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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 in a complex mixture. Sensitivity of detection of Blepharisma from a complex mixture of protozoa immobilised on FTA paper. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Amplified products from a range of areas of FTA paper were loaded on the gel. These areas contained the following equivalent number of Blepharisma cells: 2.48 cells (lane 3), 1.24 cells (lane 4), 0.62 cells (lane 5). The amplification products run in lane 6 contained the same area of FTA paper as in lane 4 except that they were taken from the mixed culture of protozoa which did not contain Blepharisma. Lane 1 contained pure Blepharisma DNA, immobilised on FTA paper, as a positive control for the PCR reaction while Lane 2 contained Paramecium caudatum DNA, on FTA paper, as a negative control. Larger areas of FTA paper (eg lane 3) consistently inhibited the PCR reaction.
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Figure 4: Sensitivity of detection in a complex mixture. Sensitivity of detection of Blepharisma from a complex mixture of protozoa immobilised on FTA paper. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Amplified products from a range of areas of FTA paper were loaded on the gel. These areas contained the following equivalent number of Blepharisma cells: 2.48 cells (lane 3), 1.24 cells (lane 4), 0.62 cells (lane 5). The amplification products run in lane 6 contained the same area of FTA paper as in lane 4 except that they were taken from the mixed culture of protozoa which did not contain Blepharisma. Lane 1 contained pure Blepharisma DNA, immobilised on FTA paper, as a positive control for the PCR reaction while Lane 2 contained Paramecium caudatum DNA, on FTA paper, as a negative control. Larger areas of FTA paper (eg lane 3) consistently inhibited the PCR reaction.

Mentions: To investigate whether Whatman FTA paper could be used as a useful medium for testing samples for Blepharisma, we micropipetted 5 μl of a Blepharisma culture containing known numbers of cells onto FTA paper and allowed to dry overnight according to the instructions. The number of Blepharisma cells was calculated for each area of FTA paper. The results showed that we could clearly detect an amplification product when the area of FTA paper was equivalent to 1.25 Blepharisma cells but not from an equivalent of 0.625 Blepharisma cells. (Figure 3). Repeated experiments demonstrated that, on average, this technique could be used to detect levels of Blepharisma down to the equivalent of a single cell. Larger samples of the FTA paper consistently inhibited the PCR reaction (eg. Figure 3, lanes 3 and 4). To investigate the robustness of the FTA detection system, we investigated the sensitivity of the system using a larger water sample volume which also contained an excess of other organisms. A 1 ml culture mixture was made which contained Paramecium caudatum, Euglena gracilis, Stentor coeruleus and a known number of Blepharisma japonicum (99 cells/ml). The tube was centrifuged for 10 minutes at 10000 rpm in a microcentrifuge to concentrate the organisms and the pellet was resuspended in 50 μl of water. 5 μl samples were spotted onto the FTA paper. Figure 4 depicts the results of an amplification using known proportions of the FTA paper. Detection was achieved from an area of FTA paper equivalent to 0.62 Blepharisma cells. This showed that, even in the presence of other organisms, this system could detect levels of Blepharisma down to just less than a single cell equivalent. A parallel sample in which the Blepharisma was replaced with an equivalent volume of water showed no amplification (Figure 4 lane 6). Several experiments, identical to those described in Figure 4, were conducted in which water taken from natural sources (River Irwell, Salford; local ponds and a canal near Salford) was used to make up the 1 ml mixed cultures of Blepharisma, Stentor and Paramecium. In all cases the results showed that Blepharisma could be specifically detected and that no inhibition was achieved using real environmental samples.


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 in a complex mixture. Sensitivity of detection of Blepharisma from a complex mixture of protozoa immobilised on FTA paper. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Amplified products from a range of areas of FTA paper were loaded on the gel. These areas contained the following equivalent number of Blepharisma cells: 2.48 cells (lane 3), 1.24 cells (lane 4), 0.62 cells (lane 5). The amplification products run in lane 6 contained the same area of FTA paper as in lane 4 except that they were taken from the mixed culture of protozoa which did not contain Blepharisma. Lane 1 contained pure Blepharisma DNA, immobilised on FTA paper, as a positive control for the PCR reaction while Lane 2 contained Paramecium caudatum DNA, on FTA paper, as a negative control. Larger areas of FTA paper (eg lane 3) consistently inhibited the PCR reaction.
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Related In: Results  -  Collection

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Figure 4: Sensitivity of detection in a complex mixture. Sensitivity of detection of Blepharisma from a complex mixture of protozoa immobilised on FTA paper. Lanes denoted M contain molecular size markers – figures refer to sizes in base pairs. Amplified products from a range of areas of FTA paper were loaded on the gel. These areas contained the following equivalent number of Blepharisma cells: 2.48 cells (lane 3), 1.24 cells (lane 4), 0.62 cells (lane 5). The amplification products run in lane 6 contained the same area of FTA paper as in lane 4 except that they were taken from the mixed culture of protozoa which did not contain Blepharisma. Lane 1 contained pure Blepharisma DNA, immobilised on FTA paper, as a positive control for the PCR reaction while Lane 2 contained Paramecium caudatum DNA, on FTA paper, as a negative control. Larger areas of FTA paper (eg lane 3) consistently inhibited the PCR reaction.
Mentions: To investigate whether Whatman FTA paper could be used as a useful medium for testing samples for Blepharisma, we micropipetted 5 μl of a Blepharisma culture containing known numbers of cells onto FTA paper and allowed to dry overnight according to the instructions. The number of Blepharisma cells was calculated for each area of FTA paper. The results showed that we could clearly detect an amplification product when the area of FTA paper was equivalent to 1.25 Blepharisma cells but not from an equivalent of 0.625 Blepharisma cells. (Figure 3). Repeated experiments demonstrated that, on average, this technique could be used to detect levels of Blepharisma down to the equivalent of a single cell. Larger samples of the FTA paper consistently inhibited the PCR reaction (eg. Figure 3, lanes 3 and 4). To investigate the robustness of the FTA detection system, we investigated the sensitivity of the system using a larger water sample volume which also contained an excess of other organisms. A 1 ml culture mixture was made which contained Paramecium caudatum, Euglena gracilis, Stentor coeruleus and a known number of Blepharisma japonicum (99 cells/ml). The tube was centrifuged for 10 minutes at 10000 rpm in a microcentrifuge to concentrate the organisms and the pellet was resuspended in 50 μl of water. 5 μl samples were spotted onto the FTA paper. Figure 4 depicts the results of an amplification using known proportions of the FTA paper. Detection was achieved from an area of FTA paper equivalent to 0.62 Blepharisma cells. This showed that, even in the presence of other organisms, this system could detect levels of Blepharisma down to just less than a single cell equivalent. A parallel sample in which the Blepharisma was replaced with an equivalent volume of water showed no amplification (Figure 4 lane 6). Several experiments, identical to those described in Figure 4, were conducted in which water taken from natural sources (River Irwell, Salford; local ponds and a canal near Salford) was used to make up the 1 ml mixed cultures of Blepharisma, Stentor and Paramecium. In all cases the results showed that Blepharisma could be specifically detected and that no inhibition was achieved using real environmental samples.

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