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PCR of alphavirus in mosquitoes

ROBERT COELEN robert at arbo.microbiol.uwa.edu.au
Thu Aug 12 11:47:18 EST 1993


after having spent a short while in Indonesia on a project involved in 
Jembrana disease of cattle (a retrovirus), I am back in the land of internet 
connections. A PhD student in my lab, Loryn Sellner, is trying to develop a 
PCR system for detection of alphaviruses in mosquitoes. We are quite a long 
way down the track in that we have effectively a one tube, nested RT-PCR for 
one such virus. The sensitivity is at the handful of RNA molecules level. 
Unfortunately, processing more than 20 mosquitoes in a 1 ml buffer leads to 
inhibition, despite the guanidium or RNAzol B extraction of the mosquito 
grind. We would like to get to the 1000 mosquitoes/reasonable volume stage 
(possibly at the level of 200 mosquitoes/ml). Does anyone know of a 
non-mechanical way of 'digesting' the mosquitoes so that their 'guts' become 
liberated ? Any thoughts on this matter would be appreciated.

Finally, I am sorry I can't be at ICV, I hope those of you who go get a chance 
to link up !! And please make some noise about INTERNET, in particular of 
course about bionet.virology
Kindest regards,

Dr R.J. Coelen                             phone 61 - 9 - 389 3915
Dept of Microbiology                       fax   61 - 9 - 389 2912
University of Western Australia            snail Nedlands, 6009
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