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Dayalbagh Educational Institute (2014)

Genetic Analysis of Selected Mosquito Vectors Using Random Amplified Polymorphic DNA RAPD Marker in Agra Region

Gupta, Shivani

Titre : Genetic Analysis of Selected Mosquito Vectors Using Random Amplified Polymorphic DNA RAPD Marker in Agra Region

Auteur : Gupta, Shivani

Université de soutenance : Dayalbagh Educational Institute

Grade : Doctor of Philosophy (PhD) in Zoology 2014

Résumé partiel
The population genetic structure refers to the distribution of genetic variation within and among populations of a species which can affect both disease transmission and the success of methods used for control. Therefore, understanding genetic difference within and among the mosquito vector species requires an extensive survey of larval microhabitats along with an elucidation of the possible maintenance of observed intra and interpopulation genetic polymorphism. The present research work highlights an epidemiological surveillance of selected mosquito vectors in their larval microhabitats with special emphasis on genetic analysis using RAPD-PCR markers. Three methods were used viz. SDS method, DNAzol method and DNeasy tissue kit (Qiagen). For each method, genomic DNA was extracted from single, five and ten freshly emerged IV instar mosquito larvae. The methods were evaluated for efficiency and economy and SDS lysis method satisfied these criteria. Optimization of RAPD protocols is the most crucial step for getting reproducible patterns of RAPD-PCR profile. In order to optimize the RAPD analysis several PCR variables (DNA quantity- 20ng, MgCl2 conc.- 2.5mM, primer conc.- 5pmol, Taq DNA polymerase-0.5U, dNTPs-0.4mM, number of amplification cycles- 40, annealing temperatures- 36ºC) were standardized. Initially 140 primers were screened with laboratory reared Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi in order to select a few RAPD primers which produced reproducible fingerprints for the study of field populations of these mosquito species collected from various larval habitats. Out of 140 primers screened, twenty four revealed apparent, consistent and discrete banding patterns in laboratory strains of these mosquitoes. These primers are, for Cx. quinquefasciatus : OPA02, OPM02, OPM04, OPR08 ; Ae. aegypti : OPA07, OPA09, OPAB01, OPM02 ; Ae. albopictus : OPA11, OPB04, OPAB1, OPC-15, OPM11, OPS01, OPS10 ; An. stephensi and An. culicifacies : OPA11, OPA12, OPA18, OPB16, OPC12, OPC14, OPC19, OPR01, OPM15. These primers were selected by visually analyzing gel pictures on the basis of their ability to amplify DNA fragments from mosquito genomic DNA and resolution on gel with less background smear and brighter bands. The data set was analysed using population genetic softwares POPGENE (Version 1.32), MEGA (Version 5.2) and AMOVA using GenAlEx (Version 6.5). Présentation et version intégrale (Shodhganga)

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