scholarly journals An application of PCR-RFLP species identification assay for environmental DNA detection

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7597
Author(s):  
Takeshi Igawa ◽  
Teruhiko Takahara ◽  
Quintin Lau ◽  
Shohei Komaki
Keyword(s):  
Water Samples ◽  
Nuclear Power ◽  
Spatial Scales ◽  
Environmental Dna ◽  
Rana Japonica ◽  
Detection Approach ◽  
Pcr Rflp ◽  
Static Water ◽  
Cost Efficient ◽  
The Cost

Recent advancement of environmental DNA (eDNA) methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by applying quantitative PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA detection using these approaches can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers in starting eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear power plant where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

scholarly journals An application of PCR-RFLP species identification assay for environmental DNA detection

2019 ◽  
Author(s):  
Takeshi Igawa ◽  
Teruhiko Takahara ◽  
Quintin Lau ◽  
Shohei Komaki
Keyword(s):  
Water Samples ◽  
Nuclear Power ◽  
Spatial Scales ◽  
Environmental Dna ◽  
Detection Approach ◽  
Pcr Rflp ◽  
Recent Advancement ◽  
Static Water ◽  
Cost Efficient ◽  
The Cost

Recent advancement of eDNA methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by using eDNA. However, the cost of the eDNA detection can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers foraying into eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (R. japonica, R. ornativentris, and R. tagoi tagoi) in various spatial scales including an area close to the Fukushima Nuclear Power Plant (FNPP) where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

scholarly journals An application of PCR-RFLP species identification assay for environmental DNA detection

2019 ◽  
Author(s):  
Takeshi Igawa ◽  
Teruhiko Takahara ◽  
Quintin Lau ◽  
Shohei Komaki
Keyword(s):  
Water Samples ◽  
Nuclear Power ◽  
Spatial Scales ◽  
Environmental Dna ◽  
Detection Approach ◽  
Pcr Rflp ◽  
Recent Advancement ◽  
Static Water ◽  
Cost Efficient ◽  
The Cost

Recent advancement of eDNA methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by using eDNA. However, the cost of the eDNA detection can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers foraying into eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (R. japonica, R. ornativentris, and R. tagoi tagoi) in various spatial scales including an area close to the Fukushima Nuclear Power Plant (FNPP) where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

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 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.

scholarly journals Comparison of LPWAN Technologies: Cost Structure and Scalability

2021 ◽  
Author(s):  
Mohammad Istiak Hossain ◽  
Jan I. Markendahl
Keyword(s):  
Communication Technology ◽  
Service Providers ◽  
Communication Technologies ◽  
Cost Structure ◽  
Small Scale ◽  
Area Network ◽  
Wide Area Network ◽  
Generic Framework ◽  
Cost Efficient ◽  
The Cost

AbstractSmall-scale commercial rollouts of Cellular-IoT (C-IoT) networks have started globally since last year. However, among the plethora of low power wide area network (LPWAN) technologies, the cost-effectiveness of C-IoT is not certain for IoT service providers, small and greenfield operators. Today, there is no known public framework for the feasibility analysis of IoT communication technologies. Hence, this paper first presents a generic framework to assess the cost structure of cellular and non-cellular LPWAN technologies. Then, we applied the framework in eight deployment scenarios to analyze the prospect of LPWAN technologies like Sigfox, LoRaWAN, NB-IoT, LTE-M, and EC-GSM. We consider the inter-technology interference impact on LoRaWAN and Sigfox scalability. Our results validate that a large rollout with a single technology is not cost-efficient. Also, our analysis suggests the rollout possibility of an IoT communication Technology may not be linear to cost-efficiency.

scholarly journals Cost Analysis of a Novel Method for Ecological Compensation—A Study of the Translocation of Dead Wood

2021 ◽  
Vol 13 (11) ◽  
pp. 6075
Author(s):  
Ola Lindroos ◽  
Malin Söderlind ◽  
Joel Jensen ◽  
Joakim Hjältén
Keyword(s):  
Cost Efficiency ◽  
Dead Wood ◽  
Road Transportation ◽  
Ecological Value ◽  
Ecological Compensation ◽  
Work Time ◽  
Delivery Times ◽  
Novel Method ◽  
Cost Efficient ◽  
The Cost

Translocation of dead wood is a novel method for ecological compensation and restoration that could, potentially, provide a new important tool for biodiversity conservation. With this method, substrates that normally have long delivery times are instantly created in a compensation area, and ideally many of the associated dead wood dwelling organisms are translocated together with the substrates. However, to a large extent, there is a lack of knowledge about the cost efficiency of different methods of ecological compensation. Therefore, the costs for different parts of a translocation process and its dependency on some influencing factors were studied. The observed cost was 465 SEK per translocated log for the actual compensation measure, with an additional 349 SEK/log for work to enable evaluation of the translocation’s ecological results. Based on time studies, models were developed to predict required work time and costs for different transportation distances and load sizes. Those models indicated that short extraction and insertion distances for logs should be prioritized over road transportation distances to minimize costs. They also highlighted a trade-off between costs and time until a given ecological value is reached in the compensation area. The methodology used can contribute to more cost-efficient operations and, by doing so, increase the use of ecological compensation and the benefits from a given input.

14C Distribution in Atmospheric and Aquatic Environments Around Qinshan Nuclear Power Plant, China

Radiocarbon ◽  
2014 ◽  
Vol 56 (3) ◽  
pp. 1107-1114 ◽  
Author(s):  
Zhongtang Wang ◽  
Dan Hu ◽  
Hong Xu ◽  
Qiuju Guo
Keyword(s):  
Power Plant ◽  
Nuclear Power Plant ◽  
Water Samples ◽  
Nuclear Power ◽  
Dissolved Inorganic Carbon ◽  
Tap Water ◽  
Surface Seawater ◽  
Pressurized Water ◽  
Specific Activities ◽  
Water Reactors

Atmospheric CO2 and aquatic water samples were analyzed to evaluate the environmental 14C enrichment due to operation of the Qinshan nuclear power plant (NPP), where two heavy-water reactors and five pressurized-water reactors are employed. Elevated 14C-specific activities (2–26.7 Bq/kg C) were observed in the short-term air samples collected within a 5-km radius, while samples over 5 km were close to background levels. The 14C-specific activities of dissolved inorganic carbon (DIC) in the surface seawater samples ranged from 196.8 to 206.5 Bq/kg C (average 203.4 Bq/kg C), which are close to the background value. No elevated 14C level in surface seawater was found after 20 years of operation of Qinshan NPP, indicating that the 14C discharged was well diffused. The results of the freshwater samples show that excess 14C-specific activity (average 17.1 Bq/kg C) was found in surface water and well water samples, while no obvious 14C increase was found in drinking water (groundwater and tap water) compared to the background level.

scholarly journals Visual multiple cross displacement amplification for the rapid identification of S. agalactiae immediately from vaginal and rectal swabs

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xueqin Cheng ◽  
Zhiqian Dou ◽  
Jing Yang ◽  
Dexi Liu ◽  
Yulong Gu ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Clinical Specimen ◽  
Rapid Identification ◽  
Sample Collection ◽  
Special Equipment ◽  
Multiple Cross ◽  
Rectal Swabs ◽  
Cost Efficient ◽  
The Cost ◽  
Positive Results

AbstractStreptococcus agalactiae (S. agalactiae) is an important pathogen that can lead to neonatus and mother infection. The current existing techniques for the identification of S. agalactiae are limited by accuracy, speed and high-cost. Therefore, a new multiple cross displacement amplification (MCDA) assay was developed for test of the target pathogen immediately from vaginal and rectal swabs. MCDA primers screening were conducted targeting S. agalactiae pcsB gene, and one set of MCDA primers with better rapidity and efficiency was selected for establishing the S. agalactiae-MCDA assay. As a result, the MCDA method could be completed at a constant temperature of 61 °C, without the requirement of special equipment. The detection limit is 250 fg (31.5 copies) per reaction, all S. agalactiae strains displayed positive results, but not for non-S. agalactiae strains. The visual MCDA assay detected 16 positive samples from 200 clinical specimen, which were also detected positive by enrichment/qPCR. While the CHROMagar culture detected 6 positive samples. Thus, the MCDA assay is prefer to enrichment/qPCR and culture for detecting S. agalactiae from clinical specimen. Particularly, the whole test of MCDA takes about 63.1 min, including sample collection (3 min), DNA preparation (15 min), MCDA reaction (45 min) and result reporting (6 s). In addition, the cost was very economic, with only US$ 4.9. These results indicated that our S. agalaciae-MCDA assay is a rapid, sensitive and cost-efficient technique for target pathogen detection, and is more suitable than conventional assays for an urgent detection, especially for 'on-site' laboratories and resource-constrained settings.

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