Use of bentonite-coated activated carbon for improving the sensitivity of RT-qPCR detection of norovirus from vegetables and fruits: The ISO 15216-1:2017 standard method extension

Authors: TANG, MENGXUAN - Jiangxi Agricultural University; LIAO, NINBAO - Jiangxi Agricultural University; Tian, Peng; SHEN, KAISHENG - Jiangxi Agricultural University; LIU, CHENGWEI - Jiangxi Agricultural University; RUAN, LU - Jiangxi Agricultural University; WU, GUOPING - Jiangxi Agricultural University

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/2022
Publication Date: 10/18/2022
Citation: Tang, M., Liao, N., Tian, P., Shen, K., Liu, C., Ruan, L., Wu, G. 2022. Use of bentonite-coated activated carbon for improving the sensitivity of RT-qPCR detection of norovirus from vegetables and fruits: The ISO 15216-1:2017 standard method extension . Food Microbiology. 110. Article 104165. https://doi.org/10.1016/j.fm.2022.104165.
DOI: https://doi.org/10.1016/j.fm.2022.104165

Interpretive Summary: Fruit and vegetable-related human norovirus (HuNoV) outbreaks are becoming prevalent worldwide. Detection of HuNoV in food samples has been a challenge due to the assay inhibitors present in washing from fruits and vegetables. In collaboration with scientists at Jiangxi Agricultural University, we developed a rapid and sensitive method to remove the inhibitors present in washing from testing fruits and vegetables. Bentonite coated activated carbon (BCA) exhibited a high degree of porosity and an extended surface area to adsorb the assay inhibitors. After optimization the conditions of BCAC used to remove the inhibitors, this method was applied for detection of surrogates of HuNoV as wells as surrogates (MS2, Murine norovirus-1, Tulane virus) spiked in fruits and vegetables. The recovery rates of surrogates were significantly higher than those without BCAC treatment, and the average detection limits of these viruses were reduced by approximately 10 to 100 folds. The recovery of HuNoV in in lettuce and cherry tomatoes was four and seven times higher than that without BCAC, respectively. Therefore, BCAC treatment is a rapid, sensitive method to remove inhibitors and has the potential to be used to detect low numbers of food-borne viruses in fruits and vegetables.

Technical Abstract: Produce-related foodborne outbreaks are becoming increasingly prevalent worldwide. In plant tissues, various compounds, including polysaccharides, phenolic compounds, and chlorophyll, can inhibit RT-PCR detection of viruses. In this study, we developed a highly sensitive RT-qPCR in combination with the bentonite-coated activated carbon (BCAC) assay for detection of norovirus from fruits and vegetables, which could be completed within 7 h and was about 10–100 fold more sensitive than the standard procedures (ISO 15216-1:2017). The extraction efficiencies of three surrogate viruses (MS2, MNV-1, and TV) from five fresh produce (lettuce, cherry tomato, blueberry, strawberry, and spinach) were higher with BCAC treatment than those of control groups, ranging from 17.82% to 98.60%. The average detection limit of these viruses using the BCAC-RTqPCR method was stable at an average of 102 PFU/g or GC/g. Finally, this BCAC-RT-qPCR method was applied for detection of human norovirus GII.4 spiked onto lettuce and cherry tomato. The viral extraction efficiencies were up to 53.43% and 95.56%, respectively, which is almost four and seven times better than those without BCAC. Therefore, the BCAC-RT-qPCR method can be used to detect low levels of foodborne viruses from produce.