibitor removal, with sample tolerance exceeding 4g for depurated shellfish.
- Achieved high virus recovery rates (31% oyster, 17% mussel) and a detection limit of <10 PFU of poliovirus per 5g of shellfish.
- Successfully detected naturally occurring human enteroviruses in polluted field samples.
Conclusions:
- The developed RT-PCR method provides sensitive and reliable detection of enteroviruses in shellfish.
- This approach is adaptable for detecting other RNA viruses, such as Hepatitis A virus and Norovirus, in shellfish.
- The method enhances food safety by enabling robust viral pathogen monitoring in seafood.