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Digital RT-PCR method for hepatitis A virus and norovirus quantification in soft berries
Audrey Fraisse1, Coralie Coudray-Meunier1, Sandra Martin-Latil1
1Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
International Journal of Food Microbiology
|December 14, 2016
Summary
Detecting enteric viruses in soft berries is crucial for public health. This study improved detection methods by adding a purification step and using digital RT-PCR, enhancing accuracy and efficiency for food safety analysis.
Area of Science:
- Foodborne Pathogen Detection
- Virology
- Analytical Chemistry
Background:
- Raw fruits, especially soft berries, are increasingly linked to outbreaks of enteric viruses like norovirus and hepatitis A.
- Current detection methods require high sensitivity due to the low infectious doses and concentrations of these viruses in food samples.
- Existing reference methods (ISO/TS 15216-1; 15216-2) need enhancement for reliable enteric virus detection in soft fruits.
Purpose of the Study:
- To improve the reference method for detecting enteric viruses in soft fruits.
- To evaluate the impact of an additional RNA purification step on virus detection.
- To assess the utility of microfluidic digital RT-PCR for absolute quantification of viral genomes.
Main Methods:
- Incorporation of an additional purification step post-RNA extraction in the reference method.
- Application of microfluidic digital RT-PCR for absolute quantification of enteric viral genomes.
- Comparison of enhanced method performance against standard protocols for soft fruit samples.
Main Results:
- The enhanced purification step significantly reduced PCR inhibition, improving viral detection sensitivity.
- Microfluidic digital RT-PCR enabled accurate absolute quantification and facilitated control validation with pure RNA extracts.
- Both modifications collectively improved viral extraction efficiencies and overall reliability of enteric virus detection in soft berries.
Conclusions:
- An additional PCR inhibitor removal step is easily integrated into routine methods for enhanced enteric virus detection.
- Absolute quantification using digital RT-PCR offers a standardized and precise alternative for quantifying enteric viruses in foodstuffs.
- These improvements contribute to more robust food safety measures against viral contamination in soft fruits.
