Development Of Polymerase Chain Reaction-Based Assays For Detection Of Human Malaria
| dc.contributor.author | BOBOGARE, ALBINO | |
| dc.date.accessioned | 2016-07-19T01:42:15Z | |
| dc.date.available | 2016-07-19T01:42:15Z | |
| dc.date.issued | 1997-05 | |
| dc.description.abstract | The nested polymerase chain reaction (peR) assay described by Singh et al. (1996) for epidemiological studies used simple sample collection and DNA template extraction methods for detection of malaria. Its sensitivity had not yet been determined, and the initial objectives of this study were to determine and to improve the sensitivity of this method. The sensitivity of the assay was determined to be 6 parasites per J.lI of blood. When tested on field samples for Plasmodium !alciparum, P. vivax and P. malariae, the nested peR assay was able to detect 9 more samples as positive and 8 more mixed infections compared to microscopy, reinforcing the benefits of nested peR assays for epidemiological studies. The nested peR assay for detection of malaria parasites involves the use of a pair of genus-specific primers for the initial (nest one) peR followed by 4 pairs of species-specific oligonucleotides in four separate nest two amplifications. For malaria epidemiological studies when many samples need to be screened in a short time, it would be more rapid and less costly if the samples could be initially screened by nested peR with genus-specific primers, and only positive samples subjected to subsequent speciation by peR. Therefore, the third objective of this study was to design and test two pairs of Plasmodium genus-specific peR primers which could detect all 4 human Plasmodium species in a nested peR assay, and could also provide template for species-specific nest two peR. Primers for peR assays were designed based on the Plasmodium species genes coding for the small subunit ribosomal RNA. The genus-specific PCR primers designed, enabled detection of all 4 human malaria species, producing a band of 240 bp in nest two amplification reactions. The assay conditions were optimised and 6 parasites per J.11 of blood could be detected, making this method more sensitive than microscopy for malaria detection. The product of nest one when used as DNA template in nest two reactions with species-specific primers produced species-specific bands for each of the 4 human malaria species. When tested on field samples, results were comparable with the nested peR method of Singh et al. (1996), particularly in determining parasite prevalence and mixed infections. When compared with microscopy, it detected 7 more samples as positive and 6 more samples as mixed infections, indicating that the method is more sensitive than routine microscopical examination. The nested PCR method described is a labour saving, rapid and highly sensitive method which will be valuable for obtaining accurate epidemiological data in large scale studies of malaria. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/2289 | |
| dc.subject | Development Of Polymerase Chain Reaction-Based Assays | en_US |
| dc.subject | For Detection Of Human Malaria | en_US |
| dc.title | Development Of Polymerase Chain Reaction-Based Assays For Detection Of Human Malaria | en_US |
| dc.type | Thesis | en_US |