Detection and Quantification of Noroviruses in Shellfish

Abstract

Noroviruses (NoVs) are the most common viral agents of acute gastroenteritis in humans, and high concentrations of NoVs are discharged into the environment. As these viruses are very resistant to inactivation, the sanitary consequences are contamination of food, including molluscan shellfish. There are four major problems with NoV detection in shellfish samples: low levels of virus contamination, the difficulty of efficient virus extraction, the presence of interfering substances that inhibit molecular detection, and NoV genetic variability. The aims of this study were to adapt a kit for use with a method previously shown to be efficient for detection of NoV in shellfish and to use a one step real-time reverse transcription-PCR method with addition of an external viral control. Comparisons of the two methods using bioaccumulated oysters showed that the methods reproducibly detected similar levels of virus in oyster samples. Validation studies using naturally contaminated samples also showed that there was a good correlation between the results of the two methods, and the variability was more attributable to the level of sample contamination. Magnetic silica very efficiently eliminated inhibitors, and use of extraction and amplification controls increased quality assurance. These controls increased the confidence in estimates of NoV concentrations in shellfish samples and strongly supported the conclusion that the results of the method described here reflected the levels of virus contamination in oysters. This approach is important for food safety and is under evaluationfor European regulation.

Comprehensive Analysis of a Norovirus-Associated Gastroenteritis Outbreak, from the Environment to the Consumer

“…The direct detection of viral human pathogens has become the most reliable manner for documenting viral contamination of shellfish, and sensitive methods are now available (4,7,23). Since the development of molecular methods, the inclusion of quality controls has become a priority and has been facilitated by the use of real-time RT-PCR, which allows sample-to-sample comparisons.…”

“…Evaluation for the presence of RT-PCR inhibitors was performed by the coamplification of 2.5 l of each NA extract with 2.5 l containing 100 copies of GI or GII RNA internal controls in separate experiments (23). The amplification of RNA indicated that no more than partial inhibition was present; no adjustments to the quantitative estimates were made for samples with partial inhibition.…”

“…To be included in the quantitative analysis, all wells had to yield a C T value of Յ41. The final concentration was then determined on the basis of the NA volume analyzed (5 l of 100 l of NA extract) and the measured weight of the DT (1.5 g was analyzed) (23).…”

“…The efficiency of the virus extraction procedures was determined for each extraction by seeding 10 4 50% tissue culture-infective doses of mengovirus prior to sample processing and determining the amount of mengovirus recovered by rRT-PCR, as described previously (7,23). The NoV concentrations were then corrected for virus loss during extraction by dividing the final norovirus concentration (uncorrected) by the mean mengovirus extraction efficiency.…”

“…Virus was then concentrated by polyethylene glycol 6000 (Sigma, St. Quentin, France) precipitation (4). Viral NAs were extracted with a Nuclisens kit (bioMérieux, France), suspended in 100 l of elution buffer with 20 U of RNase inhibitor (Invitrogen, France), and analyzed immediately or kept frozen at Ϫ80°C (23).…”