A single temperature amplification technique applied to the detection of citrus tristeza viral RNA in plant nucleic acid extracts

• Published in: Journal of virological methods Vol. 47; no. 1; pp. 141 - 151
• Main Authors: Lair, S.V., Mirkov, T.E., Dodds, J.A., Murphy, M.F.
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.04.1994; Amsterdam Elsevier; New York, NY
• Subjects: 3SR TM; Agronomy. Soil science and plant productions; ARN; BARK; Base Sequence; Biological and medical sciences; CITRUS; Citrus - microbiology; Citrus tristeza virus; CLOSTEROVIRUS; CLOSTEROVIRUSES; CORTEZA; ECORCE; Enzyme-Linked Immunosorbent Assay; FEUILLE; Fundamental and applied biological sciences. Psychology; Generalities. Techniques. Transmission, epidemiology, ecology. Antiviral substances, control; HOJAS; Isothermal RNA amplification; LEAVES; METHODE; METHODS; METODOS; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Phytopathology. Animal pests. Plant and forest protection; Plant Viruses - isolation & purification; Plant viruses and viroids; Polymerase Chain Reaction; RNA; RNA, Double-Stranded - genetics; RNA, Viral - genetics; RNA, Viral - isolation & purification; Sensitivity and Specificity; Temperature; Time Factors; Isothermal RNA amplification; Citrus tristeza virus; 3SR TM; Plant pathogen; RNA; In situ; Rutaceae; Method; Virus; Amplification; Citrus; Dicotyledones; Angiospermae; Spermatophyta; Detection; Closterovirus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/0166-0934(94)90073-6

A procedure for the successful detection of citrus tristeza virus (CTV) RNA in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. The method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. The CTV viral RNA is then specifically amplified using a single temperature RNA Self-Sustained Sequence Replication technique (3SR TM) performed at 42°C for 60 minutes. The amplified negative-sense viral RNA product can subsequently be detected by fixing a portion of the reaction mixture onto a nylon membrane and hybridizing with positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Denatured viral specific dsRNA was also a suitable template for the specific detection of CTV.