Detection Of Avian Influenza Virus And Newcastle Disease Virus By Duplex RT-PCR Technique

Avian influenza virus (AIV) and Newcastle disease virus (NDV) are pathogens of social and economical importance known to be disseminated throughout the world by migratory birds. Many measures have been taken to control the introduction of these viruses into new environments, however, the epidemics of the two diseases are frequently reported to occur in poultry flocks in recently years, therefore, the new method of prevention and detection for the two viruses are in urgently needed. Virus isolation and conventional serological method are time consuming, and are comparatively low in sensitivity. To improve the efficacy and the sensitivity of different diagnosis for NDV and AIV, a duplex reverse transcription-polymerase chain reaction (RT-PCR) technique was established in the present study. To do this, two sets of primers were designed according to the sequences of NDV and AIV published in GeneBank. One pair of primer was used to amplify the NP gene of NDV and the other was utilized for amplification of M gene of AIV. Two fragments, 550 bp and 200 bp in length, were expected to be amplified, respectively.After the two sets of primers were combined and used for amplification trials, the conditions of PCR for detection of the two viruses were optimized by orthogonal assay. Based on the single RT-PCR assay that detected each of the two viruses respectively, a duplex RT-PCR system for both of them in one tube was established to distinguish the two viruses.The specificity test demonstrated that all samples which contained NDV and AIV, could be amplified by the duplex RT-PCR technique using these two sets primers, yielding two specific bands of NDV 550 bp and AIV 200 bp, while the detection resultshowed negative for IB and EDS. 32 of 52 samples were tested positive in RT-PCR, whereas only 27 were positive in separate cultivation for the virus. The results suggested that the established duplex RT-PCR system was much more sensitive, specific, rapidly and could be applied for early clinical diagnosis of AIV and NDV. Furthermore, it could also be considered to be used on the research of pathogenesis of the two viruses.