Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus

Archives of Virology
Zhujun ZhangXiufan Liu

Abstract

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of vari...Continue Reading

References

Oct 9, 2002·Journal of Virological Methods·Caroline FernandezAgnès Gautheret-Dejean
Oct 25, 2003·Avian Diseases·E SpackmanD L Suarez
Aug 31, 2007·Journal of Virological Methods·Chi Y WangHung J Liu
Oct 17, 2013·PLoS Pathogens·Suxiang TongRuben O Donis
Dec 6, 2016·Cell Host & Microbe·Yuhai BiGeorge Fu Gao

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Methods Mentioned

BETA
PCR

Software Mentioned

ABI 7500
Primer Express
System SDS
DNA Star
7500

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