RESUMO
Molecular techniques based on the polymerase chain reaction (PCR) can provide rapid and sensitive diagnosis of plum pox virus (PPV), the causal agent of the devastating 'sharka' disease of stone fruit trees. The present study compared routine polymerase chain reaction (PCR) procedures against a new system, PCR-ELISA (Boehringer Mannheim), which enables immunoenzymatic detection of PCR products. The results show that this hybridisation system ensures fast and more sensitive detection of PPV associated with stone fruit trees and herbaceous hosts. Strain-specific capture probes were also designed to identify the two major PPV isolates, D and M, without subsequent restriction fragment length polymorphism analysis of the PCR products. Optimisation of all parameters involved in the PCR-ELISA procedure are discussed and its advantages reported.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vírus Eruptivo da Ameixa/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Eletroforese em Gel de Ágar , Frutas/virologia , Vírus Eruptivo da Ameixa/química , DNA Polimerase Dirigida por RNA , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Peach latent mosaic viroid (PLMVd) is widely distributed (approximately 55%) in peach germplasm from Europe, Asia, North America, and South America. PLMVd, or a closely related viroid, was occasionally detected in cherry, plum, and apricot germplasm from countries in Europe or Asia. The cherry isolate of PLMVd is 337 nucleotides in length and is 91 to 92% homologous to PLMVd isolates from peach. Molecular hybridization experiments demonstrated that PLMVd is not related to the agent of peach mosaic disease. PLMVd was readily transmitted (50 to 70%) by contaminated blades to green shoots and lignified stems of peach GF-305 plants. These results indicate that the viroid may be transmitted in orchards with contaminated pruning equipment.
RESUMO
A dot-immunobinding assay was adapted on enhanced chemiluminescence (DIBA-ECL), which employs luminol, a cyclic diacylhydrazide, as substrate for horseradish peroxidase conjugated with a secondary antibody, for the diagnosis of grapevine closteroviruses I and III. The sensitivity of DIBA-ECL was also compared to other immunoenzymatic methods. DIBA-ECL proved to be at least 16 times more sensitive than the dot-immunobinding assay using chloronaphthol/diaminobenzidine mixture as a substrate, which was at least twice as sensitive as DAS-ELISA, DAS-indirect avidin-biotin complex ELISA, and dot-immunobinding assay, using alkaline phosphatase as enzyme. Optimisation of all parameters involved in the DIBA-ECL procedure and its advantages are discussed.
