Purpose: The purpose of this study was to detect norovirus in the American Oyster (Crassostrea virginica) using two real-time rt-qPCR methods, and compare the virus recovery rates.
Methods: Live oysters from commercial harvesting areas along Louisiana Gulf Coast were obtained on a biweekly basis. About 5 g of digestive diverticula homogenate from 10-12 oysters was digested in PBS (100 µg/ml proteinase-K). The extracted RNA was analyzed using two real-time RT-PCR assays (A and B), established by others. In response to a norovirus outbreak at Cameron Parish, LA (January, 2013) samples from the suspected contaminated area and a stool sample from an infected individual were analyzed. The virus recovery was determined by spiking oyster homogenate with a 20% aliquot of norovirus stock prepared from a GII positive stool specimen (7.5 log (genomic copies)/ml).
Results: No norovirus was detected in any of the oyster samples (n = 8). The stool sample of the infected individual was positive for GII. Analyzing the serial dilution of the stock showed both methods were linear from the range of 2.2 to 3.9 log (genomic copies)/µl of PCR reaction (R2 = 0.989 and 0.997, P > 0.05) with PCR efficiencies of 80% and 92% for methods A and B, respectively. Low and variable recoveries were found for methods A and B (4.4 ± 1.7 and 11.3 ± 2.8 percent, respectively; P < 0.05).
Significance: Low recoveries were obtained that was probably due to matrix effect of the samples, and indicates the need for improving concentration methods for detecting low copy numbers of norovirus in the oysters.