The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity.

Autor: Stoppel SM; Institute of Marine Research, Section for Seafood Hazards, Nordnesgaten 50, Bergen 5005, Norway. Electronic address: sarah.stoppel@hi.no., Lunestad BT; Institute of Marine Research, Section for Seafood Hazards, Nordnesgaten 50, Bergen 5005, Norway., Myrmel M; Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Elizabeth Stephansens vei 15, Ås 1430, Norway.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2024 Jun; Vol. 327, pp. 114919. Date of Electronic Publication: 2024 Mar 24.
DOI: 10.1016/j.jviromet.2024.114919
Abstrakt: Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID 50 ). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID 50 reductions of 0.3, 4.4 and 5.9 log 10 were observed. UV exposure (40/100/1000 mJ/cm 2 ) resulted in 1.1, 2.5 and 5.9 log 10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log 10 . After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID 50 . On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log 10 from TCID 50 . For UV light, long-range PCR was closest to TCID 50 results. Long-range reductions deviated from TCID 50 by ≤0.1 log 10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log 10 from TCID 50 . After chlorination the molecular methods repeatedly deviated from TCID 50 by >1.0 log 10 , Overall, each method needs to be further optimized for the individual types of inactivation treatment.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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