RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates. Um método foi desenvolvido para a detecção do Apple stem grooving virus (ASGV), baseado na transcrição reversa do RNA viral e na polimerização em cadeia do DNA (RT-PCR), usando os oligonucleotídeos iniciadores ASGV4F-ASGV4R, localizados na região do gene da RNA polimerase, sendo os produtos de amplificação detectados por hibridização "sandwich", em placas de poliestireno, através de reação colorimétrica. A RT-PCR foi realizada com a "Titan™ RT-PCR System", complexo enzimático formado pela AMV, responsável pela transcrição do RNA viral em cDNA e as DNA polimerases PWO e Taq, usadas para a etapa de PCR, utilizando-se para isso extrato bruto de folhas e casca do caule de macieira (Malus domestica). A detecção dos produtos de amplificação RT-PCR foi realizada hibridizando esses produtos com uma sonda de captura marcada com biotina, ligada à placa de ELISA previamente sensibilizada com estreptavidina, e uma sonda de revelação marcada com digoxigenina. O complexo foi detectado utilizando-se um