Foodborne norovirus is a major cause of human disease. Difficulties in culture of Norovirus (NoV) in simple in vitro systems are a major obstacle to studies that could aid in risk management.
The use of cultivable NoV surrogates combined with the reverse transcription real-time polymerase chain reaction (RT-qPCR) as a quantification method has been widely applied in NoV studies. In our preliminary studies, the quantification of MS2 (as NoV surrogate) by normal RT-qPCR assay (without pre-treatment) over-estimated the number of viruses surviving after heat treatment.
This study aimed to improve the RT-qPCR assay as a viral enumeration method by applying an enzymatic pre-treatment that allows the quantification of surviving viable MS2 and to determine whether this improved assay could be applied to viral particle inactivation studies involving heat or chlorination as viral denaturants.
MS2 bacteriophage (MS2) was used as a NoV surrogate. A plaque assay was used to evaluate the performance of RT-qPCR as an enumeration method. RNase alone, and RNase followed by RNasin, or TaqI were used as enzymatic pre-treatments prior to the RNA extraction. The RNA extracted from surviving MS2 heated at 60°C for 120 min. The best pre-treatment was added to the RT-qPCR enumeration protocol in subsequent MS2 inactivation studies involving heat treatment (72°C for 2.5, 5, 10, 20 and 40 min) or chlorination (1, 2, 4, 8 and 16 ppm) for 5 min at 25°C.
The normal RT-qPCR result was comparable to the plaque assay for non-treated MS2 but underestimated MS2 surviving after the heat treatments. RT-qPCR with RNase followed by RNasin (RNase+RNasin) pre-treatment showed better performance than RNase alone or TaqI, and produced results comparable to the plaque assay to quantify surviving MS2 after heat treatment. Hence, an RT-qPCR assay with RNase+RNasin pre-treatment was applied for MS2 inactivation studies. The assay produced results consistent with the plaque assay for both high temperature and chlorine dioxide inactivation treatments, and shows good potential to be applied in inactivation studies of NoV in foods.