Standardization of a duplex RT-PCR for the detection of Influenza A and Newcastle disease viruses in migratory birds

Influenza A and Newcastle disease viruses are pathogens of social and economical importance known to be disseminated throughout the world by migratory birds. Many efforts have been made to control the introduction of these viruses into new environments, and complete world surveillance has yet to be achieved. Virus isolation and immunofluorescence techniques are time consuming, have inherent limited sensitivity and present a lack of host cells permissive universally to all Influenza A viruses. In this paper, the use of a duplex RT-PCR is described capable of sorting out any NDV and Influenza A virus strain simultaneously in oral and cloacal swab specimens. This method includes fluorescent detection of amplicons that provide accurate analysis of many DNA fragments within one base discrimination. Reference viruses were used for standardization of the assay and samples from wild-type viruses were screened, with four positive results for Influenza A detected in migratory birds captured in the state of São Paulo. This screening test can be considered a first step for further studies of these viruses circulating in avian species in Brazil, and hopefully will contribute to broaden the sample spectrum from wild birds, leading to a better understanding of these viruses and their participation in the southeastern region of the country.