Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters

Rodríguez, Roberto; Thie, Lauren; Gibbons, Christopher D.; Sobsey, Mark D.

doi:10.1016/j.jviromet.2012.01.009

Cited by 37 publications

(15 citation statements)

References 30 publications

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“…Additives, such as bovine serum albumin (BSA) and T4 gene 32 protein, have been shown to be effective in reducing 16). Sample purification techniques, such as gel chromatography, cation-exchange resins, and DAX-8 resins, have been reported to be effective (17,18). Further, sample dilution technique has been used to improve detection efficiency (3, 6).…”

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confidence: 99%

Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

Hata

Katayama

Furumai

2015

Appl Environ Microbiol

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bReverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log 10 -unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10-to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances. Human enteric viruses are etiological agents that can cause clinical symptoms, such as diarrhea and vomiting. Feces and vomit from infected individuals contain a substantial amount of viruses that contaminate the water environment. Consumption of improperly treated drinking water or contaminated environmental water during recreational activity leads to waterborne infections (1, 2).Quantitative detection of viruses present in water has been carried out worldwide in studies on the fate of viruses in the environment and potential viral infection risk (3-5). The detection of viruses in a water sample is commonly carried out using a PCR assay following virus concentration and nucleic acid extraction steps due to its superior rapidity, specificity, and sensitivity. However, the reliability of the PCR-based assay occasionally has come into question, as substances present in original samples or additives used during sample processing interfere with the nucleic acid extraction and reverse transcription (RT)-PCR (6-8). Even though several virus concentration methods have been developed, none of them can exclude the inhibitory substances completely (9).Polyvalent cations and some organic substances, such as beef extract constituents and humic acids, are known to inhibit RT-PCR (8, 10). Often, the virus detection efficiency is determined by spiking a known amount of viruses or nucleic acids in a sample and recovering them (6, 7, 11). In the worst case, the concentrations of viruses were underestimated by...

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confidence: 99%

Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

Hata

Katayama

Furumai

2015

Appl Environ Microbiol

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“…Although numerous approaches to remove inhibitors have been developed, no approach is effective for all water matrices and virus types 6,34,35 , making the use of internal controls designed to estimate the level of inhibition essential. The hepatitis G reagent used in this method satisfies this requirement by providing a constant level of viral RNA in all reactions and an RT-qPCR assay for estimating inhibition.…”

Section: Discussionmentioning

confidence: 99%

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

Fout¹,

Cashdollar²,

Griffin³

et al. 2016

JoVE

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EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

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“…In addition to spin protocol in kit and the regular washing steps, glycogen co-precipitation was employed to remove any environmental inhibitors present in the samples while PCR inhibition was assessed by dilution series [29,30]. Finally all extracts were stored at −70°C until use.…”

Section: Methodsmentioning

confidence: 99%

FRNA Bacteriophages as Viral Indicators of Faecal Contamination in Mexican Tropical Aquatic Systems

et al. 2017

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A particular challenge to water safety in populous intertropical regions is the lack of reliable faecal indicators to detect microbiological contamination of water, while the numerical relationships of specific viral indicators remain largely unexplored. The aim of this study was to investigate the numerical relationships of FRNA-bacteriophage genotypes, adenovirus 41, and human adenoviruses (HADV) in Mexican surface water systems to assess sewage contamination. We studied the presence of HADV, HADV41 and FRNA bacteriophage genotypes in water samples and quantified by qPCR and RT-qPCR. Virus and water quality indicator variances, as analyzed by principal component analysis and partial least squared regression, followed along the major percentiles of water faecal enterococci. FRNA bacteriophages adequately deciphered viral and point source water contamination. The strongest correlation for HADV was with FRNA bacteriophage type II, in water samples higher than the 50th percentiles of faecal enterococci, thus indicating urban pollution. FRNA bacteriophage genotypes I and III virus indicator performances were assisted by their associations with electrical conductivity and faecal enterococci. In combination, our methods are useful for inferring water quality degradation caused by sewage contamination. The methods used have potential for determining source contamination in water and, specifically, the presence of enteric viruses where clean and contaminated water have mixed.

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Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters

Cited by 37 publications

References 30 publications

Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

FRNA Bacteriophages as Viral Indicators of Faecal Contamination in Mexican Tropical Aquatic Systems

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