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Title: Improved high-throughput method for molecular identification of Culex mosquitoes

Author
item ROCHLIN, ILIA - SUFFOLK CTY DPT HLTH SERV
item SANTORIELLO, MICHAEL - SUFFOLK CTY DPT HLTH SERV
item MAYER, RICHARD
item CAMPBELL, SCOTT - SUFFOLK CTY DPT HLTH SERV

Submitted to: Journal of the Mosquito Control Association
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2007
Publication Date: 12/1/2007
Citation: Rochlin, I., Santoriello, M.P., Mayer, R.T., Campbell, S.R. 2007. Improved high-throughput method for molecular identification of Culex mosquitoes. Journal of the Mosquito Control Association. 23 (4):488-491,2007

Interpretive Summary: Field collections of mosquitoes using traps often result in difficulties in identifying the species of mosquitoes because of damaged specimens. A modified and improved DNA isolation protocol was developed using proteinase K digestion to permit high throughput screening of a large number of Culex (mosquito) specimens for species identification using polymerase chain reaction (PCR) without hampering arbovirus surveillance efforts. The modified proteinase K method successfully identified 91% to 100 % of the Culex samples compared to a 0% to 50% success rate for methods without a proteinase K treatment.

Technical Abstract: Culex pipiens, Cx. restuans, and Cx. salinarius play important and most likely different roles in transmission of West Nile Virus (WNV) in the northeastern United Sates. While Cx. pipiens and Cx. restuans are considered the main enzootic vectors of WNV, Cx. salinarius may be involved in epizootic cycles due to its broader host preferences. Accurate morphological identification of field-collected Culex specimens may be difficult, and therefore the New York State Department of Health arbovirus surveillance program allows combined Cx. pipiens and Cx. restuans in pools tested for WNV. We have developed a modified and improved DNA isolation protocol using proteinase K digestion to permit high throughput screening of a large number of Culex specimens for species identification using polymerase chain reaction (PCR) without hampering arbovirus surveillance efforts. Proteinase K digestion of legs from individual Culex mosquitoes was performed and used for PCR amplification with previously described species specific ribosomal DNA primers. Using these rDNA primers, our modified proteinase K method successfully identified 91% to 100 % of the Culex samples compared to a 0% to 50% success rate for methods without a proteinase K treatment.