Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens

Sun, Angela; Stanton, Jo‐Ann L.; Bergquist, Peter L.; Sunna, Anwar

doi:10.3390/microorganisms9112367

Cited by 4 publications

(4 citation statements)

References 55 publications

(60 reference statements)

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“…The same protocol, however, revealed significantly less diversity following in situ testing, where the Qiagen protocol showed much higher fish detection (7.4 times higher). A similar effect was reported by Sun et al (2021). In that study, qPCR variability following DNA extraction by PDQeX was initially evident but optimization using an acid wash removed the effect and resulted in over 90% recovery rates.…”

Section: Discussionsupporting

confidence: 90%

“…In that study, qPCR variability following DNA extraction by PDQeX was initially evident but optimization using an acid wash removed the effect and resulted in over 90% recovery rates. Sun et al (2021) also showed that capture substrate has a significant impact on DNA recovery with polycarbonate filters outperforming nitrocellulose filters. These findings point to the PDQeX system requiring further optimization before it could be used reliably for in situ passive filtration methods to monitor marine biomes.…”

Section: Discussionmentioning

confidence: 98%

“…Sample dropout could be addressed by optimizing eDNA enrichment through longer lysis periods or by increasing the thermophilic proteinases and mesophilic cell wall degrading enzymes or including other enhancing reagents, such as acid treatment (Sun et al, 2021). Additionally, the choice of the most efficient passive sampler, for example, one that binds/absorbs the highest DNA quantity but releases it easily during the extraction reaction (see Sun et al, 2021), may achieve higher DNA yields. The ability to extract nucleic acids in a fast and efficient manner while in the field is valuable as in‐field extractions may help reduce the risk of DNA degradation during storage (Bowers et al, 2021) and will aid the acquisition of near real‐time data using point‐of‐need detection technologies such as desktop PCR methods, Loop‐mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), or similar approaches (Bowers et al, 2021; Zirngibl et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…Significantly, target taxa were partially detected using PDQeX even in our field trial, suggesting that with further optimization to improve DNA yields from the PDQeX extractions, detection rates might improve. Sample dropout could be addressed by optimizing eDNA enrichment through longer lysis periods or by increasing the thermophilic proteinases and mesophilic cell wall degrading enzymes or including other enhancing reagents, such as acid treatment (Sun et al, 2021). Additionally, the choice of the most efficient passive sampler, for example, one that binds/absorbs the highest DNA quantity but releases it easily during the extraction reaction (see Sun et al, 2021), may achieve higher DNA yields.…”

Section: In Situ Edna Extractionmentioning

confidence: 99%

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Moving environmental DNA (eDNA) technologies from benchtop to the field using passive sampling and PDQeX extraction

et al. 2022

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Environmental DNA (eDNA) metabarcoding has shown great promise as an effective, non‐invasive monitoring method for marine biomes. However, long filtration times and the need for state‐of‐the‐art laboratories are restricting sample replication and in situ species detections. Methodological innovations, such as passive filtration and self‐contained DNA extraction protocols, have the potential to alleviate these issues. We explored the implementation of passive sampling and a self‐contained DNA extraction protocol by comparing fish diversity obtained from active filtration (1 L; 0.45 μm cellulose nitrate [CN] filters) to five passive substrates, including 0.45 μm CN filters, 5 μm nylon filters, 0.45 μm positively charged nylon filters, artificial sponges, and fishing net. Fish diversity was then compared between the PDQeX Nucleic Acid Extractor and the conventional Qiagen DNeasy Blood & Tissue protocol. Experiments were conducted in both a controlled mesocosm and in situ at the Portobello Marine Laboratory, New Zealand. No significant differences in fish diversity were observed among active filtration and more porous passive materials (artificial sponges and fishing net) for both the mesocosm and harbor waters. For the in situ comparison, all passive filter membranes detected a significantly lower number of fish species, resulting from partial sample drop‐out. While no significant differences in fish eDNA signal diversity were observed between either DNA extraction methods in the mesocosm, the PDQeX system was less effective at detecting fish for the in situ comparison. Our results demonstrate that a passive sampling approach using porous substrates can be effectively implemented to capture eDNA from seawater, eliminating vacuum filtration processing. The large variation in efficiency observed among the five substrate types, however, warrants further optimization of the passive sampling approach for routine eDNA applications. The PDQeX system can extract high‐abundance DNA in a mesocosm with further optimization to detect low‐abundance eDNA from the marine environment.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: In Situ Edna Extractionmentioning

confidence: 99%

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Moving environmental DNA (eDNA) technologies from benchtop to the field using passive sampling and PDQeX extraction

et al. 2022

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Adapted method for rapid detection and quantification of pathogen Campylobacter jejuni from environmental water samples

Sun

Mirzayans

Piggott

et al. 2023

FEMS Microbiology Ecology

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Building on a previously developed workflow for rapid and sensitive pathogen detection by qPCR, this work has established a sample treatment strategy that produces consistent quantification efficiencies (QEs) for Campylobacter jejuni against a complex and highly variable sample matrix from a suburban river. The individual treatments most effective at minimising the inhibitory effects of the sample matrix were pH buffering with HEPES (50 mM, pH 5.7) and addition of the surfactant Tween 20 (2% v/v). Unexpectedly, sample acidification (pH 4–5) resulting from the use of aged Tween 20 that had undergone partial hydrolysis, appeared to play a key role in enhancing QE. This effect could be replicated by direct pH adjustment with dilute hydrochloric acid and may be linked to the solubilisation and removal of inhibitory particles at an acidic pH. While the effectiveness of each individual treatment method varied, a combined treatment of either HEPES buffer + Tween 20, or direct pH adjustment + Tween 20, consistently produced QEs of 60–70% and up to 100%, respectively, over a sampling period of one year. The consistency and scalability of this workflow make it a suitable alternative to culture-based ISO methods for detecting Campylobacter spp.

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Efficacy of a membrane concentration method combined with real-time PCR for detection of Giardia and Cryptosporidium in drinking water

Jingyi,

Ping,

Jian

et al. 2023

Letters in Applied Microbiology

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The occurrence of Giardia and Cryptosporidium (oo)cysts in drinking source water poses a serious public health risk. Here, we established a method that combines membrane concentration and real-time polymerase chain reaction (PCR) to quantify Giardia and Cryptosporidium in drinking water. The water samples were filtered through a cellulose membrane to collect Giardia and Cryptosporidium, and then nucleic acids were extracted. Specific primers and probes were designed and synthesized according to the gph gene sequence of Giardia and 18S rRNA gene sequence of Cryptosporidium. The concentrations of the two targets were determined using real-time PCR technology. The sensitivity, specificity, and stability of the method were evaluated. Our findings revealed that the detection limits of real-time PCR method for detecting Giardia and Cryptosporidium were 0.926 and 0.65 copy/µL, respectively; the spiked recovery rates were above 60% and 38%, respectively, and relative standard deviations were under 0.95% and 2.26%, respectively. Therefore, this effective procedure based on the membrane concentration method and real-time PCR will be useful for detecting Giardia and Cryptosporidium in drinking water for purpose of continuous environmental monitoring.

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Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens

Cited by 4 publications

References 55 publications

Moving environmental DNA (eDNA) technologies from benchtop to the field using passive sampling and PDQeX extraction

Moving environmental DNA (eDNA) technologies from benchtop to the field using passive sampling and PDQeX extraction

Adapted method for rapid detection and quantification of pathogen Campylobacter jejuni from environmental water samples

Efficacy of a membrane concentration method combined with real-time PCR for detection of Giardia and Cryptosporidium in drinking water

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