Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts

A procedure was developed for simultaneous detection of plant RNA viruses and of plant RNA, as a control. RT-PCR amplification with primers designed for the detection of the plant mRNAs encoding malate dehydrogenase (MDH) and the large subunit of ribulose bisphosphate carboxylase oxygenase (RubiscoL) was used for the development of a plant extraction procedure that consistently yields extracts that can be amplified. The control amplification was used successfully on extracts from cane, leaf and/or bud tissues from grapevine, apple, raspberry, strawberry, peach, apricot, plum and wheat. Multiplex RT-PCR conditions were established for the simultaneous detection in grapevine extracts of either arabis mosaic virus, rupestris stem pitting associated virus and malate dehydrogenase mRNA, or grapevine virus A, grapevine virus B, grapevine leafroll associated virus-3, and RubiscoL mRNA.