Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring

Crain, Chad; Kezer, Keith; Steele, S.; Owiti, Judith; Rao, Sphoorthy; Victorio, Maria; Austin, Brett; Volner, Alon; Draper, William M.; Griffith, John F.; Steele, Joshua A.; Seifert, Marva

doi:10.1016/j.mimet.2021.106206

Cited by 5 publications

(6 citation statements)

References 26 publications

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“…2 and Supplementary Table ST1 summarize the published comparisons of the analytical performance of dPCR and qPCR for health-related water microbiology. Many earlier studies have reported that dPCR and qPCR measurements harmonize well, and both can be reliably used for monitoring various microorganisms in water (Rothrock et al, 2013;Cao et al, 2015;Cao et al, 2016;Verhaegen et al, 2016;Singh et al, 2017;Ibekwe et al, 2020;Crain et al, 2021). However, qPCR and dPCR methods can have considerable differences in performance characteristics, as the enumeration approaches between these methods are fundamentally different (Table 2).…”

Section: Qpcr and Dpcr Comparisonsmentioning

confidence: 97%

“…Many earlier studies used dPCR and qPCR in parallel to measure the known concentration of Legionella pneumophila in seeded drinking water (Falzone et al, 2020), human norovirus, and human adenovirus in blackwater and greywater (Jahne et al, 2020), human rotavirus in different types of surface water (Rački et al, 2014a), human adenoviruses in river water (Kishida et al, 2014), Naegleria fowleri in river water (Xue et al, 2018), Cyanobacteria in river water (Te et al, 2015), Vibrio parahaemolyticus in river water (Lei et al, 2020), Shiga toxin-producing E. coli in bovine feces (Verhaegen et al, 2016;Singh et al, 2017), E. coli O157 and Listeria monocytogenes in drinking water (Bian et al, 2015), Enterococcus spp. and sewage-associated marker gene Bacteroides HF183 in bathing water (Cao et al, 2015;Wang et al, 2016;Crain et al, 2021), Cryptosporidium oocysts in sheep, cattle, and humans fecal samples (Yang et al, 2014), SARS-CoV-2 RNA in wastewater (Ahmed et al, 2022b;Ahmed et al, 2022c;Ciesielski et al, 2021;Flood et al, 2021;Dumke et al, 2021). Each of these studies reported greater analytical sensitivity of dPCR compared to qPCR.…”

Section: Limit Of Detection and Quantificationmentioning

confidence: 99%

“…The published literature indicates that during such applications, well-optimized dPCR assays are highly sensitive and resilient to inhibition and can provide improved analytical performance compared to qPCR for the typical user. For example, after laboratory validation, the San Diego County Department of Environmental Health sought the approval of ddPCR for regulatory monitoring of FIB in recreational waters in California, USA (Crain et al, 2021). The United States Environmental Protection Agency has also approved ddPCR for enumerating Enterococcus genome copies for regulatory monitoring of recreational beaches along with current reference methods (USEPA, 2020).…”

Section: Limitations Of Dpcrmentioning

confidence: 99%

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Application of digital PCR for public health-related water quality monitoring

Tiwari

Ahmed

Oikarinen

et al. 2022

Science of The Total Environment

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Section: Qpcr and Dpcr Comparisonsmentioning

confidence: 97%

Section: Limit Of Detection and Quantificationmentioning

confidence: 99%

Section: Limitations Of Dpcrmentioning

confidence: 99%

See 1 more Smart Citation

Application of digital PCR for public health-related water quality monitoring

Tiwari

Ahmed

Oikarinen

et al. 2022

Science of The Total Environment

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“…While a national database has been established for SARS-CoV-2 WBE in the United States (CDC-NWSS), there remains no standardized protocol, and laboratories are often limited by the equipment already available to them. Interlaboratory comparisons have shown one or more orders of magnitude differences in results reported by groups using different methods (3)(4)(5). While much work has focused on the comparison of viral concentration and RNA extraction methods from wastewater, there is limited data to specifically address differences introduced by the choice of quantification platform.…”

Section: Introductionmentioning

confidence: 99%

“…With the increased adoption of digital PCR platforms, there have been several extensive comparisons of ddPCR and qPCR applied for sensitive quantification of microorganisms in the environment (3)(4)(5). As SARS-CoV-2 WBE developed, many researchers performed methodological comparisons that included side-by-side tests of ddPCR and qPCR for SARS-CoV-2 gene targets (7)(8)(9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

Comparison of RT-qPCR and Digital PCR Methods for Wastewater-Based Testing of SARS-CoV-2

Hinkle

Greenwald

Metzger

et al. 2022

Preprint

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Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular targets in populations, using wastewater as a pooled sample. We compared the sensitivity, susceptibility to inhibition, and quantification of reverse transcription quantitative PCR (RT-qPCR), microfluidic well digital RT-PCR (RT-dPCR), and droplet digital RT-PCR (RT-ddPCR) measurements of SARS-CoV-2 (N1 gene target) and Pepper Mild Mottle Virus (PMMoV) RNA in 40 wastewater RNA extracts. All three methods were highly sensitive, but appeared less accurate at very low concentrations. Lower inhibition was observed for RT-ddPCR than RT-qPCR with both SARS-CoV-2 and PMMoV targets, but inhibition appeared to be mitigated by dilution of template RNA. The concentrations of N1 and PMMoV from all three methods were significantly correlated (Pearson's r=0.97-0.98 for N1 and r=0.89-0.93 for PMMoV), although RT-qPCR reported higher concentrations than digital methods. Taken together, this study provides support for the application of all three methods in wastewater-based epidemiology, with additional guidelines for the use of RT-qPCR.

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Seems fishy: environmental DNA impacts on sketa22 quality control in salmonidae dominated waterbodies using qPCR and ddPCR

Hart,

Tardani,

Ruetz

et al. 2023

Environ. Res. Commun.

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Globally, water resources used for recreation and drinking water are threatened by fecal pollution. These pollutants can cause gastrointestinal illness and environmental degradation. Additionally, most sources of fecal pollution are non-point sources stemming from multiple species. Identifying these sources is vital to categorizing the exposure risk from contact and improving remediation efforts. A common technique to provide species-specific information for fecal source identification is microbial source tracking (MST). MST quantifies DNA of host or host-associated microorganisms through polymerase chain reaction (PCR) technologies such as quantitative PCR (qPCR) or droplet digital PCR (ddPCR). MST techniques have been implemented globally and are used for routine monitoring. In the United States (US), the US Environmental Protection Agency has provided several approved standard PCR methods for MST and other recreational water quality applications. These methods have specified quality controls including sample processing controls (SPC) and assessments for sample inhibition. A standard SPC used in EPA methods involves spiking samples with salmon testes DNA (nominally originating from Chum Salmon, Oncorhynchus keta and quantifying them using Sketa22, a genus specific TaqManTM assay). This quality control (QC) behaves similarly to the microbial species being monitored. MST testing in Fall 2022 indicated elevated Sketa22 recoveries and re-analysis of samples indicated the detection of external Salmonidae DNA on both qPCR and ddPCR platforms. Our research was designed to identify the cause of this interference. Results indicate that the primer probe set may react with wild Salmonidae DNA. Analyzing the Sketa22 sequence using BLAST indicated matches with many species of Salmonidae present in the sampled stream system. Consequently, further research is required to identify the effectiveness of Sketa22 as a QC when native and migratory Salmonidae are present. General recommendations are provided to account for excess ambient Salmonidae DNA.

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Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring

Cited by 5 publications

References 26 publications

Application of digital PCR for public health-related water quality monitoring

Application of digital PCR for public health-related water quality monitoring

Comparison of RT-qPCR and Digital PCR Methods for Wastewater-Based Testing of SARS-CoV-2

Seems fishy: environmental DNA impacts on sketa22 quality control in salmonidae dominated waterbodies using qPCR and ddPCR

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