Dual quantitative PCR assay for identification and enumeration of Karlodinium veneficum and Karlodinium armiger combined with a simple and rapid DNA extraction method

Toldrà, Anna; Andrée, Karl B.; Fernández-Tejedor, Margarita; Diogène, Jorge; Campàs, Mònica

doi:10.1007/s10811-018-1446-x

Cited by 28 publications

(11 citation statements)

References 40 publications

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“…Karlodinium -specific primers [ 42 ] were also utilized for differentiating amplicons obtained from DNA extracts of bloom samples (seawater DNA extracts) by comparing melt curve profiles of previously sequenced strains to the melt curves obtained from seawater extracts ( Table 1 ). Amplifications were performed in an ABI 7300 (Life Technologies, Carlsbad, CA, USA) in a 20 µL volume containing 0.5 µM of each primer and 1× concentration of SYBR Green reaction mix (Ref# 4364344; Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean)

et al. 2017

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Abstract:A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

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Section: Methodsmentioning

confidence: 99%

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean)

et al. 2017

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“…In our study, we used the recently developed Biomeme system for DNA extraction and portable real-time qPCR (Thomas et al, 2019) that allowed us to process samples in the field or laboratory immediately after collection, in contrast to previous studies that have used traditional bench-top procedures and instruments in the lab. Prior studies have found similar DNA extraction efficiency between the Biomeme system and other common methods (Rudko et al, 2018;Toldrà et al, 2018). Others have found similar eDNA detection levels between the Biomeme extraction and qPCR methods (end-to-end) and conventional laboratory procedures (Nguyen et al, 2018;Thomas et al, 2019), but one study did find lower detection rates using the Biomeme system due to both a higher rate of inhibition and lower sensitivity (Sepulveda et al, 2018).…”

Section: Water Sample Water Samplementioning

confidence: 97%

Efficacy of environmental DNA sampling to detect the occurrence of endangered coho salmon (Oncorhynchus kisutch) in Mediterranean‐climate streams of California's central coast

et al. 2020

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Analysis of environmental DNA (eDNA) has emerged as an important tool for investigating the occurrence and distribution of fish and other species in aquatic environments. However, in lotic systems, uncertainty remains about how environmental factors influence the downstream transport, degradation, and dilution of eDNA, and hence how detection probability varies with distance from the eDNA source. We conducted cage experiments and paired eDNA and snorkel surveys to evaluate the potential for eDNA methods to detect endangered coho salmon in Mediterraneanclimate streams of the Santa Cruz Mountains, California. Juvenile coho salmon at two densities were placed in cages, and water samples were collected and analyzed (qPCR) at seven locations from 10 to 1,000 m downstream. Although we detected coho salmon eDNA up to 1,000 m downstream of the cage, the vast majority of detections were within 200 m, and detections at greater distances occurred only at the higher fish density. In field collections from 21 stream reaches across the Santa Cruz Mountains, eDNA methods and snorkeling produced identical outcomes in terms of reach-level detection of coho salmon. Probability of occurrence in a water sample and detection in a qPCR replicate both increased with observed fish density; however, even at the lowest densities, a protocol of three water samples and two qPCR replicates resulted in a >90% cumulative probability of detection. Overall, our studies suggest that detection probability decreased rapidly with distance in Santa Cruz Mountain streams, likely because low stream discharges, slow transport velocities, and strong interaction between the water column and stream substrate result in rapid degradation or settling of eDNA. Our findings thus support the use of eDNA methods for detecting rare species, but also indicate that local conditions strongly influence transport dynamics and hence need to be considered when developing survey designs.

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“…Afterwards, 50 mL of culture were harvested at the exponential phase by centrifugation (4300 g, 20 min). The resulting cell pellet was processed for DNA extraction using the phenol/chloroform/isoamylalcohol extraction (PCI) protocol according to Toldrà et al [88]. Genomic DNA was quantified and checked for its purity using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).…”

Section: Molecular Identificationmentioning

confidence: 99%

Further Advance of Gambierdiscus Species in the Canary Islands, with the First Report of Gambierdiscus belizeanus

Tudó

Gaiani

Varela

et al. 2020

Toxins

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Ciguatera Poisoning (CP) is a human food-borne poisoning that has been known since ancient times to be found mainly in tropical and subtropical areas, which occurs when fish or very rarely invertebrates contaminated with ciguatoxins (CTXs) are consumed. The genus of marine benthic dinoflagellates Gambierdiscus produces CTX precursors. The presence of Gambierdiscus species in a region is one indicator of CP risk. The Canary Islands (North Eastern Atlantic Ocean) is an area where CP cases have been reported since 2004. In the present study, samplings for Gambierdiscus cells were conducted in this area during 2016 and 2017. Gambierdiscus cells were isolated and identified as G. australes, G. excentricus, G. caribaeus, and G. belizeanus by molecular analysis. In this study, G. belizeanus is reported for the first time in the Canary Islands. Gambierdiscus isolates were cultured, and the CTX-like toxicity of forty-one strains was evaluated with the neuroblastoma cell-based assay (neuro-2a CBA). G. excentricus exhibited the highest CTX-like toxicity (9.5–2566.7 fg CTX1B equiv. cell−1) followed by G. australes (1.7–452.6.2 fg CTX1B equiv. cell−1). By contrast, the toxicity of G. belizeanus was low (5.6 fg CTX1B equiv. cell−1), and G. caribaeus did not exhibit CTX-like toxicity. In addition, for the G. belizeanus strain, the production of CTXs was evaluated with a colorimetric immunoassay and an electrochemical immunosensor resulting in G. belizeanus producing two types of CTX congeners (CTX1B and CTX3C series congeners) and can contribute to CP in the Canary Islands.

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Dual quantitative PCR assay for identification and enumeration of Karlodinium veneficum and Karlodinium armiger combined with a simple and rapid DNA extraction method

Cited by 28 publications

References 40 publications

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean)

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean)

Efficacy of environmental DNA sampling to detect the occurrence of endangered coho salmon (Oncorhynchus kisutch) in Mediterranean‐climate streams of California's central coast

Further Advance of Gambierdiscus Species in the Canary Islands, with the First Report of Gambierdiscus belizeanus

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