scholarly journals Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

2021 ◽  
Author(s):  
Suwimon Taengphu ◽  
Pattanapon Kayansamruaj ◽  
Yasuhiko Kawato ◽  
Jerome Delamare-Deboutteville ◽  
Chadag Vishnumurthy Mohan ◽  
...  
Keyword(s):  
Water Samples ◽  
Detection System ◽  
Environmental Dna ◽  
Pond Water ◽  
Disease Forecasting ◽  
Early Disease ◽  
River Water Samples ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Detection And Quantification

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is an important virus responsible for die-off of farmed tilapia globally. Detection and quantification of the virus from environmental DNA/RNA (eDNA/eRNA) using pond water represents a potential, noninvasive routine approach for pathogen monitoring and early disease forecasting in aquaculture systems. Here, we report a simple iron flocculation method for viral concentration from water combined with a newly developed hydrolysis probe quantitative RT-qPCR method for detection and quantification of TiLV. The RT-qPCR method targeting a conserved region of TiLV genome segment 9 has a detection limit of 10 viral copies per uL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 +/- 3.3% of virus from water samples spiked with viral cultures. During disease outbreak cases from an open-caged system and a closed hatchery system, both tilapia and water samples were collected for detection and quantification of TiLV. The results revealed that TiLV was detected from both clinically sick fish and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms e.g. river water samples from affected cages (8.50 x 102 to 2.79 x 104 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 x 102 to 3.53 x 103 copies/L) from affected and unaffected ponds of the hatchery. In summary, this study suggests that the eRNA detection system using iron flocculation coupled with probe based-RT-qPCR is feasible for concentration and quantification of TiLV from water. This approach might be useful for noninvasive monitoring of TiLV in tilapia aquaculture systems and facilitating appropriate decisions on biosecurity interventions needed.

scholarly journals Environmental DNA enables detection of terrestrial mammals from forest pond water

2016 ◽  
Author(s):  
Masayuki Ushio ◽  
Hisato Fukuda ◽  
Toshiki Inoue ◽  
Kobayashi Makoto ◽  
Osamu Kishida ◽  
...  
Keyword(s):  
Water Samples ◽  
Cervus Nippon ◽  
Environmental Dna ◽  
Terrestrial Ecosystems ◽  
Illumina Miseq ◽  
Pcr Primers ◽  
Pond Water ◽  
Universal Primers ◽  
Taxonomic Assignment ◽  
Terrestrial Mammals

Terrestrial animals must have frequent contact with water to maintain their lives, implying that environmental DNA (eDNA) originating from terrestrial animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. After verifying the primers’ usefulness in silico and using water samples from zoo cages of animals with known species compositions, we collected five 500-ml water samples from ponds in two cool-temperate forests in Hokkaido, northern Japan. Using eDNA extracted from the water samples, we constructed amplicon libraries using MiMammal primers for Illumina MiSeq sequencing. MiMammal metabarcoding yielded a total of 75,214 reads, which we then subjected to data pre-processing and taxonomic assignment. We thereby detected species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Previous applications of the eDNA metabarcoding approach have mostly been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even in forest mammal biodiversity surveys.

An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water

2012 ◽  
Vol 39 (7) ◽  
pp. 629 ◽  
Author(s):  
Zachary H. Olson ◽  
Jeffrey T. Briggler ◽  
Rod N. Williams
Keyword(s):  
Water Samples ◽  
Effective Means ◽  
Environmental Dna ◽  
Cost Effective ◽  
Negative Control ◽  
Specificity And Sensitivity ◽  
Genetic Sampling ◽  
Conservation Concern ◽  
Species Specific ◽  
Eastern Hellbender

Context Environmental DNA, or eDNA, methods are a novel application of non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. eDNA methods have the potential to revolutionise the study of rare or endangered taxa. Aims We evaluated the efficacy of eDNA sampling to detect populations of an amphibian of conservation concern, the eastern hellbender (Cryptobranchus a. alleganiensis), indirectly from their aquatic environments. Methods We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods in silico and in laboratory trials. In the field, we collected water samples from three sites with known densities of hellbenders, and from one site where hellbenders do not occur. We filtered water samples, extracted DNA from filters, and assayed the extraction products for hellbender DNA by using polymerase chain reaction (PCR) and gel electrophoresis. Key results Our methods detected hellbenders at densities approaching the lowest of reported natural densities. The low-density site (0.16 hellbenders per 100 m2) yielded two positive amplifications, the medium-density site (0.38 hellbenders per 100 m2) yielded eight positive amplifications, and the high-density site (0.88 hellbenders per 100 m2) yielded 10 positive amplifications. The apparent relationship between density and detection was obfuscated when river discharge was considered. There was no amplification in any negative control. Conclusion eDNA methods may represent a cost-effective means by which to establish broad-scale patterns of occupancy for hellbenders. Implications eDNA can be considered a valuable tool for detecting many species that are otherwise difficult to study.

scholarly journals Filtration extraction method using microfluidic channel for measuring environmental DNA

2021 ◽  
Author(s):  
Takashi Fukuzawa ◽  
Yuichi Kameda ◽  
Hisao Nagata ◽  
Naofumi Nishizawa ◽  
Hideyuki Doi
Keyword(s):  
Water Sample ◽  
River Water ◽  
Dna Extraction ◽  
Water Samples ◽  
Extraction Method ◽  
Microfluidic Channel ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Laboratory Analysis ◽  
River Water Samples

The environmental DNA (eDNA) method, which is widely applied for biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, Biryu-Chip (BC), and a filtration/extraction method, BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of the extracted DNA was obtained with 1/20–1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we could perform on-site measurements of eDNA within 30 min using a mobile PCR device. Using the BC method, filtration and extraction could be performed easily and quickly. The PCR results obtained by the BC method were similar to those obtained by the Sterivex method. The BC method required fewer steps and therefore, the risk of DNA contamination could be reduced. When combined with a mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from a few 10 mL of the water sample, even on-site.

scholarly journals Utilizing environmental DNA for wide-range distributions of reproductive area of an invasive terrestrial toad in Ishikari river basin in Japan

2021 ◽  
Author(s):  
Hiroki Mizumoto ◽  
Osamu Kishida ◽  
Kotaro Takai ◽  
Hitoshi Araki
Keyword(s):  
Invasive Species ◽  
River Basin ◽  
Water Samples ◽  
Detection System ◽  
Environmental Dna ◽  
Watershed Scale ◽  
Efficient Tool ◽  
Wide Range ◽  
Powerful Means ◽  
In The Wild

Abstract Understanding the distribution of invasive species and their reproductive area is crucial for their managements after invasion. While catch and observation surveys are still embraced, environmental DNA (eDNA) has been increasingly utilized as an efficient tool for identifying these species in the wild. In this study, we developed an eDNA detection system for an invasive, toxic, and terrestrial toad species Bufo japonicus in Hokkaido, Japan, and applied it to their reproductive area at watershed scale. We found that our system successfully detected their eDNA not only in ponds where their larvae were observed, but also in rivers downstream of the reproductive ponds. Thus, the system provided us an opportunity to estimate watersheds that include their reproductive area by collecting downstream water samples. Applying it to the Ishikari river basin, the largest river basin in Hokkaido (c.a., 14,330 km2), we detected their eDNA at 32 out of 73 river sampling sites. They are composed of eleven sites with species observation records nearby (all the sites with observation records within a 500 m radius) and21 sites without such records. And those eDNA detections were from 14 out of 31 river systems, and they were widespread across the river basin. These results suggest that the eDNA detection system can efficiently estimate the presence of reproductive area of the terrestrial toad even from a distant downstream of the watershed, and that it provides a powerful means of detecting new reproductive area and monitoring further spread of invasive species.

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.

scholarly journals Filtration extraction method using microfluidic channel for measuring environmental DNA

2021 ◽  
Author(s):  
Fukuzawa Takashi ◽  
Yuichi Kameda ◽  
Hisao Nagata ◽  
Naofumi Nishizawa ◽  
Hideyuki Doi
Keyword(s):  
Water Sample ◽  
River Water ◽  
Dna Extraction ◽  
Water Samples ◽  
Extraction Method ◽  
Microfluidic Channel ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Laboratory Analysis ◽  
River Water Samples

The environmental DNA (eDNA) method, which is widely applied for biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, Biryu-Chip (BC), and a filtration/extraction method, BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of the extracted DNA was obtained with 1/20-1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we could perform on-site measurements of eDNA within 30 min using a mobile PCR device. Using the BC method, filtration and extraction could be performed easily and quickly. The PCR results obtained by the BC method were similar to those obtained by the Sterivex method. However, the BC method required fewer steps and therefore, the risk of DNA contamination could be reduced. When combined with a mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from 10 mL of the water sample, even on-site.

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

2020 ◽  
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

AbstractThe 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 in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex PCRs, 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 analysing dilution series of DNA extracts and 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 DNA extract. celPCR was suitable for quantifying target DNA and direct inference of DNA concentrations from RFU was possible after accounting for primer effects. Furthermore, multiplex celPCRs and dPCRs 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.

Investigation of Some Water Quality Parameters of Pond Water under Mymensingh Municipality

2015 ◽  
Vol 8 (1) ◽  
pp. 85-89
Author(s):  
F Zannat ◽  
MA Ali ◽  
MA Sattar
Keyword(s):  
Water Quality ◽  
Water Samples ◽  
Chemical Properties ◽  
Pond Water ◽  
Quality Parameters ◽  
Water Quality Parameters ◽  
Urban Region ◽  
Mn Toxicity ◽  
Ph Values ◽  
Drinking Water Standard

A study was conducted to evaluate the water quality parameters of pond water at Mymensingh Urban region. The water samples were collected from 30 ponds located at Mymensingh Urban Region during August to October 2010. The chemical analyses of water samples included pH, EC, Na, K, Ca, S, Mn and As were done by standard methods. The chemical properties in pond water were found pH 6.68 to 7.14, EC 227 to 700 ?Scm-1, Na 15.57 to 36.00 ppm, K 3.83 to 16.16 ppm, Ca 2.01 to 7.29 ppm, S 1.61 to 4.67 ppm, Mn 0.33 to 0.684 ppm and As 0.0011 to 0.0059 ppm. The pH values of water samples revealed that water samples were acidic to slightly alkaline in nature. The EC value revealed that water samples were medium salinity except one sample and also good for irrigation. According to drinking water standard Mn toxicity was detected in pond water. Considering Na, Ca and S ions pond water was safe for irrigation and aquaculture. In case of K ion, all the samples were suitable for irrigation but unsuitable for aquaculture.J. Environ. Sci. & Natural Resources, 8(1): 85-89 2015

Sign in / Sign up

Export Citation Format

Share Document