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A rapid and simple method of detection of Blepharisma japonicum using PCR and immobilisation on FTA paper.

Hide G, Hughes JM, McNuff R - BMC Ecol. (2003)

Bottom Line: Using primers designed from the alpha-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved.Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper.This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Parasite Biology, Molecular Epidemiology and Ecology, Biosciences Research Institute, School of Environment and Life Sciences, University of Salford, UK, M5 4WT. g.hide@salford.ac.uk

ABSTRACT

Background: The rapid expansion in the availability of genome and DNA sequence information has opened up new possibilities for the development of methods for detecting free-living protozoa in environmental samples. The protozoan Blepharisma japonicum was used to investigate a rapid and simple detection system based on polymerase chain reaction amplification (PCR) from organisms immobilised on FTA paper.

Results: Using primers designed from the alpha-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved. Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper.

Conclusion: This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms.

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Related in: MedlinePlus

Specifity of detection of Blepharisma japonicum. Specificity of detection of Blepharisma japonicum using gel electrophoresis. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Protozoan species: Paramecium putrinum (lane 1), Climacostomum virens (lane 2), Amoeba proteus (lane 3), Vorticella microstoma (lane 4), Colpidium campylum (lane5), Spirostomum ambiguum (lane 6), Chaos carolinensis (lane 7), Stentor coeruleus (lanes 8 – 10), Paramecium caudatum (lane 11), Blepharisma japonicum (lane 12). (Three different isolates of Stentor – different size morphs – were incorporated).
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Figure 1: Specifity of detection of Blepharisma japonicum. Specificity of detection of Blepharisma japonicum using gel electrophoresis. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Protozoan species: Paramecium putrinum (lane 1), Climacostomum virens (lane 2), Amoeba proteus (lane 3), Vorticella microstoma (lane 4), Colpidium campylum (lane5), Spirostomum ambiguum (lane 6), Chaos carolinensis (lane 7), Stentor coeruleus (lanes 8 – 10), Paramecium caudatum (lane 11), Blepharisma japonicum (lane 12). (Three different isolates of Stentor – different size morphs – were incorporated).

Mentions: PCR amplification of DNA extracted from Blepharisma japonicum using primers BLEPTUBF and BLEPTUBR produced a band which co-migrated with the 400 bp marker and was therefore consistent with the predicted fragment size of 399 base pairs (Figure 1). This product was consistently produced when other independent cultures of Blepharisma were also tested. To investigate the specificity of this amplification reaction, we extracted DNA from a further 18 species of protozoa (Table 1) which represented 3 protozoan phyla and also included a genus, Spirostomum, which is believed to be closely related to Blepharisma, [15]. In all cases no amplification of the 399 bp fragment was detected under the conditions used (Figure 1).


A rapid and simple method of detection of Blepharisma japonicum using PCR and immobilisation on FTA paper.

Hide G, Hughes JM, McNuff R - BMC Ecol. (2003)

Specifity of detection of Blepharisma japonicum. Specificity of detection of Blepharisma japonicum using gel electrophoresis. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Protozoan species: Paramecium putrinum (lane 1), Climacostomum virens (lane 2), Amoeba proteus (lane 3), Vorticella microstoma (lane 4), Colpidium campylum (lane5), Spirostomum ambiguum (lane 6), Chaos carolinensis (lane 7), Stentor coeruleus (lanes 8 – 10), Paramecium caudatum (lane 11), Blepharisma japonicum (lane 12). (Three different isolates of Stentor – different size morphs – were incorporated).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC239857&req=5

Figure 1: Specifity of detection of Blepharisma japonicum. Specificity of detection of Blepharisma japonicum using gel electrophoresis. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Protozoan species: Paramecium putrinum (lane 1), Climacostomum virens (lane 2), Amoeba proteus (lane 3), Vorticella microstoma (lane 4), Colpidium campylum (lane5), Spirostomum ambiguum (lane 6), Chaos carolinensis (lane 7), Stentor coeruleus (lanes 8 – 10), Paramecium caudatum (lane 11), Blepharisma japonicum (lane 12). (Three different isolates of Stentor – different size morphs – were incorporated).
Mentions: PCR amplification of DNA extracted from Blepharisma japonicum using primers BLEPTUBF and BLEPTUBR produced a band which co-migrated with the 400 bp marker and was therefore consistent with the predicted fragment size of 399 base pairs (Figure 1). This product was consistently produced when other independent cultures of Blepharisma were also tested. To investigate the specificity of this amplification reaction, we extracted DNA from a further 18 species of protozoa (Table 1) which represented 3 protozoan phyla and also included a genus, Spirostomum, which is believed to be closely related to Blepharisma, [15]. In all cases no amplification of the 399 bp fragment was detected under the conditions used (Figure 1).

Bottom Line: Using primers designed from the alpha-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved.Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper.This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Parasite Biology, Molecular Epidemiology and Ecology, Biosciences Research Institute, School of Environment and Life Sciences, University of Salford, UK, M5 4WT. g.hide@salford.ac.uk

ABSTRACT

Background: The rapid expansion in the availability of genome and DNA sequence information has opened up new possibilities for the development of methods for detecting free-living protozoa in environmental samples. The protozoan Blepharisma japonicum was used to investigate a rapid and simple detection system based on polymerase chain reaction amplification (PCR) from organisms immobilised on FTA paper.

Results: Using primers designed from the alpha-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved. Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper.

Conclusion: This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms.

Show MeSH
Related in: MedlinePlus