Sample preparations and RT-PCR assays for large scale detection of fruit tree viruses covered by certification standards.
Different RNA extraction methods followed by single or multiplex reverse transcription-polymerase chain reaction (RT-PCR) were examined in order to find the simplest way to detect different viruses present in single or mixed infections in fruit tree species. Samples of pome (apple) and stone fruit (peach, plum, apricot, almond) trees, naturally infected with plum pox virus, prunus necrotic ringspot virus, apple mosaic virus, prune dwarf virus and apple chlorotic leafspot virus were analysed by single or multiplex one-step RT-PCR using a mixture of primers specific for each virus. RNA was extracted from viruses by trapping virions with a mixture of antisera specific for each tested virus (IC-RT-PCR) or by a simple rapid extraction method (REM-RT-PCR). Both procedures needed no more than 0.3 g of green tissue and eliminated laborious and hazardous extraction procedures. It was possible to determine the presence of a virus or viruses in a mixed infection in a large number of samples (50-80) in one or two days.