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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.

Show MeSH
Sensitivity of detection of Blepharisma japonicum. Sensitivity of detection of Blepharisma japonicum by PCR amplification. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Dilutions of target DNA were amplified as follows: undiluted (lane 1), 1/5 (lane 2), 1/25 (lane 3), 1/125 (lane 4), 1/625 (lane 5), 1/3125 (lane 6). Lane 7 contains undiluted Paramecium caudatum DNA as a negative control.
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Figure 2: Sensitivity of detection of Blepharisma japonicum. Sensitivity of detection of Blepharisma japonicum by PCR amplification. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Dilutions of target DNA were amplified as follows: undiluted (lane 1), 1/5 (lane 2), 1/25 (lane 3), 1/125 (lane 4), 1/625 (lane 5), 1/3125 (lane 6). Lane 7 contains undiluted Paramecium caudatum DNA as a negative control.

Mentions: To investigate the sensitivity of this process for the detection of Blepharisma, we carried out serial dilutions of DNA extracted from a known number of Blepharisma which were micromanipulated into an extraction tube and counted manually prior to extraction. The results are depicted in Figure 2 which shows that an amplification product can be clearly seen in a 1:3125 dilution. This was equivalent to 0.015 of a Blepharisma cell. Detection at this level was reproducible over a number of experiments. Further dilution to 1:10000 (equivalent to 0.005 cells) occasionally produced a faint band in some experiments.


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)

Sensitivity of detection of Blepharisma japonicum. Sensitivity of detection of Blepharisma japonicum by PCR amplification. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Dilutions of target DNA were amplified as follows: undiluted (lane 1), 1/5 (lane 2), 1/25 (lane 3), 1/125 (lane 4), 1/625 (lane 5), 1/3125 (lane 6). Lane 7 contains undiluted Paramecium caudatum DNA as a negative control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC239857&req=5

Figure 2: Sensitivity of detection of Blepharisma japonicum. Sensitivity of detection of Blepharisma japonicum by PCR amplification. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Dilutions of target DNA were amplified as follows: undiluted (lane 1), 1/5 (lane 2), 1/25 (lane 3), 1/125 (lane 4), 1/625 (lane 5), 1/3125 (lane 6). Lane 7 contains undiluted Paramecium caudatum DNA as a negative control.
Mentions: To investigate the sensitivity of this process for the detection of Blepharisma, we carried out serial dilutions of DNA extracted from a known number of Blepharisma which were micromanipulated into an extraction tube and counted manually prior to extraction. The results are depicted in Figure 2 which shows that an amplification product can be clearly seen in a 1:3125 dilution. This was equivalent to 0.015 of a Blepharisma cell. Detection at this level was reproducible over a number of experiments. Further dilution to 1:10000 (equivalent to 0.005 cells) occasionally produced a faint band in some experiments.

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