Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense.
Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of Af...
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ftdoajarticles:oai:doaj.org/article:6c91ceeeb3fe4f7e8851a0cf57121d58 2023-05-15T15:10:39+02:00 Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. Zablon Kithinji Njiru Andrew Stanislaw John Mikosza Tanya Armstrong John Charles Enyaru Joseph Mathu Ndung'u Andrew Richard Christopher Thompson 2008-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0000147 https://doaj.org/article/6c91ceeeb3fe4f7e8851a0cf57121d58 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC2238707?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0000147 https://doaj.org/article/6c91ceeeb3fe4f7e8851a0cf57121d58 PLoS Neglected Tropical Diseases, Vol 2, Iss 1, p e147 (2008) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2008 ftdoajarticles https://doi.org/10.1371/journal.pntd.0000147 2022-12-31T04:46:10Z Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was approximately 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 2 2 e147 |
institution |
Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Zablon Kithinji Njiru Andrew Stanislaw John Mikosza Tanya Armstrong John Charles Enyaru Joseph Mathu Ndung'u Andrew Richard Christopher Thompson Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was approximately 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions. |
format |
Article in Journal/Newspaper |
author |
Zablon Kithinji Njiru Andrew Stanislaw John Mikosza Tanya Armstrong John Charles Enyaru Joseph Mathu Ndung'u Andrew Richard Christopher Thompson |
author_facet |
Zablon Kithinji Njiru Andrew Stanislaw John Mikosza Tanya Armstrong John Charles Enyaru Joseph Mathu Ndung'u Andrew Richard Christopher Thompson |
author_sort |
Zablon Kithinji Njiru |
title |
Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. |
title_short |
Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. |
title_full |
Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. |
title_fullStr |
Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. |
title_full_unstemmed |
Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. |
title_sort |
loop-mediated isothermal amplification (lamp) method for rapid detection of trypanosoma brucei rhodesiense. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2008 |
url |
https://doi.org/10.1371/journal.pntd.0000147 https://doaj.org/article/6c91ceeeb3fe4f7e8851a0cf57121d58 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 2, Iss 1, p e147 (2008) |
op_relation |
http://europepmc.org/articles/PMC2238707?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0000147 https://doaj.org/article/6c91ceeeb3fe4f7e8851a0cf57121d58 |
op_doi |
https://doi.org/10.1371/journal.pntd.0000147 |
container_title |
PLoS Neglected Tropical Diseases |
container_volume |
2 |
container_issue |
2 |
container_start_page |
e147 |
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1766341631118671872 |