RESUMO
Standard molecular methods for plant virus diagnosis require the purification of RNA or DNA extracts from a large number of samples, with sufficient concentration and quality for their use in PCR, RT-PCR, or qPCR analysis. Most methods are laborious and use either hazardous and/or costly chemicals. A previously published protocol for RNA isolation from several plant species yields high amounts of good quality RNA-DNA mixture in a simple, safe and inexpensive manner. In the present work, this method was tested to obtain RNA-DNA extracts from leaves of tomato, potato and three species of citrus, and was compared with two commercial kits. The results demonstrated that this protocol offers at least comparable nucleic acid quality, quantity and purity to those provided by commercial phenol-based or spin column systems and that are suitable to be used in PCR, RT-PCR and qPCR for virus and viroid detection. Because of its easy implementation and the use of safe and inexpensive reagents, it can be easily implemented to work in plant virus and viroid detection in different plant species.
Assuntos
DNA Viral/isolamento & purificação , Folhas de Planta/genética , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , Plantas/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Viroides/genética , Viroides/isolamento & purificação , Citrus/genética , Citrus/virologia , DNA de Plantas/isolamento & purificação , DNA Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Folhas de Planta/virologia , Plantas/virologia , RNA de Plantas/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/normas , Solanum tuberosum/genética , Solanum tuberosum/virologiaRESUMO
A simple, low-cost hybridization assay using a universal DIG-labeled riboprobe for the rapid detection and identification of coleus viroids is presented. An octamer of 32-nucleotide sequence derived from the central conserved region (CCR) of viroids in the genus Coleviroid was used to develop a universal cRNA probe (8-central-conserved-region probe, 8CCR probe) for coleus viroids. Dot-blot hybridization assays demonstrated that the sensitivity of this probe was similar to specific probes for each CbVd, and Northern hybridization results revealed that at least four coleus viroids could be distinguished readily and simultaneously using the 8CCR probe. Batch detection assay showed that hybridization using the 8CCR probe can identify coleus viroids rapidly and effectively. This rapid and low-cost molecular hybridization technique is an effective way to survey the occurrence of coleus viroids, and has reference for the detection of other viroids and possibly viruses.
Assuntos
Hibridização de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos/genética , Viroides/genética , Viroides/isolamento & purificação , Virologia/métodos , Coleus/virologia , Sensibilidade e Especificidade , Fatores de Tempo , Virologia/economiaRESUMO
A Monte Carlo method was used to test the extent of sequence similarity among viroids, satellite RNAs, and hepatitis delta virus. This analysis revealed that there is insufficient sequence similarity among these pathogens to support the hypothesis that they have a common evolutionary origin. Furthermore, while definite patterns of sequence similarity were observed among some viroids, there was a clear lack of overall similarity, indicating that a monophyletic origin for even this group cannot be reliably supported from sequence data alone.