Purpose: To develop a DNA hybridization method to confirm presumptively positive RT-qPCR results obtained from environmental samples screened for evidence of human norovirus (HuNoV) contamination.
Methods: RT-PCR (traditional and quantitative) was applied to RNA extracted using a silica-based guanadinium isothionate method, followed by amplification using primers JJV2F and COG2R targeting the ORF1-ORF2 junction (region B) of the HuNoV genome. Probe Ring2P was used for qRT-PCR (45 cycles) and hybridization. After optimization of the hybridization, serial dilutions of stool samples positive for HuNoV were subjected to RT-qPCR and traditional RT-PCR followed by dot blot hybridization to compare assay detection limits. Both methods were applied to a subsample of 20 environmental samples collected from daycare facilities in the Carolinas previously tested for evidence of HuNoV contamination using RT-qPCR.
Results: As applied to serially diluted HuNoV-positive stool, RT-qPCR Ct values ranged from a low of 22 to a high of 38. Detection limit of the RT-qPCR method was identical to that of RT-PCR followed by hybridization. When both methods were applied to environmental samples all 10 samples previously screened negative for HuNoV by RT-qPCR were also negative by hybridization. However, 10 samples previously classified as presumptively positive for HuNoV based on Ct values ranging from 36-44 were in fact negative by hybridization.
Significance: The inability to confirm samples in the Ct range of 35-45 suggests cautious interpretation of RT-qPCR results and further highlights the need for confirmation. DNA hybridization is a viable method to confirm the status of such samples when sequencing is not possible.