ISHS Acta Horticulturae 781: XX International Symposium on Virus and Virus-Like Diseases of Temperate Fruit Crops - Fruit Tree Diseases
DEVELOPMENT OF ROUTINE DUPLEX RT-PCR TESTS FOR CERTIFICATION OF FRUIT TREE MULTIPLICATION MATERIAL |
| Authors: | S. Massart, S. Roussel, J. Kummert, O. Dutrecq, M.H. Jijakli |
| Keywords: | RT-PCR, certification, duplex PCR, PDV, PNRSV, ApMV, ACLSV, ASPV, ASGV |
| Abstract:
In phytosanitary certification, there is currently a need for the development of reliable, sensitive and rapid tests for the routine detection of ilarviruses and latent viruses of fruit trees throughout the year. The simultaneous detection of several viruses in a single PCR tube is a promising but challenging way which can reduce the cost of the analysis. So, we have developed three duplex RT-PCR protocols for the simultaneous detection of two viruses: ASPV-ASGV, ACLSV-ApMV and PDV-PNRSV. The development of a multiplex detection protocol requires numerous steps: adequate virus combination, optimisation of the PCR mix, limit of detection in comparison with the single detection protocol, adequate integration in the certification process, intra-laboratory validation. We described this step-by-step development of duplex protocols with special emphasis on Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV). The three duplex protocols were successfully validated in our laboratory. There are currently validated through a European ring-test including 10 partners. | |
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