Molecular detection of infectious human norovirus
Afolayan, Olamide Tolulope
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Human norovirus (HuNoV) is responsible for an estimated 5.5 million illness per year in the U.S., which is about 58% of all foodborne illnesses attributed to known pathogens (Scallan et al., 2011b). The most common means of detection of HuNoV is through the use of RT-PCR, which signifies presence of the viral genomic material in a tested sample, but does not distinguish between infectious and non-infectious HuNoV. The aim of this study was to determine how sample properties such as virus titer, strain, suspension matrix, and inactivation methods influence the reliability of potential methods for detecting infectious HuNoV. Two possible methods of detecting infectious HuNoV; enzymatic pre-treatment with Proteinase K and RNase A, and the use of porcine gastric mucin (PGM) binding, each method prior to RT-PCR, were evaluated against these sample properties. The result of the enzymatic pre-treatment study showed that virus titer and suspension matrix impacted estimation of infectious HuNoV and observations also varied from one viral strain to another. Compared to plaque assay in a cultivable surrogate (MNV-1), there was no correlation in estimation of infectivity; while plaque assay estimated up to 6 log reduction in infectivity after heat treatment, the enzyme pre-treatment method estimated a maximum of 2.94 log reduction in viral RNA. Evaluation of the PGM-binding method shows variation in estimation of infectivity across inactivation methods and viral strains. By subjecting virus samples to PGM-binding prior to RT-PCR, complete elimination of RT-PCR signals was observed in virus samples treated to a lev/SDS (0.5% levulinic acid + 0.1% sodium dodecyl sulphate) sanitizer, while positive RT-PCR signals were observed in virus samples heat treated at 99oC for 1 min. Non-specific binding of virus samples to testing surfaces was also observed, a variable not considered or evaluated in previous studies. The impact of food matrix and RNA extraction methods on RT-qPCR inhibition and norovirus recovery was also investigated. This study suggests that the efficiency of each RNA extraction method in reducing PCR inhibition varied with the sample matrix being tested in GII.4 samples (p<0.0001), but sample matrix was not a significant (p=0.1675) contributor in GI.1 signal inhibition.