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 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 Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples

Plant Methods ◽  
2014 ◽  
Vol 10 (1) ◽  
Author(s):  
Nejc Rački ◽  
Tanja Dreo ◽  
Ion Gutierrez-Aguirre ◽  
Andrej Blejec ◽  
Maja Ravnikar
Keyword(s):  
Reverse Transcriptase ◽  
Water Samples ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Pcr Inhibitors ◽  
Plant Soil ◽  
Soil And Water

scholarly journals Primer evaluation and development of a droplet digital PCR protocol targeting amoA genes for the quantification of Comammox in lakes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Manuel Harringer ◽  
Albin Alfreider
Keyword(s):  
Water Column ◽  
Lake Water ◽  
Water Samples ◽  
Target Genes ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Minor Importance ◽  
Sequencing Analysis ◽  
Α Subunit ◽  
Wide Range

AbstractTo date, little is known about the ecological significance of Comammox (COMplete AMMonia OXidizers) Nitrospira in the water column of freshwater lakes. Water samples collected along depth profiles were used to investigate the distribution of Comammox in 13 lakes characterized by a wide range of physicochemical properties. Several published primers, which target the α-subunit of the ammonia monooxygenase, generated non-specific PCR products or did not amplify target genes from lake water and other habitats. Therefore, a new primer set has been designed for specific detection of Comammox in lakes. The high specificity of the PCR assay was confirmed by sequencing analysis. Quantification of Comammox amoA genes in lake water samples based on droplet digital PCR (ddPCR) revealed very low abundances (not exceeding 85 amoA copies ml−1), which suggest that Comammox is of minor importance for the nitrification process in the water column of the study sites. Surprisingly, samples taken from the sediment/water-interface along an oxygen gradient in dimictic Piburger See showed Comammox abundances three to four magnitudes higher than in the pelagic realm of the lake, which indicates a preference of Comammox to a particle-attached lifestyle.

scholarly journals Transformation of Contaminants of Emerging Concern (CECs) during UV-Catalyzed Processes Assisted by Chlorine

Catalysts ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1432
Author(s):  
Edyta Kudlek
Keyword(s):  
Hydrogen Peroxide ◽  
Water Samples ◽  
Water Environment ◽  
Toxicity Assessment ◽  
Contaminants Of Emerging Concern ◽  
Post Processing ◽  
Processing Water ◽  
Living Organisms ◽  
Trace Concentrations ◽  
By Products

Every compound that potentially can be harmful to the environment is called a Contaminant of Emerging Concern (CEC). Compounds classified as CECs may undergo different transformations, especially in the water environment. The intermediates formed in this way are considered to be toxic against living organisms even in trace concentrations. We attempted to identify the intermediates formed during single chlorination and UV-catalyzed processes supported by the action of chlorine and hydrogen peroxide or ozone of selected contaminants of emerging concern. The analysis of post-processing water samples containing benzocaine indicated the formation of seven compound intermediates, while ibuprofen, acridine and β-estradiol samples contained 5, 5, and 3 compound decomposition by-products, respectively. The number and also the concentration of the intermediates decreased with the time of UV irradiation. The toxicity assessment indicated that the UV-catalyzed processes lead to decreased toxicity nature of post-processed water solutions.

scholarly journals Droplet digital PCR quantification of norovirus and adenovirus in decentralized wastewater and graywater collections: Implications for onsite reuse

2020 ◽  
Vol 169 ◽  
pp. 115213 ◽  
Author(s):  
Michael A. Jahne ◽  
Nichole E. Brinkman ◽  
Scott P. Keely ◽  
Brian D. Zimmerman ◽  
Emily A. Wheaton ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr

scholarly journals Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

2021 ◽  
Author(s):  
Christian Schulze ◽  
Anne-Catrin Geuthner ◽  
Dietrich Mäde
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Bread Wheat ◽  
Common Wheat ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Food Fraud ◽  
Single Genome ◽  
Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

scholarly journals Longitudinal Circulating Levels of miR-23b-3p, miR-126-3p and lncRNA GAS5 in HCC Patients Treated with Sorafenib

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 813
Author(s):  
Michele Manganelli ◽  
Ilaria Grossi ◽  
Manuela Ferracin ◽  
Paola Guerriero ◽  
Massimo Negrini ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Healthy Individuals ◽  
Roc Curve Analysis ◽  
Multikinase Inhibitor ◽  
Sorafenib Treatment ◽  
The Third ◽  
Circulating Levels ◽  
Systemic Drug

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The multikinase inhibitor sorafenib is a systemic drug for unresectable HCC. The identification of molecular biomarkers for the early diagnosis of HCC and responsiveness to treatment are needed. In this work, we performed an exploratory study to investigate the longitudinal levels of cell-free long ncRNA GAS5 and microRNAs miR-126-3p and -23b-3p in a cohort of 7 patients during the period of treatment with sorafenib. We used qPCR to measure the amounts of GAS5 and miR-126-3p and droplet digital PCR (ddPCR) to measure the levels of miR-23b-3p. Patients treated with sorafenib displayed variable levels of GAS5, miR-126-3p and miR-23b-3p at different time-points of follow-up. miR-23b-3p was further measured by ddPCR in 37 healthy individuals and 25 untreated HCC patients. The amount of miR-23b-3p in the plasma of untreated HCC patients was significantly downregulated if compared to healthy individuals. The ROC curve analysis underlined its diagnostic relevance. In conclusion, our results highlight a potential clinical significance of circulating miR-23b-3p and an exploratory observation on the longitudinal plasmatic levels of GAS5, miR-126-3p and miR-23b-3p during sorafenib treatment.

Selective pre-enrichment method to lessen time needed to recover Salmonella from commercial poultry processing samples

2021 ◽  
Vol 99 ◽  
pp. 103818
Author(s):  
Surendra Rasamsetti ◽  
Mark Berrang ◽  
Nelson A. Cox ◽  
Nikki W. Shariat
Keyword(s):  
Enrichment Method ◽  
Poultry Processing ◽  
Commercial Poultry
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