Lymphatic filariasis is a very painful and disfiguring helminth disease caused by the tissue nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. This parasitosis is considered a Neglected Tropical Disease and it is a major public health burden for 72 tropical countries of Africa, Southeast Asia, the Caribbean, and South America. Despite the effectiveness of many control programs, there remains the need to develop new pharmacological agents to treat lymphatic filariasis, as most programs rely on a limited variety of drugs that are expensive, logistically difficult to obtain, and can lead to drug resistance. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology is currently being used to develop a genetic toolkit and methods for the study of transcriptional and post-transcriptional regulation in all lifecycle stages and tissues of B. malayi. To genotype transgenic parasites, it is necessary to detect the genomic DNA of B. malayi in a non-invasive way, since the invasive sampling of the parasite damages its cuticle resulting in death. This study presents the development of specific nested primers to amplify Y chromosomal DNA from the DNA present in the media of the molting parasites. Knowing the sex of the larvae will allow us to optimize the sex ratio for the backcrossing of transgenic parasites. B. malayi third stage infective L3 were cultured with 1x105 Bovine Embryo Skeletal Muscle (BESM) cells/well and Minimal Essential Media (MEM) containing 20% fetal bovine serum (FBS). Following the collection of the molting media for day 8, DNA was extracted using the DNeasy® Blood & Tissue Kit and the Dynabeads™ M-280 Streptavidin method. A single distinct band of approximately 210-220 bp was obtained from molting media samples that contained one singe L3 larvae using the NPRx2 and NPSet1 nested primers. A single distinct band of approximately 420 bp was obtained from the same media samples using the RPS12UTRF2 and the RPS12UTRR2 nested primers. Amplification from both the shp2 and BmRPS12 genes demonstrated that the genomic DNA present in the media of molting parasites can be used to non-invasively genotype individual larvae. Furthermore, the Tag on Y (ToY) assay can then be used to determine the sex of the cultured parasites. This study provides important tools to genotype and to optimize the sex ratio for the backcrossing of transgenic parasites.
|Advisor:||Unnasch, Thomas R.|
|Commitee:||Ntumngia, Francis, De, Sai Lata|
|School:||University of South Florida|
|School Location:||United States -- Florida|
|Source:||MAI 82/2(E), Masters Abstracts International|
|Subjects:||Public health, Epidemiology, Parasitology|
|Keywords:||B. malayi, Nested PCR, Primer development, Y chromosome marker gene|
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