Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence

Abstract

The application of the polymerase chain reaction DNA amplification technique to the detection and typing of isolates of maize streak virus (MSV) and other related geminiviruses of grasses is described. The oligonucleotide primers used for amplification were 17-mers which contained a number of degeneracies. An approximately 250 base pair fragment was amplified from all geminivirus-infected grass and cereal samples tested. The amplification reaction was specific, working down to a concentration of 50 fg/ml of MSV-specific plasmid-cloned DNA and with a 10(-9) dilution of MSV-infected maize DNA extract. DNA could also be amplified from distantly related geminiviruses, including two different sugarcane viruses, digitaria streak virus and another as yet uncharacterized virus of a Panicum sp. Amplified DNA from a Mauritian sugarcane isolate (SSV-M) was cloned and sequenced. Sequence comparison and phylogenetic analysis showed that this sequence differed sufficiently from the analogous region of other geminiviruses for SSV-M to be considered a distinct virus. The use of the polymerase chain reaction for the amplification of gemini- and other virus genomes or genomic fragments for typing, mapping, phylogenetic analysis and taxonomy is discussed.