scholarly journals Multiple cyanotoxin congeners produced by sub-dominant cyanobacterial taxa in riverine cyanobacterial and algal mats

2019 ◽  
Author(s):  
Laura T. Kelly ◽  
Keith Bouma-Gregson ◽  
Jonathan Puddick ◽  
Rich Fadness ◽  
Ken G. Ryan ◽  
...  
Keyword(s):  
Green Alga ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Wilcoxon Test ◽  
Cladophora Glomerata ◽  
Chain Reaction ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Polymerase Chain ◽  
Russian River

AbstractBenthic cyanobacterial proliferations in rivers are have been reported with increasing frequency worldwide. In the Eel and Russian rivers of California, more than a dozen dog deaths have been attributed to cyanotoxin toxicosis since 2000. Periphyton proliferations in these rivers comprise multiple cyanobacterial taxa capable of cyanotoxin production, hence there is uncertainty regarding which taxa are producing toxins. In this study, periphyton samples dominated by the cyanobacterial genera Anabaena spp. and Microcoleus spp. and the green alga Cladophora glomerata were collected from four sites in the Eel River catchment and one site in the Russian River. Samples were analysed for potential cyanotoxin producers using polymerase chain reaction (PCR) in concert with Sanger sequencing. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry, and anatoxin quota determined using droplet digital PCR. Sequencing indicated Microcoleus sp. and Nodularia sp. were the putative producers of anatoxins and nodularins, respectively, regardless of the dominant taxa in the mat. Anatoxin concentrations in the mat samples varied from 0.1 to 18.6 μg g−1 and were significantly different among sites (p < 0.01, Wilcoxon test); however, anatoxin quotas were less variable (< 5-fold). Dihydroanatoxin-a was generally the most abundant variant in samples comprising 38% to 71% of the total anatoxins measured. Mats dominated by the green alga C. glomerata contained both anatoxins and nodularin-R at concentrations similar to those of cyanobacteria-dominated mats. This highlights that even when cyanobacteria are not the dominant taxa in periphyton, these mats may still pose a serious health risk and indicates that more widespread monitoring of all mats in a river are necessary.

scholarly journals Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events

2020 ◽  
Author(s):  
Tigst Demeke ◽  
Monika Eng ◽  
Michelle Holigroski ◽  
Sung-Jong Lee
Keyword(s):  
Zero Tolerance ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Genetically Engineered ◽  
Droplet Digital Pcr ◽  
Chain Reaction ◽  
Low Level ◽  
Polymerase Chain ◽  
Reliable Quantification ◽  
Detection And Quantification

Abstract Low-level detection and quantification of genetically engineered (GE) traits with polymerase chain reaction (PCR) is challenging. For unapproved GE events, any level of detection is not acceptable in some countries because of zero tolerance. Droplet digital PCR (ddPCR) has been successfully used for absolute quantification of GE events. In this study, reliability of low level quantification of GE events with ddPCR was assessed using a total of 50, 100, 200, 400, and 600 ng DNA spiked at 0.01% and 0.1% concentration levels. Genetically engineered canola (GT73 and MON88302 events) and soybean (A2704-12 and DP305423 events) events were used for the study. For samples spiked at 0.1% level, reliable quantification was achieved for the four GE events using 50 or 100 ng DNA. Few target droplets were generated for 0.01% spiked GE samples using 50 and 100 ng DNA. Increasing the amount of DNA for ddPCR generated more number of target droplets. For GE canola events, the use of 400 and 600 ng DNA for ddPCR resulted in saturation. The use of multiple wells of 200 ng DNA (instead of 400 and 600 ng per well) helped to overcome the saturation problem. Overall, the use of high amount of DNA for ddPCR was helpful for the detection and quantification of 0.01% GE samples.

A rapid nucleic acid concentration measurement system with large-field-of-view for droplet digital PCR microfluidic chip

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Jinrong Shen ◽  
Jihong Zheng ◽  
Zhenqing Li ◽  
Yourong Liu ◽  
Fengxiang Jing ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Field Of View ◽  
Large Field ◽  
Chain Reaction ◽  
Effective Technique ◽  
Nucleic Acid Concentration ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Droplet digital polymerase chain reaction(ddPCR) is an effective technique for the absolute quantification of target mucleic acid unparalleled sensitivity. However, current commerical ddPCR device for the detection of the gene...

scholarly journals Quantitative real-time polymerase chain reaction (PCR) and droplet digital PCR duplex assays for detectingZostera marinaDNA in coastal sediments

2018 ◽  
Vol 16 (4) ◽  
pp. 253-264 ◽  
Author(s):  
Masami Hamaguchi ◽  
Hiromori Shimabukuro ◽  
Masakazu Hori ◽  
Goro Yoshida ◽  
Tomoko Terada ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Coastal Sediments ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1270
Author(s):  
Anna Cutarelli ◽  
Andrea Fulgione ◽  
Pasquale Fraulo ◽  
Francesco Paolo Serpe ◽  
Pasquale Gallo ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Buffalo Milk ◽  
Cow Milk ◽  
Mozzarella Cheese ◽  
Chain Reaction ◽  
New Methods ◽  
Protected Designation Of Origin ◽  
Commission Regulation ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Buffalo mozzarella cheese is one of the most appreciated traditional Italian products and it is certified as a Protected Designation of Origin (PDO) product under the European Commission Regulation No. 1151/2012. It is obtained exclusively from buffalo milk. If made from cow milk, or a mixture of buffalo and cow milk, buffalo mozzarella cheese does not qualify as a PDO product. In order to maximize their profits, some producers market buffalo mozzarella that also contains cow milk as a PDO product, thus defrauding consumers. New methods for revealing this fraud are therefore needed. One such method is the droplet digital Polymerase Chain Reaction (ddPCR). Thanks to its high precision and sensitivity, the ddPCR could prove an efficacious means for detecting the presence of cow milk in buffalo mozzarella cheese that is marketed as a PDO product. ddPCR has proved able to detect the DNA of cow and/or buffalo milk in 33 buffalo mozzarella cheeses labelled as PDO products, and experimental evidence could support its application in routine analyses.

scholarly journals A New Specimen for Syphilis Diagnosis: Evidence by High Loads of Treponema pallidum DNA in Saliva

2020 ◽  
Author(s):  
Cuini Wang ◽  
Zhixiang Hu ◽  
Xin Zheng ◽  
Meiping Ye ◽  
Chunjie Liao ◽  
...  
Keyword(s):  
Treponema Pallidum ◽  
Digital Pcr ◽  
Secondary Syphilis ◽  
Clearance Time ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Detection Rates ◽  
Polymerase Chain ◽  
Pcr Targeting ◽  
Latent Syphilis

Abstract Background DNA from many pathogens can be detected in saliva. However, the presence and quantity of Treponema pallidum DNA in patients with syphilis in saliva is unknown. Methods 234 patients with syphilis with different stages and 30 volunteers were enrolled. Paired saliva and plasma samples were collected from all participants. Consecutive saliva samples from 9 patients were collected every 4 hours following treatment. Treponema pallidum DNA in samples was determined by nested polymerase chain reaction (PCR) and droplet digital PCR targeting polA and Tpp47. Results Treponema pallidum DNA detection rates in saliva and plasma were 31.0% (9/29) and 51.7% (15/29) in primary syphilis (P = .11), 87.5% (63/72) and 61.1% (44/72) in secondary syphilis (P &lt; .001), 25.6% (21/82) and 8.5% (7/82) in latent syphilis (P = .004), and 21.6% (11/51) and 5.9% (3/51) in symptomatic neurosyphilis (P = .021), respectively. Median (range) loads of Tpp47 and polA in saliva were 627 (0–101 200) and 726 (0–117 260) copies/mL, respectively, for patients with syphilis. In plasma, however, loads of Tpp47 and polA were low: medians (range) of 0 (0–149.6) and 0 (0–176) copies/mL, respectively. Loads of T. pallidum DNA in saliva during treatment fluctuated downward; the clearance time was positively correlated with the loads of T. pallidum DNA before treatment. Conclusions Collection of saliva is noninvasive and convenient. The high loads of T. pallidum DNA in saliva and reduction after treatment indicated that saliva can be not only a diagnostic fluid for syphilis but also an indicator of therapeutic effectiveness.

scholarly journals Digital PCR as an Emerging Tool for Monitoring of Microbial Biodegradation

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 706 ◽  
Author(s):  
Yiqi Cao ◽  
Miao Yu ◽  
Guihua Dong ◽  
Bing Chen ◽  
Baiyu Zhang
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Functional Genes ◽  
Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Quantification Method ◽  
Monitoring Technique ◽  
Microbial Biodegradation ◽  
Polymerase Chain ◽  
Pcr Techniques

Biodegradation of contaminants is extremely complicated due to unpredictable microbial behaviors. Monitoring of microbial biodegradation drives us to determine (1) the amounts of specific degrading microbes, (2) the abundance, and (3) expression level of relevant functional genes. To this endeavor, the cultivation independent polymerase chain reaction (PCR)-based monitoring technique develops from endpoint PCR, real-time quantitative PCR, and then into novel digital PCR. In this review, we introduce these three categories of PCR techniques and summarize the timely applications of digital PCR and its superiorities than qPCR for biodegradation monitoring. Digital PCR technique, emerging as the most accurately absolute quantification method, can serve as the most promising and robust tool for monitoring of microbial biodegradation.

scholarly journals Development and Evaluation of Droplet Digital PCR Assay for the Detection of Reticuloendotheliosis Virus in Attenuated Vaccines

2021 ◽  
Author(s):  
Fanfeng Meng ◽  
Zhihao Ren ◽  
Yixin Wang ◽  
Peng Zhao ◽  
Guozhong Zhang
Keyword(s):  
Low Dose ◽  
Digital Pcr ◽  
Conventional Polymerase Chain Reaction ◽  
Droplet Digital Pcr ◽  
Reticuloendotheliosis Virus ◽  
Polymerase Chain Reaction Detection ◽  
Dot Blot ◽  
Pcr Assay ◽  
Live Virus ◽  
Polymerase Chain

Abstract Background: The use of Reticuloendotheliosis virus (REV) from contaminated live virus vaccine is suspected to be one of the most important causes of massive outbreaks of Reticuloendotheliosis in China. Methods: In this study, we established a droplet digital PCR (ddPCR) detection method for REV and compared its sensitivity to different methods to detect REV contamination in a vaccine. Results: The results indicated that both quantitative PCR and dot-blot methods could detect REV contamination at a dose of 1 TCID50/1,000 feathers, whereas ddPCR could detect REV contamination at a dose of 0.1 TCID50/1,000 feathers, which is 1,000-fold more sensitive than conventional polymerase chain reaction detection (102 TCID50/1000 feathers). ddPCR not only exhibited the highest sensitivity but also proved extremely intuitive, especially to detect REV contamination in vaccines.Conclusions: The ddPCR method established in this study to detect REV contamination in vaccines can effectively detect and quantify low-dose REV contamination. This provides a new method for the rapid detection of REV contamination in various samples, especially vaccines.

67 INHIBITION OF 5α-REDUCTASE DURING LATE GESTATION IN THE MARE

2016 ◽  
Vol 28 (2) ◽  
pp. 163
Author(s):  
M. Wynn ◽  
E. Legacki ◽  
A. Conley ◽  
S. Loux ◽  
A. Esteller-Vico ◽  
...  
Keyword(s):  
Mixed Model ◽  
Late Gestation ◽  
Wilcoxon Test ◽  
Serum Concentrations ◽  
Gestational Length ◽  
Time Treatment ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
High Concentrations ◽  
And Control

During the latter half of pregnancy in the mare, circulating concentrations of progesterone (P4) are low and a major bioactive progestogen, 5α-dihydroprogesterone (DHP), is present in high concentrations. DHP is formed through the activity of 5α-reductase, which converts P4 to DHP. Further metabolites of DHP have been attributed to fetal and myometrial quiescence. The purpose of this study was to examine the effects of a 5α-reductase inhibitor (dutasteride) on P4 metabolism and onset of parturition. Pregnant mares (n = 5) were treated with dutasteride (0.01 mg kg–1; IM), and control mares (n = 4) received vehicle alone from 300 to 320 days of gestation or until foaling. Serum concentrations of P4 and DHP were determined with liquid chromatography/tandem mass spectrometry (LC/MS-MS) daily for 9 days preceding parturition. Endocrine data were analysed with a random effects mixed model with time, treatment (TRT), and time × TRT interaction. Gestational data were analysed with a Wilcoxon test. Although there was a significant effect of time on P4 and DHP, there was no effect of TRT or time × TRT on these progestogens. For the ratio of DHP/P4, there were significant effects of time, TRT, and time × TRT interaction such that the ratio of DHP/P4 was greater in the control than dutasteride-treated mares. Birth weight and gestational length were not different (P > 0.2) between the dutasteride-treated and control mares, although placental weights were greater (P < 0.05) in dutasteride-treated mares. Because the ratio of DHP/P4 was suppressed in dutasteride-treated mares, it appears that dutasteride was active in late gestational mares. Although gestational length and neonatal weights were not different between groups, placentas from dutasteride-treated mares were heavier than those from control mares. The reason for the increase in placental weights was not determined.

scholarly journals Detection of fish allergen by droplet digital PCR

2019 ◽  
Vol 7 (4) ◽  
Author(s):  
Cinzia Daga ◽  
Simona Cau ◽  
Maria Giovanna Tilocca ◽  
Barbara Soro ◽  
Aldo Marongiu ◽  
...  
Keyword(s):  
18S Rrna ◽  
Annealing Temperature ◽  
Pcr Primers ◽  
Digital Pcr ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Chain Reaction ◽  
European Regulation ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Fish is one of fourteen allergens that must be highlighted on the label within the ingredients list. It should be noted that the European regulation, is very restrictive to allergens with zero tolerance. Therefore it is important to establish sensitive and specific methods for detecting fish allergen. Applicability to detect and quantify fish allergen by droplet digital polymerase chain reaction (ddPCR) has been evaluated in this work. Genomic DNA of three fish species belonging to the most common fish families were analyzed. PCR primers were designed to amplify a 166 bp region of the 18S rRNA gene. Comparative studies were performed to establish the optimal primer and probe concentrations.  Annealing temperature was determined by using thermal gradient. The results have shown good applicability of the optimized 18S rRNA gene-method to detect and quantify small amounts of the target in all samples analyzed. However, validation studies are needed in order to apply ddPCR technology for routine allergens analysis.  

scholarly journals Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

2020 ◽  
Vol 21 (9) ◽  
pp. 3141 ◽  
Author(s):  
Nicoletta Coccaro ◽  
Giuseppina Tota ◽  
Luisa Anelli ◽  
Antonella Zagaria ◽  
Giorgina Specchia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Residual Disease ◽  
Hematologic Malignancies ◽  
Digital Pcr ◽  
Future Perspectives ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Generation Sequencing ◽  
Minimal Residual

The digital polymerase chain reaction (dPCR) is considered to be the third-generation polymerase chain reaction (PCR), as it yields direct, absolute and precise measures of target sequences. dPCR has proven particularly useful for the accurate detection and quantification of low-abundance nucleic acids, highlighting its advantages in cancer diagnosis and in predicting recurrence and monitoring minimal residual disease, mostly coupled with next generation sequencing. In the last few years, a series of studies have employed dPCR for the analysis of hematologic malignancies. In this review, we will summarize these findings, attempting to focus on the potential future perspectives of the application of this promising technology.

Sign in / Sign up

Export Citation Format

Share Document