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Enzymatic and viability RT ‐ qPCR assays for evaluation of enterovirus, hepatitis A virus and norovirus inactivation: Implications for public health risk assessment
Author(s) -
Monteiro S.,
Santos R.
Publication year - 2018
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13568
Subject(s) - enterovirus , norovirus , virology , viability assay , infectivity , biology , murine norovirus , rnase p , microbiology and biotechnology , real time polymerase chain reaction , virus , cell culture , rna , biochemistry , gene , genetics
Aim To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR ( RT ‐ qPCR ) pretreatment to discriminate between infectious and noninfectious enteric viruses. Methods and Results Enterovirus (EntV), norovirus (NoV) GII .4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT ‐ qPCR alone, (iii) RT ‐ qPCR assay preceded by RN ase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT ‐ qPCR . In addition, heat‐inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD‐ RT ‐ qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RN ase. RD‐ RT ‐ qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT ‐ qPCR , with the exception of Triton X‐100. Conclusion The use of surfactant/RD‐ RT ‐ qPCR , although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. Significance and Impact of the Study Surfactant/RD‐ RT ‐ qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses.