A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates
<p>Abstract</p> <p>Background</p> <p>Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2008-10-01
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| Series: | Malaria Journal |
| Online Access: | http://www.malariajournal.com/content/7/1/222 |
| _version_ | 1828493332313014272 |
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| author | Weissenbach Matthew E Shoue Doug A Balu Bharath Maher Steven P Adams John H |
| author_facet | Weissenbach Matthew E Shoue Doug A Balu Bharath Maher Steven P Adams John H |
| author_sort | Weissenbach Matthew E |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies.</p> <p>Methods</p> <p>Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite <it>18s rRNA </it>gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis.</p> <p>Results</p> <p>The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity.</p> <p>Conclusion</p> <p>The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.</p> |
| first_indexed | 2024-12-11T11:36:29Z |
| format | Article |
| id | doaj.art-48a3ee96084e4f49bfd332db98b15ed9 |
| institution | Directory Open Access Journal |
| issn | 1475-2875 |
| language | English |
| last_indexed | 2024-12-11T11:36:29Z |
| publishDate | 2008-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Malaria Journal |
| spelling | doaj.art-48a3ee96084e4f49bfd332db98b15ed92022-12-22T01:08:44ZengBMCMalaria Journal1475-28752008-10-017122210.1186/1475-2875-7-222A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter platesWeissenbach Matthew EShoue Doug ABalu BharathMaher Steven PAdams John H<p>Abstract</p> <p>Background</p> <p>Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies.</p> <p>Methods</p> <p>Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite <it>18s rRNA </it>gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis.</p> <p>Results</p> <p>The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity.</p> <p>Conclusion</p> <p>The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.</p>http://www.malariajournal.com/content/7/1/222 |
| spellingShingle | Weissenbach Matthew E Shoue Doug A Balu Bharath Maher Steven P Adams John H A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates Malaria Journal |
| title | A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates |
| title_full | A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates |
| title_fullStr | A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates |
| title_full_unstemmed | A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates |
| title_short | A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates |
| title_sort | highly sensitive pcr based method for the detection of it plasmodium falciparum it clones in microtiter plates |
| url | http://www.malariajournal.com/content/7/1/222 |
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