A Gene Expression Study of the Alternatively Spliced b20 Gene in the Filarial Parasite Brugia malayi
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Lymphatic filaraisis, a disease caused by the parasitic nematode Brugia malayi and transmitted by mosquitoes, infects approximately 130 million people in eighty-three countries in the tropical/sub-tropical belt. B. malayi b20 (Bmb20), an ortholog of the nematode-specific gene Onchocerca volvulus b20 (Ovb20), has been identified as an alternatively-spliced gene, with at least four splice variants identified by plaque-screenings of B. malayi cDNA libraries. The purpose of the following work is to evaluate the transcription patterns of Bmb20 splice variants in the various life cycle stages of the B. malayi parasite using both RT-PCR and quantitative RT-PCR methods. The evaluation of Bmb20 splice variants may result in the identification of a splice variant form specific to the thrid larval (L3) of B. malayi. This L3-specific form could be used as a target in a diagnostic assay to identify L3 infective mosquitoes and thus evaluate the transmission potential in endemic regions, a necessary component of the Global Programme to Eliminate Lymphatic Filariasis. Eight b20 splice variants were tested using RT-PCR and none appeared to be L3-specific, though two forms did appear to be upregulated in the L3 stage. Quantitative RT-PCR experiments with one of these forms, b20-l, indicates that with respect to most life cycle stages, the b20-l variant is upregulated in L3. However, further studies are necessary in order to assess the level of expression in comparison to other larval stages. This study illustrated that RT-PCR and qRT-PCR are useful methods in the detection of gene expression.