Simultaneous detection of eight viruses and two viroids affecting stone fruit trees by using a unique polyprobe
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The use of riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the simultaneous detection of up to six different pathogens using a non-radioactive molecular hybridization procedure. In the present report, we describe the development of a unique polyprobe (poly10) with the capacity to detect viruses and viroids commonly found infecting fruit trees. The poly10 covers eight viruses: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV), Plum bark necrosis and stem pitting-associated virus (PBNSPaV) and two viroids: Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). Poly10 is comparable to the individual riboprobes in terms of end-point dilution limit and specificity, allowing the detection of up to 2.5 picograms of viral or viroidal RNA. However, the polyprobe requires a hybridization temperature of 60°C instead of the standard 68°C. The validation of the new simultaneous detection strategy was confirmed by the analysis of 60 field samples, which came from seven different hosts. The use of the polyprobe as an alternative to other routinely used detection methods is discussed.
KeywordsMultiplex Non-radioactive molecular hybridization Stone fruit tree viruses and viroids Dig-RNA probe
A. P. M. is the recipient of a JAE-Pre contract from the Consejo Superior de Investigaciones Científicas (CSIC). We thank L. Corachán for her excellent technical assistance. This work was supported by grant BIO2008-03528 from the Spanish granting agency DGICYT and by grant ACOMP/2010/214 from the Generalitat Valenciana.
- Astruc, N., Marcos, J. F., Macquaire, G., Candresse, T., & Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. European Journal of Plant Pathology, 102, 837–846.CrossRefGoogle Scholar
- Matic, S., Sánchez-Navarro, J. A., Mandic, B., Myrta, A., & Pallás, V. (2008). Tracking three ilarviruses in stone fruit trees throughout the year by ELISA and tissue-printing hybridization. Journal of Plant Pathology, 90, 137–141.Google Scholar
- Saade, M., Aparicio, F., Sánchez-Navarro, J. A., Herranz, M. C., Myrta, A., Di-Terlizzi, B., et al. (2000). Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridisation and multiplex reverse-transcription polymerase chain reaction. Phytopathology, 90, 1330–1336.PubMedCrossRefGoogle Scholar