Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

Gibson, Kristen E.; Schwab, Kellogg J.; Spencer, Susan K.; Borchardt, Mark A.

doi:10.1016/j.watres.2012.04.030

Cited by 126 publications

(101 citation statements)

References 47 publications

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“…In some situations it may be adequate to identify sample containing inhibitors from their flat curves or from the complete absence of amplification products [44]. Inhibition makes interpretation of public health risk of enteric virus pathogen difficult, because inhibition can result in underestimating exposure and consequently health risk [5].…”

Section: Detection Of Viruses In Watermentioning

confidence: 99%

“…There is therefore need to have multiple or supplementary indicators which include enteric viruses and if possible to directly monitor the levels of viral pathogens in surface waters, irrigation water, sewage effluent as well as treated drinking water for public health safety and quality assessment [5]. However due to the low concentration of viruses in water matrices and presence of inhibitors, efficient concentration methods from large quantities are essential [5,6].…”

mentioning

confidence: 99%

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Adsorption-Elution Techniques and Molecular Detection of Enteric Viruses from Water

Ruhanya

Kabego

Gichana

2016

JHVRV

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It is generally accepted by municipalities and public health authorities that monitoring microbial water quality is performed by enumerating faecal coliforms. These bacterial indicators of faecal contamination are used because of the assumption that they have the same behaviour in the water as enteric pathogens, which is not true with regards to viruses. Therefore, there is need for a supplementary viral indicator or to directly detect viruses from water. Detection of viruses in water is complicated because of occurrence of viruses in low numbers. There is need for concentration of the viruses from large volumes water to aliquots that can be used in PCR or cell culture. We present the adsorptionelution techniques for the recovery and concentration of enteric viruses from water and subsequent detection by quantitative real time RT-PCR/PCR.

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Section: Detection Of Viruses In Watermentioning

confidence: 99%

mentioning

confidence: 99%

Adsorption-Elution Techniques and Molecular Detection of Enteric Viruses from Water

Ruhanya

Kabego

Gichana

2016

JHVRV

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“…This molecular method allows for sensitive and specific detection and quantification of SaVs (Chan et al 2006) but its application can be hampered by the presence of inhibitory compounds in the sample. Inhibitors are often co-concentrated and extracted with the target nucleic acid and can interfere with the RT and/or PCR amplification processes (Gibson et al 2012). Inhibition of the assay can either result in a shift in the real-time PCR cycle threshold (Ct) value, thus adversely affecting quantification, or inhibit amplification completely.…”

Section: Sapoviruses Have Been Quantitatively Detected and Characterimentioning

confidence: 99%

“…Various ACs have been developed to validate amplification in a RT-qPCR assay (Costafreda et al 2006;Diez-Valcarce et al 2011;Gibson et al 2012) and these can be classified as external amplification controls (EACs) or internal amplification controls (IACs). The EAC approach involves performing two separate reactions for each sample.…”

Section: Sapoviruses Have Been Quantitatively Detected and Characterimentioning

confidence: 99%

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Application of a Competitive Internal Amplification Control for the Detection of Sapoviruses in Wastewater

et al. 2012

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Virus recovery by tangential flow filtration: A model to guide the design of a sample concentration process

Lasareishvili

Shi

Wang

et al. 2020

Biotechnology Progress

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A simple model is developed to describe the instantaneous (r v) and cumulative (R v) recovery of viruses from water during sample concentration by tangential flow filtration in the regime of constant water recovery, r. A figure of merit, M = r v r, is proposed as an aggregate performance metric that captures both the efficiency of virus recovery and the speed of sample concentration. We derive an expression for virus concentration in the sample as a function of filtration time with the rate-normalized virus loss, η = 1− rv r , as a parameter. A practically relevant case is considered when the rate of virus loss is proportional to the permeation-driven mass flux of viruses to the membrane: dm ad dt Q p C f Q p C p. In this scenario, the instantaneous recovery is constant, the cumulative recovery is decreasing as a power function of time, R v = 1− Q p V 0 t η , η mediates the trade-off between r and r v , and M is maximized at r = r opt = 1 2 η. The proposed model can guide the design of the sample concentration process and serve as a framework for quantification and interlaboratory comparison of experimental data on virus recovery.

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Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

Cited by 126 publications

References 47 publications

Adsorption-Elution Techniques and Molecular Detection of Enteric Viruses from Water

Adsorption-Elution Techniques and Molecular Detection of Enteric Viruses from Water

Application of a Competitive Internal Amplification Control for the Detection of Sapoviruses in Wastewater

Virus recovery by tangential flow filtration: A model to guide the design of a sample concentration process

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