Lymphatic filariasis (LF) is an incapacitating disease caused by three filarial nematodes belonging to the family Onchocercidae, namely Brugia timori, Wuchereria bancrofti and Brugia timori. An estimated 90% of lymphatic filariasis cases globally are caused by Wuchereria bancrofti. To evaluate the success of the Global Program to eliminate Lymphatic Filariasis it is essential to monitor the frequency of larval infection in the mosquito vector. Molecular methods to detect Wuchereria bancrofti DNA in mosquitoes have been in existence since 1996. However these methods have not been widely adopted due to the high cost associated with them and the inability of these assays to distinguish between immature and infectious stages in the mosquito vector. The overall aim of this project was to modify, as previously described in literature, the Laney real time PCR assay to permit it to be used in an end point Reverse Transcriptase (RT)-PCR ELISA format. The endpoint PCR-ELISA uses inexpensive conventional thermocyclers and inexpensive reagents and probes. To accomplish this overall goal the specific objectives were to produce a positive control RNAs for Wb-cut-1.2 L3 specific RT-PCR and Wb-TPH RT-PCR that detects any stage of the parasite, and to adapt the detection of both transcripts to a PCR-ELISA format. Positive RNA controls were prepared and purified using template cDNA made available through FR3, subsequent development and optimization of the RT PCR ELISA was achieved through the adaptation of the Onchocerca volvulus O150 PCR ELISA protocol.
We found a 16-fold difference in the limit of detection between the ELISA assay and conventional end point RT-PCR when we did a 2-fold dilution series of PCR products for both Wb-Cut-1.2 and Wb-TPH. This indicates that our assay was 16 times more sensitive than the use of regular agarose gel electrophoresis to analyze PCR products. The limit of detection with ELISA and gel analysis were comparable when a 10-fold dilution series of the positive control RNA template was done. The RT-PCR ELISA takes a day to complete and up to three 96 well plates a day can be processed compared to the limited number of samples that can be analyzed by gel electrophoresis a day. It is anticipated that our assay will be used in the molecular xenomonitoring of Wuchereria bancrofti providing earlier time-point assessments of LF infection in endemic areas. In areas that were once endemic, our diagnostic tool will play a pivotal role in monitoring LF resurgence.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-7530 |
| Date | 01 July 2016 |
| Creators | Mzizi, Nompumelelo Mzizi |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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