|Riassunto in Inglese:|
Fruit trees are affected by a large number of different and economically important viruses and viroids. Once a virus or viroid infects a plant, the progress of infection cannot be prevented or controlled. Therefore, to slow down the spread of viruses in the field, it is important to detect these viruses in their early stage of infection and eliminate the infected plants. Recently, diagnostic methods have been developed to improve the sensitivity and reduce the processing time and cost. Thus, efforts have been directed to achieve the simultaneous detection of several viruses in a single assay. Many of these techniques are PCR-based with the associated problem of false positive results. These may be due to contamination or non-specific amplification. Steps to reduce the incidents of false positive results increase the value of these techniques for routine diagnosis. In spite of the fact that molecular hybridization is a less common methodology in diagnostic laboratories, this technique offers great advantages when compared to PCR-based methods. For example, molecular hybridization allows for near-total absence of contamination problems and a much greater flexibility. Indeed, its specificity can be adjusted to make the hybridization test as specific or as general as required.
The potential of molecular hybridization as a diagnostic tool in Plant Virology was first demonstrated for the detection of viroids for which no serological method could be used due to the lack of protein component in their structural constituents. The technique was later applied to plant viruses. The basic principles of molecular hybridization are beyond the scope of this chapter and will only be briefly discussed here. Several aspects affecting the different steps of the molecular hybridization technique (which include the synthesis of the labeled probe, sample preparation, hybridization and detection) have been described in previous reviews. Molecular hybridization is based on the specific interaction between complementary purine and pyrimidine bases forming A-T and G-C base pairs, which results in a stable hybrid formed by part (or the totality) of the nucleic acid sequence of the pathogen to be detected (target molecule) and by the labeled complementary sequence (probe). The stability of the hybrid depends upon the number of hydrogen bonds formed, and upon both electrostatic and hydrophobic forces. Electrostatic forces rely on the phosphate molecules of the nucleic acid backbone, whereas hydrophobic interactions are maintained between the staggered bases.