Establishment of a bank of positive controls for diagnosis of quarantine viruses and viroids in Brazil through PCR and RT-PCR

Abstract

The analysis of plant materials such as seeds, seedlings and other units for vegetative propagation that are imported, before planting in the country, routinely transported across international borders, is essential to decrease the imminent risk of introducing exotic pathogens, considered A1 pests. However, this requires the use of reliable positive controls, to prevent any false positives and/or negative results, causing undesirable consequences. The aims of this study were to set up a bank of positive controls constituted, in most cases, by the cDNA of the viral capsid gene cloned into commercial plasmid and propose diagnostic primers for PCR and RT-PCR assays. Genomic fragments of 41 quarantine viruses and two quarantine viroids in Brazil, as well as 12 regulated non-quarantine viruses, were cloned in plasmids, to be used as a positive control in diagnostic RT-PCR and PCR tests. Besides being a safer and more economical alternative, this bank will avoid the use of virus-infected tissue or total nucleic acid, which would still be infectious. The correct usage of this kind of positive control would eliminate the risk of incidental introduction of exotic viruses in Brazil.