Dengue virus detection, cultivation

[Ronald Matias]

I am very much interested in detecting the four serotypes of dengue virus 
by RT-PCR. I am having problems with the protocols I am following from 
published papers. Could someone suggest a workable protocol in 
applying RT-PCR to the detection of Dengue virus. I will be most grateful 
for the details of the primer sequence, PCR conditions as well as buffer 
compositions. I have recently obtained C6/36 Aedes albopictus cells as 
well as BHK-21 cells. The former cell line has been shown to be an 
excellent host for dengue. Could someone suggest protocols for dengue 
virus isolation and culture. I know that these are probably standard 
techniques but I really have tried my best but could not detect nor isolate 
the virus. I am however able to detect antiviral antibodies in the serum of 
patients clinically diagnosed dengue. We have numerous cases of 
clinically diagnosed dengue and we want to confirm these by RT-PCR and 
eventually isolate the virus for further studies such as sequencing. I will 
be most grateful for anyone who can help me with this request and will 
keep them posted with our results. I am open to collaborative research 
work. I have in my disposal cell culture facilities, a transmission electron 
microcope, an automated laser induced fluorescence DNA sequencer, a 
DNA synthesizer, thermocycler and small equipments for nucleic acid and 
protein electrophoresis. We have established a link between several 
hospitals so I have access to clinicians who diagnose dengue patients
Thank you