A single-tube multiplex reverse transcription-polymerase chain reaction for detection and differentiation of vesicular stomatitis Indiana 1 and New Jersey viruses in insects

A multiplex single-tube reverse transcription—polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10—30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.