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

Leifels, Mats; Cheng, Dan; Sozzi, Emanuele; Shoults, David C.; Wuertz, Stefan; Mongkolsuk, Skorn; Sirikanchana, Kwanrawee

doi:10.1016/j.wroa.2020.100080

Cited by 55 publications

(48 citation statements)

References 73 publications

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“…A viral viability molecular assay was carried out on heat-inactivated and gamma-irradiated SARS-CoV-2 recovered from plastic, glass and steel surfaces following the swabbing method A (based on ISO 15216:1 standard norm). The viability RT-qPCR assay was assessed using both heat-inactivated and gamma-irradiated SARS-CoV-2 because the mode of viral inactivation may influence the performance of this technique ( Leifels et al, 2020 ; Randazzo et al, 2018 ). Platinum chloride (IV) (PtCl 4 ) was dissolved in DMSO at 1 M and then diluted in water to a final 50 mM ready-to-use stock.…”

Section: Methodsmentioning

confidence: 99%

“…Platinum compounds such as platinum chloride (IV) (PtCl 4 ) present some advantages over other photoactivable viability dyes such as propidium monoazide (PMA) and ethidium monoazide (EMA) ( Elizaquível et al, 2014 ; Puente et al, 2020 ; Randazzo et al, 2018a , 2018b ) as they are not sensitive to visible light and are more affordable from an economic perspective (Karami et al, 2014; Soejima and Iwatsuki, 2016). Moreover, photoactivation of azo dyes may be prevented in complex matrices, such as wastewater and molluscs, due to the turbidity, suspended solid and density of concentrated samples ( Leifels et al, 2020 ; Randazzo et al, 2016 , 2018a ).…”

Section: Introductionmentioning

confidence: 99%

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Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR

Cuevas-Ferrando

Girón-Guzmán

Falcó

et al. 2022

Environmental Research

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The ongoing coronavirus 2019 (COVID-19) pandemic constitutes a concerning global threat to public health and economy. In the midst of this pandemic scenario, the role of environment-to-human COVID-19 spread is still a matter of debate because mixed results have been reported concerning SARS-CoV-2 stability on high-touch surfaces in real-life scenarios. Up to now, no alternative and accessible procedures for cell culture have been applied to evaluate SARS-CoV-2 infectivity on fomites. Several strategies based on viral capsid integrity have latterly been developed using viability markers to selectively remove false-positive qPCR signals resulting from free nucleic acids and damaged viruses. These have finally allowed an estimation of viral infectivity. The present study aims to provide a rapid molecular-based protocol for detection and quantification of viable SARS-CoV-2 from fomites based on the discrimination of non-infectious SARS-CoV-2 particles by platinum chloride (IV) (PtCl 4 ) viability RT-qPCR. An initial assessment compared two different swabbing procedures to recover inactivated SARS-CoV-2 particles from fomites coupled with two RNA extraction methods. Procedures were validated with human (E229) and porcine (PEDV) coronavirus surrogates, and compared with inactivated SARS-CoV-2 suspensions on glass, steel and plastic surfaces. The viability RT-qPCR efficiently removed the PCR amplification signals from heat and gamma-irradiated inactivated SARS-CoV-2 suspensions that had been collected from specified surfaces. This study proposes a rapid viability RT-qPCR that discriminates non-infectious SARS-CoV-2 particles on surfaces thus helping researchers to better understand the risk of contracting COVID-19 through contact with fomites and to develop more efficient epidemiological measures.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR

Cuevas-Ferrando

Girón-Guzmán

Falcó

et al. 2022

Environmental Research

View full text Add to dashboard Cite

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“…Moreover, an assumption of infectious ratio values applied to the monitored DNA measurements (e.g., by molecular qPCR assays) contributes another uncertainty factor to the concentrations of infectious viruses to inform QMRA. Viral infectivity measurements, including cell culture and qPCR with azo-dye pretreatment methods (Ko et al, 2003;Leifels et al, 2019Leifels et al, , 2021, could directly provide infectious concentrations of viruses for risk analysis. The differential decay characteristics of the HAdV40/41 and MST markers (i.e., crAssphage and HPyVs) in receiving water may also result in under-or over-estimation of the yearly infection risk (Greaves et al, 2020;McMinn et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Human Fecal Pollution Monitoring and Microbial Risk Assessment for Water Reuse Potential in a Coastal Industrial–Residential Mixed-Use Watershed

et al. 2021

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Rapid economic development has caused industrial expansion into residential communities, leading to higher fecal pollution loads that could be discharged into aquatic environments. However, little is known regarding the potential microbial impact on human health. This study investigated microbial contamination from coastal industrial–residential community areas in nine sampling sites in waterways during three dry events. A general microbial source tracking (MST) marker, GenBac3, was detected in all samples from all three events, indicating continuing fecal pollution in the area, mostly from human sewage contamination. This was shown by the human-specific genetic marker crAssphage (88.9%) and human polyomavirus (HPyVs; 92.6%) detection. Enteric human adenovirus (HAdV40/41) showed three positive results only from residential sites in the first event. No spatial difference was observed for MST markers and traditional fecal indicators (total coliforms and Escherichia coli) in each event. Still, a significantly lower abundance of GenBac3, HPyVs, and total coliforms in the first sampling event was detected. Spearman’s rho analysis indicated a strong correlation among certain pairs of microbial parameters. Multivariate analysis revealed two clusters of samples separated by land use type (industrial vs. residential). According to factor analysis of mixed data, the land use parameter was more associated with physicochemical parameters (i.e., salinity, conductivity, water temperature, and dissolved oxygen). A Quantitative Microbial Risk Assessment (QMRA) was then conducted to estimate the annual infection risks of HAdV40/41 for non-potable water reuse purposes using predicted concentrations from crAssphage and HPyVs. The highest risks (95th percentiles) were ranked by food crop irrigation, aquaculture, and toilet flushing, at 10–1, 10–2, and 10–3 per person per year (pppy). Required treatment levels to achieve a 10–4 pppy annual infection risk were estimated. QMRA-based water treatment scenarios were suggested, including chlorination for toilet flushing reuse and depth filtration prior to chlorination for aquaculture and food crop irrigation. Microbial monitoring combined with a QMRA could provide better insights into fecal pollution patterns and the associated risks, facilitating effective water quality management and appropriate prior treatments for water reuse.

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“…To overcome the limitations of infectivity assays, modified RT-qPCR assays have been developed for specific quantification of infectious virions, for example, by pretreatment with intercalating dyes. Intercalating dyes pass through damaged viral protein capsids (corresponding to inactivated virions), where they bind to nucleic acid and inhibit amplification, but cannot pass through intact capsids (infectious virions), allowing amplification to continue [44,45]. Inactivation methods that do not cause capsid damage are not accounted for (e.g.…”

Section: Quantification Of Coronaviruses From Environmental Samplesmentioning

confidence: 99%

Porous surfaces: stability and recovery of coronaviruses

et al. 2021

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The role of indirect contact in the transmission of SARS-CoV-2 is not clear. SARS-CoV-2 persists on dry surfaces for hours to days; published studies have largely focused on hard surfaces with less research being conducted on different porous surfaces, such as textiles. Understanding the potential risks of indirect transmission of COVID-19 is useful for settings where there is close contact with textiles, including healthcare, manufacturing and retail environments. This article aims to review current research on porous surfaces in relation to their potential as fomites of coronaviruses compared to non-porous surfaces. Current methodologies for assessing the stability and recovery of coronaviruses from surfaces are also explored. Coronaviruses are often less stable on porous surfaces than non-porous surfaces, for example, SARS-CoV-2 persists for 0.5 h–5 days on paper and 3–21 days on plastic; however, stability is dependent on the type of surface. In particular, the surface properties of textiles differ widely depending on their construction, leading to variation in the stability of coronaviruses, with longer persistence on more hydrophobic materials such as polyester (1–3 days) compared to highly absorbent cotton (2 h–4 days). These findings should be considered where there is close contact with potentially contaminated textiles.

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

Cited by 55 publications

References 73 publications

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR

Human Fecal Pollution Monitoring and Microbial Risk Assessment for Water Reuse Potential in a Coastal Industrial–Residential Mixed-Use Watershed

Porous surfaces: stability and recovery of coronaviruses

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