scholarly journals Detection of fish pathogen Saprolegnia parasitica in environmental DNA samples by droplet digital PCR

2021 ◽  
Vol 4 ◽  
Author(s):  
Dora Pavić ◽  
Anđela Miljanović ◽  
Uršula Prosenc-Zmrzljak ◽  
Rok Košir ◽  
Dorotea Grbin ◽  
...  
Keyword(s):  
Water Samples ◽  
Genomic Dna ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Economic Losses ◽  
Internal Transcribed Spacer Region ◽  
Saprolegnia Parasitica ◽  
Oomycete Pathogen ◽  
Total Dna

Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections of freshwater animals. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, a disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop a droplet digital PCR (ddPCR) assay for the detection and quantification of S. parasitica in environmental DNA samples. Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The specificity of primers was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA (as negative control). The primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (e.g. Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Next, the limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. The determined sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of the reaction mixture. Assay performance was further assessed with environmental DNA samples isolated from water from the trout farms and natural environments, as well as (ii) biofilm from the host surface (swab samples). Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin and eggs of the rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle of signal crayfish (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA. Saprolegnia parasitica was detected in 76 % of water samples (16/21) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Regarding the swab samples, S. parasitica load was significantly higher in diseased trout than in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (average agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had signs of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load than swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (agent level A5 vs. A4, respectively). In conclusion, our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environments. This would help in a better understanding of S. parasitica ecology and its effects on the host populations.

scholarly journals Quantification of Zoonotic Bacterial Pathogens within Commercial Poultry Processing Water Samples Using Droplet Digital PCR

2013 ◽  
Vol 03 (05) ◽  
pp. 403-411 ◽  
Author(s):  
Michael J. Rothrock ◽  
Kelli L. Hiett ◽  
Brian H. Kiepper ◽  
Kim Ingram ◽  
Arthur Hinton
Keyword(s):  
Water Samples ◽  
Bacterial Pathogens ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Poultry Processing ◽  
Commercial Poultry ◽  
Processing Water

scholarly journals Development of a droplet digital PCR for detection and quantification of porcine epidemic diarrhea virus

2020 ◽  
Vol 32 (4) ◽  
pp. 572-576 ◽  
Author(s):  
Wei W. Cao ◽  
Dong S. He ◽  
Zhen J. Chen ◽  
Yu Z. Zuo ◽  
Xun Chen ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Fold Increase ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Economic Losses ◽  
Swine Industry ◽  
Diarrhea Virus ◽  
Viral Loads ◽  
Promising Tool ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea, a disease caused by porcine epidemic diarrhea virus (PEDV), results in large economic losses to the global swine industry. To manage this disease effectively, it is essential to detect PEDV early and accurately. We developed a sensitive and accurate droplet digital PCR (ddPCR) assay to detect PEDV. The optimal primer-to-probe concentration and melting temperature were identified as 300:200 nM and 59.2°C, respectively. The specificity of the ddPCR assay was confirmed by negative test results for common swine pathogens. The detection limit for the ddPCR was 0.26 copies/μL, which is a 5.7-fold increase in sensitivity compared to that of real-time PCR (rtPCR). Both ddPCR and rtPCR assays exhibited good linearity, although ddPCR provided higher sensitivity for clinical detection compared to that of rtPCR. Our ddPCR methodology provides a promising tool for evaluating the PEDV viral load when used for clinical testing, particularly for detecting samples with low-copy viral loads.

scholarly journals Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0122763 ◽  
Author(s):  
Hideyuki Doi ◽  
Kimiko Uchii ◽  
Teruhiko Takahara ◽  
Saeko Matsuhashi ◽  
Hiroki Yamanaka ◽  
...  
Keyword(s):  
Environmental Dna ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Fish Abundance

scholarly journals Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

2014 ◽  
Vol 53 (2) ◽  
pp. 699-701 ◽  
Author(s):  
Eva Malatinkova ◽  
Maja Kiselinova ◽  
Pawel Bonczkowski ◽  
Wim Trypsteen ◽  
Peter Messiaen ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Terminal Repeat ◽  
Genomic Dna Isolation ◽  
Plasmid Isolation ◽  
Hiv 1 ◽  
Accurate Quantification

Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation.

scholarly journals Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254356
Author(s):  
Bettina Thalinger ◽  
Yannick Pütz ◽  
Michael Traugott
Keyword(s):  
Capillary Electrophoresis ◽  
Water Samples ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Dilution Series ◽  
Target Dna ◽  
Cost Efficient ◽  
Detection And Quantification

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.

Proof of Principle for Mutations Monitoring Using Picoliter-Droplet Digital PCR on DNA and Living Cells: Application to Myelodysplastic Syndromes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5515-5515
Author(s):  
Hélène Guermouche ◽  
Philippe Nizard ◽  
Heng Lu ◽  
Charles Decraene ◽  
Juliette Nectoux ◽  
...  
Keyword(s):  
Cell Lines ◽  
Genomic Dna ◽  
Mutant Allele ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Living Cells ◽  
Droplet Digital Pcr ◽  
Wild Type ◽  
Allelic Discrimination ◽  
Picoliter Droplet

Abstract Introduction Myelodysplastic syndromes (MDS) are phenotypically and genotypically heterogeneous diseases with several driver mutations, which are closely related to patient prognosis. Dynamic evolution of mutations reflects the selection of subclones during disease evolution until transformation in secondary acute myeloid leukemia. Thanks to increasing knowledge in gene functions, target drugs are now available in therapeutic. However, questions remain on the impact of such treatments on malignant cells. We have previously investigated the effects of lenalidomide on clonal evolution, by monitoring variant allele frequencies (VAF) using next generation sequencing (NGS) in non-del5q MDS patients (Chesnais et al, Blood 2015). Here, we present a rapid and ultra-sensitive method using picoliter-droplet digital PCR for mutation detection in MDS with ring sideroblasts (RS). Materials and Methods Bone marrow aspirates were obtained from MDS patients included at diagnosis in a multicentric observational trial (PHRC MDS-04, NCT02619565). Three cell lines (HL60, OCI-AML3, UKE-1) were also used to establish the specificity and the sensitivity of assays. Both frozen living cells and extracted DNA were used. Selected samples were screened for mutations in 39 genes by an NGS approach using a Personal Genome Machine® (PGM, ThermoFisher Scientific, Waltham, MA, USA). Primers and probes were designed for Taqman assays based on allelic discrimination of recurrent mutations found in DNMT3A, SF3B1, JAK2 and NRAS genes. For the detection of SF3B1 p.K700E mutation, 3 locked nucleic acids were notably added to the probes to improve specificity. Picoliter-droplet digital PCR was performed on RainDrop® Digital PCR System (RainDance™ Technologies). Results Allelic discrimination assays were validated on genomic DNA extracted from cell lines and patient samples harboring or not targeted mutations using the RainDance system. About 5.106 droplets were generated using RainDrop Source. Wild-type (WT) DNA was tested in order to assess false positive signals for each design, characterized by λFP (mean number of false positive signals), limit of blanck (LOB) and limit of detection (LOD) for all experiments. The limit of blanck (LOB) defined here the highest number of droplets corresponding to apparent droplets containing mutated amplicons while testing wild type DNA. The limit of detection (LOD) was the lower number of droplets which can be distinguish from LOB while testing DNA with very low concentration of mutant genome. All the designed assays were also strongly approved for linearity using mixtures of mutated and WT DNA from cell lines (0.01% to 100% mutated allele frequency). Specificity, linearity and sensibility of the selected assays were validated on genomic DNA. Later on, we investigate genomic DNA of 3 MDS patients with RS and harboring JAK2 and SF3B1 mutations. For these patients, we obtained comparable results using both NGS and picoliter-droplet digital PCR in term of mutant allele burden quantification. Moreover, a triplex assay allowing mutant allele discrimination in JAK2 and SF3B1 genes was established on these patients. Further analyses were conducted on living cells harboring JAK2 or NRAS mutations. This approach was first conducted using a "home made" microfluidic system based on the detection of fluorescent probes in living cells encapsulated into agarose beeds. We obtained specific fluorescent signals corresponding to the genotypes. In parallel, an alternative method based on the QX100™Droplet Digital™PCR system (Biorad) also demonstrated the feasibility of allelic discrimination in living cells. Experiments based on frozen cells of MDS patients are currently under investigation. Conclusion This study is the first application of multi-target digital PCR used to detect and quantify somatic mutations recurrently found in MDS. Analyses of the clonal architecture determined on living cells and its evolution upon treatment in MDS patients with RS by this approach will help us to investigate the monitoring of the therapeutic response. Our study supports a proof of principle for further large-scale analyses of MDS patients at diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.

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