"Development and application of a non-radioactive nucleic acid hybridization system for the simultaneous detection of four potato pathogens".

H.E. HOPP, L. HAIM, F. BRAVO ALMONACID, A.C. TOZZINI, B. ORMAN, A.I. ARESE, N.F. CERIANI, M.V. SALADRI¬GAS, R. CELNIK, M. DEL VAS Y A.N. MENTABERRY.

JOURNAL OF VIROLOGICAL METHODS

Año: 1991 vol. 31 p. 11 - 30

0166-0934

cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding  RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strains-pecific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DASELBA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens