Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

Download Full-text

Quantitative PCR assays to detect humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge) in marine water samples

10.1101/2020.08.17.240275 ◽

2020 ◽

Author(s):

Elizabeth A. Andruszkiewicz ◽

Kevan M. Yamahara ◽

Collin J. Closek ◽

Alexandria B. Boehm

Keyword(s):

Quantitative Pcr ◽

Water Samples ◽

Humpback Whale ◽

Megaptera Novaeangliae ◽

Individual Species ◽

Uria Aalge ◽

Common Murre ◽

Assay Design ◽

Species Specific

AbstractMonitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. In order to obtain quantitative datasets for individual species, species-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre to the genus-specific shortbelly rockfish assay, to the humpback whale assay which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

Download Full-text

Development of a quantitative PCR assay for detection of redside shiner (Richardsonius balteatus) from environmental DNA

BMC Research Notes ◽

10.1186/s13104-019-4819-6 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

Marshal S. Hoy ◽

Carl O. Ostberg

Keyword(s):

Quantitative Pcr ◽

Water Samples ◽

Effective Means ◽

Environmental Dna ◽

Sympatric Species ◽

Negative Control ◽

Control Site ◽

Qpcr Assay ◽

Redside Shiner ◽

Richardsonius Balteatus

Abstract Objective A quantitative PCR (qPCR) assay for the detection of redside shiner (Richardsonius balteatus) environmental DNA (eDNA) was designed as a side product of a larger project aimed at using eDNA to determine the presence and geographic extent of native and non-native fishes in the reservoirs and associated tributaries above the three mainstem dams (Ross, Diablo, Gorge) on the Skagit River, Washington, USA. The eDNA survey results can be used to help guide additional sampling efforts that include traditional sampling methods, such as electrofishing and netting. Results The redside shiner qPCR assay (RSSCOI_540-601) was validated by testing for sensitivity using redside shiner genomic DNA from three different populations and by testing for specificity against 30 potentially sympatric species. No non-target amplification was observed in our validation tests. We then evaluated the assay on field-collected water samples where there are known populations of redside shiner and a negative control site where the target species is known to be absent. The field-collected water samples tested positive at the redside shiner sites and tested negative at the negative control site. The assay could provide resource managers with an effective means for surveying and monitoring redside shiner populations.

Download Full-text

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

Conservation Genetics Resources ◽

10.1007/s12686-017-0812-3 ◽

2017 ◽

Vol 10 (3) ◽

pp. 317-319 ◽

Cited By ~ 13

Author(s):

Patrick R. Hutchins ◽

Adam J. Sepulveda ◽

Renee M. Martin ◽

Lacey R. Hopper

Keyword(s):

Quantitative Pcr ◽

Water Samples ◽

Environmental Dna ◽

Fish Tissue ◽

Pcr Assay ◽

Tetracapsuloides Bryosalmonae ◽

Quantitative Pcr Assay

Download Full-text

The Function of Ophiocordyceps sinensis in Airway Epithelial Cell Senescence in a Rat COPD Model

Canadian Respiratory Journal ◽

10.1155/2018/6080348 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9

Author(s):

Xiaohui Ma ◽

Xingai Jiao ◽

Jinxiang Wu ◽

Jiping Zhao ◽

Yurong Xu ◽

...

Keyword(s):

Epithelial Cell ◽

Quantitative Pcr ◽

Airway Epithelial Cell ◽

Cell Senescence ◽

Airway Epithelial ◽

Airway Epithelia ◽

Real Time Quantitative Pcr ◽

Ophiocordyceps Sinensis

Ophiocordyceps sinensis (O. sinensis) seems to be able to alleviate airway epithelial cell senescence in chronic obstructive pulmonary disease (COPD). The objective of the study is to evaluate the effect of O. sinensis on airway epithelial senescence in the COPD model both in vitro and in vivo. We observed the expression of P16 and P21 in the airway epithelia of 30 patients with COPD. The optimal concentration of O. sinensis and exposure time of the cigarette smoke extract (CSE) were determined in vitro, and senescence-associated β-galactosidase (SA-β-gal) and 5-bromodeoxyuridine (BrdU) were used to evaluate the senescence and proliferation of human bronchial epithelial (16HBE) cells pretreated with O. sinensis by staining kits. COPD model rats were treated with O. sinensis at various concentrations to determine the changes in P16 and P21 expression in airway epithelial tissues. It was found that the expression levels of P16 and P21 were higher in the airway epithelia of COPD patients than those in the control group based on immunohistochemical staining, real-time quantitative PCR, and western blotting. The CSE could induce 16HBE cell senescence, and O. sinensis could alleviate CSE-induced senescence and promote the proliferation of 16HBE cells. The expression levels of P16 and P21 were also higher in the airway epithelia of COPD model rats; however, the levels of P16 and P21 in the groups treated with all concentrations of O. sinensis were obviously lower than those in the COPD model group based on real-time quantitative PCR and western blotting. In conclusion, the CSE can induce airway epithelium senescence, and O. sinensis can inhibit CSE-induced cellular senescence, both in vitro and in vivo.

Download Full-text

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus

Thrombosis and Haemostasis ◽

10.1160/th04-02-0068 ◽

2004 ◽

Vol 92 (08) ◽

pp. 317-327 ◽

Cited By ~ 4

Author(s):

Dmitri Gnatenko ◽

Yong Wu ◽

Jolyon Jesty ◽

Andrea Damon ◽

Patrick Hearing ◽

...

Keyword(s):

Factor Viii ◽

Quantitative Pcr ◽

Hemophilia A ◽

High Titer ◽

Small Nuclear Rna ◽

Genomic Integration ◽

Novel Approach ◽

Fviii Activity

SummaryWe have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIΔ761-1639). Productive replication of Ad/AAV/FVIIIΔ761-1639 in AAV repexpressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIΔ761-1639). In vitro studies using Ad/AAV/FVIIIΔ761-1639 generated ∼2-logs greater FVIII activity than mAd/FVIIIΔ761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIΔ761-1639 was injected by tail vein into three groups of hemophilia A mice (2 X 1011 vp [n = 3]; 4 X 1011 vp [n = 3]; 8 X 1011 vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16% – 51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1 – 0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (∼10 genomes/ cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of “gutted” monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

Download Full-text

Faculty Opinions recommendation of Microfluidic quantitative PCR for simultaneous quantification of multiple viruses in environmental water samples.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718891100.793527549 ◽

2017 ◽

Author(s):

Christiane Wobus

Keyword(s):

Quantitative Pcr ◽

Water Samples ◽

Environmental Water ◽

Environmental Water Samples ◽

Simultaneous Quantification

Download Full-text

Monitoring post-release survival of the northern corroboree frog, Pseudophryne pengilleyi, using environmental DNA

Wildlife Research ◽

10.1071/wr17179 ◽

2018 ◽

Vol 45 (7) ◽

pp. 620 ◽

Cited By ~ 1

Author(s):

Jack Rojahn ◽

Dianne Gleeson ◽

Elise M. Furlan

Keyword(s):

Water Samples ◽

Survey Methods ◽

Quantitative Polymerase Chain Reaction ◽

Environmental Dna ◽

Life Stages ◽

Breeding Programs ◽

Non Invasive ◽

Naturally Occurring ◽

Cause Of Failure

Context Translocations are becoming an increasingly important conservation tool to combat rising levels of species extinction. Unfortunately, many translocation efforts fail; yet, the timing and cause of failure often remain unknown. Monitoring individuals in the days and weeks following release can provide valuable information on their capacity to survive this initial hurdle. In Australia, breeding programs have been established for the endangered northern corroboree frog, Pseudophryne pengilleyi, to enable reintroduction to the wild via captive-reared individuals, typically, early life stages such as eggs or juvenile frogs that cannot be monitored via traditional survey methods that target adult frogs (e.g. shout–response). Environmental DNA (eDNA) detects trace amounts of DNA that organisms release into their environment and could provide a means to infer population persistence for wildlife releases and translocations. Aims In the present study, we aim to develop an eDNA assay capable of detecting both sexes of P. pengilleyi across multiple life stages, and use it to monitor their survival. Methods An eDNA assay was developed to target the two corroboree frog species (P. pengilleyi and P. corroboree, the southern corroboree frog) and was tested for its sensitivity and specificity in silico and in vitro. Pseudophryne pengilleyi eggs were released into three naturally occurring ponds and water samples were, subsequently, collected from each pond on several occasions over a period of 78 days. Quantitative polymerase chain reaction was used to detect P. pengilleyi eDNA from water samples. Key Results The developed assay was shown to be sensitive and specific to corroboree frogs. eDNA monitoring of reintroduced P. pengilleyi detected the species’ DNA at three of three release ponds and DNA remained detectable until at least 78 days post-release at two of three ponds. Conclusions We show how the development of a corroboree frog-specific assay allowed us to monitor the post-release survival of P. pengilleyi in naturally occurring pools. Implications eDNA surveys may provide a useful tool to monitor post-release survival of translocated populations in a non-invasive manner, with the potential to identify the timing and causes of failure. Such knowledge can be used to inform the management of translocated populations and future release strategies.

Download Full-text

In Vitro and In Vivo Bacteriolytic Activities of Escherichia coli Phages: Implications for Phage Therapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.7.2558-2569.2004 ◽

2004 ◽

Vol 48 (7) ◽

pp. 2558-2569 ◽

Cited By ~ 128

Author(s):

Sandra Chibani-Chennoufi ◽

Josette Sidoti ◽

Anne Bruttin ◽

Elizabeth Kutter ◽

Shafiq Sarker ◽

...

Keyword(s):

Escherichia Coli ◽

Drinking Water ◽

Environmental Water ◽

Gut Flora ◽

E Coli ◽

Adult Mice ◽

Fecal Recovery ◽

Gut Mucosa

ABSTRACT Four T4-like coliphages with broad host ranges for diarrhea-associated Escherichia coli serotypes were isolated from stool specimens from pediatric diarrhea patients and from environmental water samples. All four phages showed a highly efficient gastrointestinal passage in adult mice when added to drinking water. Viable phages were recovered from the feces in a dose-dependent way. The minimal oral dose for consistent fecal recovery was as low as 103 PFU of phage per ml of drinking water. In conventional mice, the orally applied phage remained restricted to the gut lumen, and as expected for a noninvasive phage, no histopathological changes of the gut mucosa were detected in the phage-exposed animals. E. coli strains recently introduced into the intestines of conventional mice and traced as ampicillin-resistant colonies were efficiently lysed in vivo by phage added to the drinking water. Likewise, an in vitro phage-susceptible E. coli strain freshly inoculated into axenic mice was lysed in vivo by an orally applied phage, while an in vitro-resistant E. coli strain was not lysed. In contrast, the normal E. coli gut flora of conventional mice was only minimally affected by oral phage application despite the fact that in vitro the majority of the murine intestinal E. coli colonies were susceptible to the given phage cocktail. Apparently, the resident E. coli gut flora is physically or physiologically protected against phage infection.

Download Full-text

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Applied and Environmental Microbiology ◽

10.1128/aem.00205-13 ◽

2013 ◽

Vol 79 (9) ◽

pp. 2891-2898 ◽

Cited By ~ 67

Author(s):

Satoshi Ishii ◽

Takahiro Segawa ◽

Satoshi Okabe

Keyword(s):

Quantitative Pcr ◽

Water Samples ◽

Simultaneous Detection ◽

Environmental Water ◽

Waterborne Pathogens ◽

Fecal Indicator ◽

Content Type ◽

Template Dna ◽

Multiple Pathogens ◽

Taqman Qpcr

ABSTRACTThe direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food- and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (generalEscherichia coli), and a process control strain in the microfluidic qPCR system. We preamplified the template DNA to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach.

Download Full-text