Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens

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...

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Detalles Bibliográficos
Autores principales: Hopp, H.E., Hain, L., Bravo Almonacid, F., Tozzini, A.C., Orman, B., Arese, A.I., Ceriani, M.F., Saladrigas, M.V., Celnik, R., del Vas, M., Mentaberry, A.N.
Formato: JOUR
Materias:
complementary dna
article
dna probe
dna rna hybridization
nonhuman
plant virus
potato
priority journal
rna virus
virus detection
Cloning, Molecular
DNA Probes
Enzyme-Linked Immunosorbent Assay
Methods
Nucleic Acid Hybridization
Plant Diseases
Plant Viruses
Potatoes
RNA, Viral
Sensitivity and Specificity
Support, Non-U.S. Gov't
Hexapoda
Insecta
Perodicticus potto
Potato leafroll virus
Potato spindle tuber viroid
Potato virus Y
RNA viruses
Solanum tuberosum
Viroids
Acceso en línea:http://hdl.handle.net/20.500.12110/paper_01660934_v31_n1_p11_Hopp
Aporte de:
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
Descripción
Sumario: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 strain-specific. 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 (DAS-ELISA). 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 in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts. © 1991.

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