Variably improved microbial source tracking with digital droplet PCR

Nshimyimana, Jean Pierre; Cruz, Mercedes Cecilia; Wuertz, Stefan; Thompson, Janelle R.

doi:10.1016/j.watres.2019.04.056

Cited by 22 publications

(25 citation statements)

References 51 publications

(72 reference statements)

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“…Overall, both ddPCR and qPCR measurements have been shown to be highly correlated in many different studies, consistent with high experimental reproducibility and repeatability for both [110]. In one study, ddPCR measurements showed better reproducibility for quantification in fecal composites, while qPCR showed a higher reproducibility for environmental and composite samples [69]. In another study, ddPCR achieved a higher reproducibility for specific species such as P. falciparum, while it showed similar sensitivity to qPCR in P. vivax [111].…”

Section: Reproducibilitysupporting

confidence: 60%

“…Some research showed that ddPCR may have better LoD but has a limitation in detecting a high number of target genes, and both qPCR and ddPCR showed a similar coefficient of determination of standards in the gyrB gene [68]. Other research with bacterial genetic markers stated that while ddPCR shows better reproducibility for marker detection in fecal composites, qPCR shows a higher sensitivity for markers with environmental and composite samples with less than 10% sensitivity difference [69]. Nevertheless, both methods rely on target amplification, so certain genetic targets may work better in one or the other technology, and marker optimization is critical to both technologies.…”

Section: Comparison Of Qpcr and Ddpcr Methods And Their Applicationsmentioning

confidence: 99%

“…The cost-effectiveness of ddPCR is one of its main concerns [41,69]. It is reported that ddPCR costs two times as much as qPCR [53], and its lesser availability has prevented distribution of ddPCR technology in developing countries since its introduction in 2011 [41].…”

Section: Costmentioning

confidence: 99%

See 2 more Smart Citations

Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Park

Rana

Sung

et al. 2021

Applied Microbiology

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With fast-growing polymerase chain reaction (PCR) technologies and various application methods, the technique has benefited science and medical fields. While having strengths and limitations on each technology, there are not many studies comparing the efficiency and specificity of PCR technologies. The objective of this review is to summarize a large amount of scattered information on PCR technologies focused on the two majorly used technologies: qPCR (quantitative polymerase chain reaction) and ddPCR (droplet-digital polymerase chain reaction). Here we analyze and compare the two methods for (1) efficiency, (2) range of detection and limitations under different disciplines and gene targets, (3) optimization, and (4) status on antibiotic resistance genes (ARGs) analysis. It has been identified that the range of detection and quantification limit varies depending on the PCR method and the type of sample. Careful optimization of target gene analysis is essential for building robust analysis for both qPCR and ddPCR. In our era where mutation of genes may lead to a pandemic of viral infectious disease or antibiotic resistance-induced health threats, this study hopes to set guidelines for meticulous detection, quantification, and analysis to help future prevention and protection of global health, the economy, and ecosystems.

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Section: Reproducibilitysupporting

confidence: 60%

Section: Comparison Of Qpcr and Ddpcr Methods And Their Applicationsmentioning

confidence: 99%

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Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Park

Rana

Sung

et al. 2021

Applied Microbiology

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“…Complete abatement of qPCR inhibition is likely unrealistic; nevertheless, recent efforts to standardize qPCR procedures for water quality assessment suggest that a set of existing mitigation practices is sufficient to render matrix interference a manageable nuisance in most applications [81, 144, 149•]. Increasing adoption of digital PCR (dPCR), a quantitative PCR approach robust to inhibition, will likely further alleviate the challenge of inhibition for routine molecular detection of fecal microbes [61,114,128,[150][151][152].…”

Section: Analytical Sensitivitymentioning

confidence: 99%

Microbial Indicators of Fecal Pollution: Recent Progress and Challenges in Assessing Water Quality

Holcomb

Stewart

2020

Curr Envir Health Rpt

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Purpose of Review Fecal contamination of water is a major public health concern. This review summarizes recent developments and advancements in water quality indicators of fecal contamination. Recent Findings This review highlights a number of trends. First, fecal indicators continue to be a valuable tool to assess water quality and have expanded to include indicators able to detect sources of fecal contamination in water. Second, molecular methods, particularly PCR-based methods, have advanced considerably in their selected targets and rigor, but have added complexity that may prohibit adoption for routine monitoring activities at this time. Third, risk modeling is beginning to better connect indicators and human health risks, with the accuracy of assessments currently tied to the timing and conditions where risk is measured. Summary Research has advanced although challenges remain for the effective use of both traditional and alternative fecal indicators for risk characterization, source attribution and apportionment, and impact evaluation.

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“…The result presented strong evidence that our method displayed high sensitivity and specificity. Different from other ddPCR detection which had narrow range (Wang et al, 2018;Zhong et al, 2018;Jahne et al, 2019;Nshimyimana et al, 2019), our method presented a wider detection range from 3.9 × 10 7 to 3.9 × 10 1 CFU/mL. Owing to approximate 80 thousand droplets produced, a narrowed detection range of ddPCR was circumvented.…”

Section: Discussionmentioning

confidence: 95%

A 4-plex Droplet Digital PCR Method for Simultaneous Quantification and Differentiation of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Based on Single Intact Cells

Lei

Wang

et al. 2020

Front. Microbiol.

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Vibrio parahaemolyticus is a significant seafood-borne pathogen, leading to serious acute gastrointestinal diseases worldwide. In this study, a reliable 4-plex droplet digital PCR (ddPCR) was successfully established and evaluated for the simultaneous detection of V. parahaemolyticus based on tlh, tdh, ureR, and orf8 in food samples using single intact cells. The targets tlh and ureR were labeled with 6-Carboxyfluorescein (FAM), and the targets tdh and orf8 were labeled with 5'-Hexachlorofluorescein (HEX). Due to reasonable proration of primers and probes corresponding into the two fluorescence channels of the ddPCR detecting platforms, the clearly separated 16 (2 4) clusters based on fluorescence amplitude were obtained. For better results, the sample hot lysis time and the cycle number were optimized. The results showed that the minimum number of "rain" and maximum fluorescence amplification were presented for precise detection in the condition of 25 min of the sample hot lysis time and 55 cycles. The sensitivity of this 4-plex ddPCR assay was 39 CFU/mL, which was in accordance with that of the conventional plate counting and was 10-fold sensitive than that of qPCR. In conclusion, the 4-plex ddPCR assay presented in this paper was a rapid, specific, sensitive, and accurate tool for the detection of V. parahaemolyticus including pandemic group strains and could be applied in the differentiation of V. parahaemolyticus in a wide variety of samples.

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Variably improved microbial source tracking with digital droplet PCR

Cited by 22 publications

References 51 publications

Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Microbial Indicators of Fecal Pollution: Recent Progress and Challenges in Assessing Water Quality

A 4-plex Droplet Digital PCR Method for Simultaneous Quantification and Differentiation of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Based on Single Intact Cells

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