A duplex RT-PCR assay for simultaneous detection of H5 subtype avian influenza viruses and velogenic Newcastle disease virus.

A duplex polymerase chain reaction assay(dRT-PCR)was developed in this study which could simultaneously ampli- fy the hemagglutinin glycoprotein (HA) gene of the AIV H5 and distinctive sequence of vNDV F gene in a single tube. The positive results could be obtained from AIV H5 and vNDV strains while no positive amplification was observed with other HA subtypes of AIV, avirulent NDV and other unrelated avian pathogens. The endpoint of detection was defined as approxi- mately 20.6fg for DNA plasmid containing HA gene of AIV H5 and 406fg for DNA plasmid containing F gene of vNDV. Compared with the single RT-PCR, the dRT-PCR assay was able to detect AIV/NDV with similar sensitivity. The whole process of detection, from sample processing to obtaining the results, could be finished within 5h. Of 24 clinical samples detected by this assay, all were AIV-H5 negative and 18 were vNDV positive. Sequencing analysis confirmed that all PCR products contained characteristic sequence of vNDV at cleavage site. NDV isolation was performed by SPF embryo from 9 clinical sam- ples randomly selected and showed a 6/9 isolation rate. Samples of livers, brains, lungs and cloacal swabs collected from 5 five-day-old SPF chicken experimentally co-infected with 100ELD50 AIV H5 and 100ELD50 vNDV were detected by the dRT-PCR, the detection rate of AIV H5 was 4/5,3/5,5/5, 4/5, the detection rate of vNDV was 3/5,5/5,4/5,3/5; while no positive samples from chicken experimentally co-infected with AIV H9N2 and LaSota, and negative control has been found using the dRT-PCR, In conclusion, the method developed in this study provides a rapid, accurate, economical and effective way to detect AIV H5 and vNDV.