dc.contributor.author	Thompson, Simone Jamika
dc.coverage.spatial	Central and North America -- United States	en_US
dc.date.accessioned	2016-05-13T18:07:23Z
dc.date.available	2016-05-13T18:07:23Z
dc.date.issued	2016-05
dc.identifier.uri	http://hdl.handle.net/10428/2175
dc.description.abstract	Thompson, Simone J. Determination of a Gene Expressed only in the Infective Larval Stage of Dirofilaria immitis. Thesis. (Biology Department: Valdosta State University, 2016). Current molecular assays for detecting Dirofilaria immitis DNA in mosquitoes by PCR cannot differentiate between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third-stage parasite larvae (L3). This over-estimates transmission risks to canines because only L3 D. immitis are capable of establishing infections in canines. I have developed an assay that detects D. immitis L3 in the vector Aedes aegypti based on a reverse transcriptase-polymerase chain reaction (RT-PCR) method that detects an L3-activated gene transcript. Potential L3 stage genes were identified using bioinformatics tools and were screened by PCR using complementary DNA (cDNA) libraries as templates. Candidate genes were screened for stage-specific expression using RNA isolated from both D. immitis UUID: 0D49F070-F616-4C29-85F8-FEC62FD3AC9B	en_US
dc.description.tableofcontents	Chapter I: INTRODUCTION……………………………………………..……………...1 Chapter II: MATERIALS AND METHODS……………………………...………….….5 Search Strategy for L3-activated Candidate Genes………………………............5 Primer Design….…………………………………………………….…….……..5 Pre-Screening of Candidate L3-activated Genes using mRNA Libraries…….....6 Parasite Infection of Mosquitoes ……………………………………..….….......8 RT-PCR Testing of Putative Target Genes against Mosquito Pools……….…....8 Time Course Screening with L3 Specific Primers…………………………..…...9 Chapter III: RESULTS…………………………………………………………….….....10 cDNA Library Multiplex RT-PCR…………………………………………........10 Conventional RT-PCR of Candidate Genes against Mosquito Pools………........11 Conventional RT-PCR of CK855471A and Ditph using Time-Course Study......11 Chapter IV: DISCUSSION……………………………………………………………....18 FOOTNOTES REFERENCES……………………………………………………....…..22 BIBLIOGRAPHY……………………..………………………………………….......….23 APPENDIX A: Valdosta State University Biosafety Committee Approval Form……....29 APPENDIX B: Molecular Genetics of Dirofilaria immitis Biosafety Manual - Biosafety Level 2………………………………………………………………….37 APPENDIX C: Isolation of Total RNA from Tissue Samples.…………………….........48 LIST OF TABLES Table 1: Candidate Gene Primer Sequences………………………….…………..…...…13 Table 2: cDNA Library PCR Screening of Candidate Genes…………..………….…….14 Table 3: RT-PCR of Candidate Genes against Mosquito Pools………………………....15 Table 4: RNA Extraction Quantity and Quality across Time-Course Study………....….16	en_US
dc.language.iso	en_US	en_US
dc.subject	Thesis, Academic	en_US
dc.subject	Biology	en_US
dc.title	Determination of a Gene Expressed only in the Infective Larval Stage of Dirofilaria immitis	en_US
dc.type	Thesis	en_US
dc.contributor.department	Biology	en_US
dc.description.advisor	Chambers, Eric W.
dc.description.committee	Blackmore, Mark S.
dc.description.committee	Elder, John F.
dc.description.degree	M.S.	en_US
dc.description.major	Biology	en_US