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The sensitivity and specificity of a rapid nucleic acid hybridization method for the detection of potato virus X in crude sap samples
Author(s) -
BAULCOMBE D.,
FLAVELL R. B.,
BOULTON R. E.,
JELLIS G. J.
Publication year - 1984
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/j.1365-3059.1984.tb01331.x
Subject(s) - potato virus x , rna , complementary dna , biology , microbiology and biotechnology , potexvirus , nucleic acid , dna , restriction enzyme , virology , gene , genetics , coat protein
A DNA copy (cDNA) of the common European strain (strain‐group 3) potato virus X RNA (PVX 3 ‐RNA) was prepared by reverse transcription of PVX‐RNA and amplified by cloning into E. coli plasmid pBR322. This DNA hybridizes with PVX‐RNA and not at all to RNA of potato virus Y. A low amount of cross‐hybridization was observed with strain‐group 2 of PVX (PVX 2 ). However, by hybridization of PVX 2 ‐RNA to restriction endonuclease fragments of PVX 3 ‐cDNA, it was demonstrated that cross‐homology was not present in all regions of the PVX genome. The cloned PVX 3 ‐cDNA was hybridized to viral RNA in crude sap samples which had been spotted on to nitrocellulose membrane. The signal, which was assayed by autoradiography, allowed the detection of 50 pg of viral RNA. This value was not adversely affected by encapsidation of the RNA, nor by components of crude sap. The sap spot hybridization was as effective as enzyme‐linked immunosorbent assay both in detection of small quantities of purified PVX 3 , and in screening of plants from a breeding programme for immunity to PVX 3 infection.