Development of multiplex RT-PCR methods for detection of arboviruses.

Objective To develop a multiplex reverse transcription-polymerase chain reaction(RT-PCR) method for the detection of dengue virus type 1 and 2,Japanese encephalitis virus and Yellow Fever virus.Methods Based on the genomes sequence analysis of these viruses,four pair of primers were designed,the specificity of the primers was checked by comparing with the GenBank DNA sequence database.Then the optimal reaction conditions of the multiplex RT-PCR were established.The specificity of the RT-PCR was tested using the homologous virus,namely the dengue virus type 3 and 4,Sindbis virus and Chikungunya virus.Results Fragments of 574,251,879 and 422 bp,corresponding to the dengue virus type 1,2,Japanese encephalitis virus and Yellow Fever virus,respectively,were seen in the multiplex RT-PCR reaction.Amplification was not seen in the control group.Conclusion Multiplex RT-PCR can be used in the rapid detection of dengue virus type 1,2,Japanese encephalitis virus and Yellow Fever virus.