Quantitative analysis of norovirus outbreak related shellfish samples using digital PCR and qPCR
Abstract
Human noroviruses are a major cause of acute gastroenteritis outbreaks worldwide infecting all ages by foodborne, waterborne and person to person transmission. Outbreaks of acute gastroenteritis caused by norovirus have been... [ view full abstract ]
Human noroviruses are a major cause of acute gastroenteritis outbreaks worldwide infecting all ages by foodborne, waterborne and person to person transmission. Outbreaks of acute gastroenteritis caused by norovirus have been linked to the consumption of bivalve molluscan shellfish following faecal contamination of growing waters in many countries, including New Zealand. Until recently, qPCR has been the only available approach for norovirus quantitation. However, qPCR has limitations due to the lack of quantifiable reference materials and reliance on standard curves. Digital PCR allows for precise and absolute quantitation of a specific target - in this case norovirus GI or GII - without need for a reference or standard curve. Digital PCR has been reported as being at least comparable to qPCR in terms of sensitivity and tolerance to PCR inhibitors in the matrix. To estimate the concentrations of norovirus in shellfish associated with norovirus outbreaks, several historical shellfish homogenates, prepared using the proteinase K digestion method, were first treated with and without RNases to remove free (non-infectious) viral genome. Following nucleic acid extraction, and the RT step to convert RNA to cDNA, samples were then tested, at least in triplicate, with both qPCR and digital PCR, and results compared. Results showed that based on the DNA plasmid standard curves qPCR alone can overestimate the concentration of norovirus and that concentrations as low as 40 viruses / gram of shellfish may cause an outbreak. The detection and accurate quantitation of norovirus in shellfish may better inform on risk to health health.
Authors
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Joanne Hewitt
(ESR Ltd)
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Dawn Croucher
(ESR Ltd)
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Laetitia Kaas
(ESR Ltd)
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Jeremie Lang
(ESR Ltd)
Topic Areas
Emerging Methods for Virus Identification , New Technology
Session
OS-11 » New Laboratory methods in shellfish safety – Part 2 (09:10 - Wednesday, 17th May, Bailey Allen 1)