Detection of Newcastle disease virus and H5-subtype Influenza virus in swiftlet houses by Multiplex reverse transcription PCR assay

The purpose of this study was to investigate the presence of Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), and Avian influenza type A, especially H5 subtype (AIV-H5) in swiftlet houses and swiftlet nest by multiplex RT-PCR (mRT-PCR). The assay used two specific primer pairs designed to detect the conserved sequence of the F gene of NDV and the HA gene of the AIV-H5, with product sizes of 282 bp and 420 bp, respectively. The mRT-PCR was established with detection limit of 25 copies/reaction for each target virus. The thermal cycle was optimized as follows: cDNA synthesis at 45ºC for 20 min, an initial denaturation at 95ºC for 5 min, followed by 35 cycles of amplification encompassing denaturation at 95ºC for 30 sec, annealing at 58ºC for 30 sec, and extension at 72ºC for 45 sec, ending by a final extension step at 72ºC for 7 min. Eighty-eight field samples including feces and swabs of the nest surface were examined and all samples were confirmed to be negative for these two viruses. The results of this study indicated that the swiftlet nests and the environment of swiftlet houses were not contaminated with NDV or AIV-H5 viruses. Moreover, the established mRT-PCR protocol had good specificity, detection limit, and can apply for routine veterinary diagnosis.