EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

Fout, G. Shay; Cashdollar, Jennifer L.

doi:10.3791/52437

Cited by 4 publications

(2 citation statements)

References 20 publications

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“…Fout and Cashdollar (2016) indicated that USEPA Method 1615 may be adapted for the detection of adenoviruses (59). It is recommended to explore modifying existing methods with secondary concentration by PEG precipitation to uphold virus recovery and minimize inactivation during sample processing and detection by culture-based methods.…”

Section: Masclaux Et Al (mentioning

confidence: 99%

Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water

McLellan

Weir

Lee

et al. 2021

Preprint

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Enteric viruses are a leading cause of waterborne illness worldwide and surveillance studies lack standardization in method selection. The most common and cost-effective approach to concentrating viruses from water samples involves virus adsorption and elution (VIRADEL) procedures, followed by secondary concentration. There is a lack of consistency in how secondary concentration methods are practiced and some methods may have better recovery for particular groups of viruses. Secondary concentration methods typically involve precipitation and the most common methods employ organic flocculation (OF) by acidification at a pH of 3.5, or precipitation by polyethylene glycol (PEG) in combination with the addition of NaCl. In this study, the recovery of coliphage MS2 using the plaque assay and human adenovirus strain 41 (HAdV41) using cell-culture and qPCR assays were evaluated by OF and PEG secondary concentration of spiked samples of wastewater, surface water, and groundwater. The recovery of MS2 and HAdV41 by PEG precipitation was significantly higher than that by OF (p<0.0001) when viruses were detected by culture based methods and marginally better when HAdV41 was enumerated by qPCR (p<0.019). The recovery of HAdV41 by qPCR ranged from 75.3% to 94.4% (n=36). The mean recovery of MS2 by OF was 4.4% (0.9%-7.7%; n=14) and ranged from 57.1% to 87.9% (n=28) for the PEG methods. The poor recovery of MS2 by OF was attributed to inactivation or poor stability at acidic conditions as MS2 were not recovered in the supernatant following OF and centrifugation. The inconsistency and lack of justification for method selection in many studies calls for a systematic study to inform guidance and standardization with respect to the application of concentration methods for various water types and viral pathogens.IMPORTANCEMS2 should not be used as a process control for methods involving acidification and culture-based detection. The dense floc produced by the PEG method may have contributed to higher recoveries as the pellet was more compact and stable than the loose pellet formed by OF. Standard methods for the detection of enteric viruses and surrogates that involve acidification could be modified with PEG precipitation to uphold virus recovery and minimize inactivation.

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Section: Masclaux Et Al (mentioning

confidence: 99%

Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water

McLellan

Weir

Lee

et al. 2021

Preprint

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“…The standard molecular methods with which the water industry may be familiar are based on quantitative polymerase chain reaction (qPCR); for example, the detection and quantification of Legionella spp./ L. pneumophila in environmental and recreational waters in the UK [12] or enterovirus, norovirus and enterococci in the USA [13, 14]. It is plausible that qPCR might overcome some of the limitations of microscopy for monitoring drinking water for Cryptosporidium, and capitalize on the presence of four sporozoites in each intact oocyst, increasing the number of targets by fourfold compared to oocyst microscopy.…”

Section: Introductionmentioning

confidence: 99%

A comparison of qPCR and microscopy for the detection and enumeration of Cryptosporidium oocysts from drinking water

Robinson

Elwin

Jones

et al. 2023

Journal of Medical Microbiology

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Introduction. Cryptosporidium presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of Cryptosporidium is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures. Hypothesis. The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR. Aim. We aimed to develop and evaluate a qPCR for the detection and enumeration of Cryptosporidium in drinking water, and to compare the assay with the standard method used in the UK. Methodology. We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for Cryptosporidium genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 Cryptosporidium oocysts in 10 l of artificially contaminated drinking water. Results. The results demonstrated that detection of Cryptosporidium by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy. Conclusions. Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for Cryptosporidium analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.

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Efficiency of Reovirus Concentration from Water with Positively Charged Filters

2017

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EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

Cited by 4 publications

References 20 publications

Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water

Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water

A comparison of qPCR and microscopy for the detection and enumeration of Cryptosporidium oocysts from drinking water

Efficiency of Reovirus Concentration from Water with Positively Charged Filters

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