Purpose: Our goal was to develop a sensitive NoV detection method for fresh produce, hands, and agricultural water that identifies sample detection inhibition.
Methods: From farms and packing facilities, we rinsed cantaloupes, jalapeños, tomatoes, and hands in a 1% peptone solution and collected agricultural water. One of each sample was spiked with NoV GI.1 or GII.2 (n=10) to optimize NoV concentration and extraction, RT-qPCR amplification, and RT-qPCR amplification inhibition analysis (using an internal amplification control [IAC]). Significant comparisons were assessed by Student’s t-test.
Results: The addition of an elution step (0.15N NaCl and pH 9.0), prior to NoV concentration, improved NoV amplification - likely by promoting NoV dissociation from rinsate filth. We found no significant difference (P=0.3) in NoV yield between two polyethylene glycol (PEG) concentrations (12 and 16 g/100 ml). Though bovine serum albumin (BSA) was essential to NoV detection during PEG concentration of low turbidity samples, there was no significant difference in yield (P=0.48) between 1, 2, 3, or 4% BSA. Further, a secondary, compared to a single, PEG concentration step improved the effective volume tested by 30-50 fold. In optimizing NoV amplification, RT-qPCR primer concentrations (GI: 1200 nM forward/ 800 nM reverse; GII: 400 nM forward/ 400 nM reverse), compared to control (25 nM all primers), improved the NoV yield by 2 CTs (P=0.01). Application of an optimized IAC (GI and GII: 1 pg/µl) to 50 agricultural samples resulted in the development of an algorithm to interpret preliminary RT-qPCR results (presence/absence; confirmed/suspected).
Significance: This method can be used to identify the NoV prevalence on produce, hands and agricultural water having low virus concentrations and matrix inhibitors.