A polymerase chain reaction assay for cucumber mosaic virus in lupin seeds [Lupinus angustifolius]
1993
Wylie, S. | Wilson, C.R. | Jones, M.G.K. (Murdoch Univ., Perth (Australia). School of Biological and Environmental Sciences) | Jones, R.A.C. (Western Australian Dept. of Agriculture, South Perth)
A Polymerase Chain Reaction (PCR) using ground dry seed samples was developed. Primers based on consensus sequences of 8 published CMV coat protein cDNAs (RNA3) of CMV subgroups 1 and 2 were used. The assay involved (1) a reverse transcription step for cDNA synthesis and (2) amplification of a specific fragment by PCR. Two methods of extracting virus from infected lupin material were used: a rapid procedure which was effective for samples with higher levels of infection, e.g. infected leaves and greater than or equal to 0.5 percent infected seed; and phenol-chloroform procedure, which led to greater sensitivity, enabling reliable detection of 0.1 percent seed infection. It detected CMV in 16 commercial seed samples (0.1-8 percent seed infection) belonging to 7 cultivars from 12 different localities. Both methods were suitable for routine testing of the flour derived from grinding dry seed. On dissection of infected seeds, CMV was detected in the cotyledons and embryo and usually in or on the testa. The PCR assay detected virus from both CMV subgroups, but only subgroup 2 was found in lupin seed samples. The 2 CMV subgroups can be distinguished by digestion of amplified DNA with the restriction enzyme EcoRI; only CMV strains of subgroup 2 are digesta to yield two fragments of size 330 to 170 bp.
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