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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todo:paper_01660934_v31_n1_p11_Hopp2023-10-03T15:03:10Z Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens 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. 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 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. Fil:Hopp, H.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Tozzini, A.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Orman, B. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ceriani, M.F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Saladrigas, M.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Celnik, R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:del Vas, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Mentaberry, A.N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_01660934_v31_n1_p11_Hopp |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 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. Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens |
| topic_facet |
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 |
| description |
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. |
| format |
JOUR |
| author |
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. |
| author_facet |
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. |
| author_sort |
Hopp, H.E. |
| title |
Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens |
| title_short |
Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens |
| title_full |
Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens |
| title_fullStr |
Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens |
| title_full_unstemmed |
Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens |
| title_sort |
development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens |
| url |
http://hdl.handle.net/20.500.12110/paper_01660934_v31_n1_p11_Hopp |
| work_keys_str_mv |
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| _version_ |
1782023638879305728 |