RAPID DIFFERENTIATION OF CLOSELY-RELATED ISOLATES OF 2 PLANT-VIRUSES BY POLYMERASE CHAIN-REACTION AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS
Dj. Barbara et al., RAPID DIFFERENTIATION OF CLOSELY-RELATED ISOLATES OF 2 PLANT-VIRUSES BY POLYMERASE CHAIN-REACTION AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS, Journal of virological methods, 55(1), 1995, pp. 121-131
Citations number
18
Categorie Soggetti
Virology,"Biochemical Research Methods","Biothechnology & Applied Migrobiology
Immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR)
followed by restriction fragment length polymorphism (RFLP) analysis
of the product has been shown to be an effective procedure for discrim
inating serologically indistinguishable isolates of two plant viruses,
raspberry bushy dwarf (RBDV) and zucchini yellow mosaic (ZYMV). For b
oth viruses, only limited sequence information was available at the ti
me of primer design, but most of the isolates which were tested could
be amplified (the one exception being a serologically quite distinct i
solate of ZYMV). Restriction endonucleases revealing diagnostic RFLPs
were readily identified. Each of two isolates of ZYMV could be detecte
d in the presence of the other and the relative proportions approximat
ely quantified by visual estimation of the relative intensity of the a
ppropriate bands. A range of isolates of different RBDV pathotypes wer
e compared; isolates were grouped in ways that accorded with their kno
wn history. Computer analysis of the published sequence from which the
primers had been derived showed the sequenced isolate to be identical
with an isolate imported from the USSR. The PCR/RFLP procedure is rap
id (it can be completed in less than 2 days), effective and will proba
bly be generally applicable to distinguishing closely related virus is
olates, even where little sequence information is available.