Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications – A systematic review

• Published in: Water research X Vol. 11; p. 100080
• Main Authors: Leifels, Mats, Cheng, Dan, Sozzi, Emanuele, Shoults, David C., Wuertz, Stefan, Mongkolsuk, Skorn, Sirikanchana, Kwanrawee
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.05.2021; Elsevier
• Subjects: azo dye; EMA; Microbial contamination; PMA; virus infectivity; Water quality; EMA; azo dye; PMA; virus infectivity; Microbial contamination; Water quality
• ISSN: 2589-9147; 2589-9147
• DOI: 10.1016/j.wroa.2020.100080

Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009–2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 μM; incubation on ice or at room temperature (20 - 25 °C) for 5–120 min; and dye activation using LED or high light (500–800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever. [Display omitted] •PMA/PMAxx have higher efficiency removing false negatives from qPCR for DNA/RNA viruses than EMA.•One size fits all pretreatment approaches are possible but lead to lower virus signal reduction.•Capsid integrity qPCR is a valuable tool to adapt existent workflows for improving risk assessment.•Azo dye pretreatment can help refine significance of qPCR during virus outbreaks.