Development of an 18S rRNA gene targeted PCR based diagnostic for the blue crab parasite Hematodinium sp.

Gruebl, Tomas; Frischer, Marc E.; Sheppard, M.; Neumann, M.; Maurer, Andreas; Lee, Richard F.

doi:10.3354/dao049061

Cited by 68 publications

(108 citation statements)

References 24 publications

(24 reference statements)

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“…PCR diagnosis has mostly been used to evaluate the presence of DNA from I used a universal primer set targeting highly conserved regions of 18S rDNA for PCR amplification. This universal primer set has been utilized as reliable primers from several studies (Gruebl et al, 2002;Troedsson et al, 2008aTroedsson et al, , 2008b. The detection limit of PNA-PCR in this study was relatively lower than those of species-specific PCR (10 3 -10 2 copies) (data not shown), which might be acceptable for a surveillance tool of parasitism because a species-specific PCR assay does not verify the existence of various parasitism infections (Burreson, 2008).…”

Section: Discussionmentioning

confidence: 91%

Development of a Denaturing High-Performance Liquid Chromatography (DHPLC) Assay to Detect Parasite Infection in Grass Shrimp Palaemonetes pugio

Cho¹

2012

Fisheries and aquatic sciences

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In developing a useful tool to detect parasitic dynamics in an estuarine ecosystem, a denaturing high-performance liquid chromatography (DHPLC) assay was optimized by cloning plasmid DNA from the grass shrimp Palaemonetes pugio, and its two parasites, the trematode Microphallus turgidus and bopyrid isopod Probopyrus pandalicola. The optimal separation condition was an oven temperature of 57.9°C and 62-68% of buffer B gradient at a flow rate of 0.45 mL/min. A peptide nucleic acid blocking probe was designed to clamp the amplification of the host gene, which increased the amplification efficiency of genes with low copy numbers. Using the DHPLC assay with wild-type genomic, the assay could detect GC Gram positive bacteria and the bopyrid isopod (P. pandalicola). Therefore, the DHPLC assay is an effective tool for surveying parasitic dynamics in an estuarine ecosystem.

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Section: Discussionmentioning

confidence: 91%

Development of a Denaturing High-Performance Liquid Chromatography (DHPLC) Assay to Detect Parasite Infection in Grass Shrimp Palaemonetes pugio

Cho¹

2012

Fisheries and aquatic sciences

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“…Furthermore, several phylogenetic analyses suggest that the SSU rRNA sequences of Hematodinium sp. (Syndiniales), a parasite of the blue crab (Gruebl et al 2002), is closely related to those of Group II. Therefore the whole Group II may corre-285 Fig.…”

Section: Discussionmentioning

confidence: 99%

Genetic diversity and habitats of two enigmatic marine alveolate lineages

Groisillier¹,

Massana²,

Valentin³

et al. 2006

Aquat. Microb. Ecol.

107

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Systematic sequencing of environmental SSU rDNA genes amplified from different marine ecosystems has uncovered novel eukaryotic lineages, in particular within the alveolate and stramenopile radiations. The ecological and geographic distribution of 2 novel alveolate lineages (called Group I and II in previous papers) is inferred from the analysis of 62 different environmental clone libraries from freshwater and marine habitats. These 2 lineages have been, up to now, retrieved exclusively from marine ecosystems, including oceanic and coastal waters, sediments, hydrothermal vents, and permanent anoxic deep waters and usually represent the most abundant eukaryotic lineages in environmental genetic libraries. While Group I is only composed of environmental sequences (118 clones), Group II contains, besides environmental sequences (158 clones), sequences from described genera (8) (Hematodinium and Amoebophrya) that belong to the Syndiniales, an atypical order of dinoflagellates exclusively composed of marine parasites. This suggests that Group II could correspond to Syndiniales, although this should be confirmed in the future by examining the morphology of cells from Group II. Group II appears to be abundant in coastal and oceanic ecosystems, whereas permanent anoxic waters and hydrothermal ecosystems are usually dominated by Group I. Based upon the similarity of partial sequences, we organized these 2 groups into clusters. The diversity of Group II (16 clusters) is wider than that of Group I (5 clusters). Two clusters from Group I have a widespread distribution and are found in all explored marine habitats. In contrast, all other clusters seem to be limited to specific marine habitats. For example, some clusters belonging to Group I and Group II are only detected in extreme environments (anoxic and hydrothermal vents), whereas many clusters from Group II have only been retrieved from coastal waters. We determined near-complete SSU rRNA gene sequences for 26 environmental clones, selected in order to obtain at least one complete sequence per cluster. Phylogenetic analyses (maximum likelihood, neighbor joining, maximum parsimony, and Bayesian reconstruction) based upon complete sequences all concurred to place both Group I and II as sister lineages of dinoflagellates. This result contradicts several published studies, which placed both groups within dinoflagellates.KEY WORDS: Alveolates · rRNA environmental sequences · Group I and Group II · Dinoflagellates · Syndiniales · BiogeographyResale or republication not permitted without written consent of the publisher

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“…(Gruebl et al 2002, Small et al 2007a. A 196 bp fragment of the SSU coding region was amplified from the Hematodinium sp.…”

Section: Methodsmentioning

confidence: 99%

Laser-assisted microdissection: a new tool for aquatic molecular parasitology

Small¹,

Sturve²,

Bignell³

et al. 2008

Dis. Aquat. Org.

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Laser-assisted microdissection (LMD) has been developed to isolate distinct cell populations from heterogeneous tissue sections, cytological preparations, or live cell samples. Downstream applications typically include gene expression studies using real-time PCR and array platforms, diagnostic PCR, and protein expression studies. LMD techniques are now commonplace in mainstream biological research and clearly have suitable applications in the field of aquatic pathology and parasitology. The present study used LMD to isolate 2 dinoflagellate parasites (Hematodinium spp.) from formalin-fixed paraffin-embedded tissue sections from 2 crustacean hosts, Cancer pagurus and Portunus trituberculatus. DNA was isolated from LMD parasite preparations, and partial regions (up to 300 bp) of the small subunit and the first internal transcribed spacer region of the rRNA gene complex from the Hematodinium spp. were PCR amplified using diagnostic primers. The amplification products were sequenced to confirm the identity of the targeted regions. The techniques, applications, and limitations of LMD to address questions in aquatic molecular pathology and parasitology are discussed.KEY WORDS: Laser-assisted microdissection · Molecular parasitology · DNA · RNA · Aquatic parasitologyResale or republication not permitted without written consent of the publisher

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Development of an 18S rRNA gene targeted PCR based diagnostic for the blue crab parasite Hematodinium sp.

Cited by 68 publications

References 24 publications

Development of a Denaturing High-Performance Liquid Chromatography (DHPLC) Assay to Detect Parasite Infection in Grass Shrimp Palaemonetes pugio

Development of a Denaturing High-Performance Liquid Chromatography (DHPLC) Assay to Detect Parasite Infection in Grass Shrimp Palaemonetes pugio

Genetic diversity and habitats of two enigmatic marine alveolate lineages

Laser-assisted microdissection: a new tool for aquatic molecular parasitology

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