Desenho de primers e clonagem do gene do capsídeo de fitovírus para uso como controles positivos na diagnose por RT-PCR

Abstract

The frequent exchange of plant material for propagation, provides a good performance of agriculture in Brazil, but exposes the country to the risk of introducing unwanted pathogens. To prevent this, the early diagnosis of pathogens of both quarantine and the non-quarantine viruses is essential. Regarding to viruses, the establishment of efficient techniques for its diagnosis has been a great challenge, due to the high yield losses they can induce and also because there is no curative methods of controlling these pathogens. One of the major constraints to the development of these methods is the lack of positive controls to check the reliability of the diagnostic technique employed, since there is always the possibility of false negatives and/or false positives results. This study aimed to contribute for the formation of a bank of positive controls for the virus diagnosis, by cloning the gene of viral capsid in plasmid and design the primer to be used in virus diagnosis by RT-PCR. Genomic fragments of 3 quarantine viruses and 8 non-quarantine viruses were cloned and sequenced. All the plasmids with the genomic viral fragments and the designed primers were tested as positive controls and worked as a secure method for using in the diagnosis of viruses tested.