Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus
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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 various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.
This work was supported by the National Key Research and Development Project of China (2016YFD0501601, 2016YFD0500202-1), the National Natural Science Foundation of China (31502076), the Jiangsu Provincial Natural Science Foundation of China (BK20150444), the National Key Technologies R&D Program of China (2015BAD12B01-3), the “Qing Lan Project” of Higher Education Institutions of Jiangsu Province, China, the “High-End Talent Support Program” of Yangzhou University, China, the Special Financial Grant from the China Postdoctoral Science Foundation (2016T90515), the Postdoctoral Science Foundation of Jiangsu Province, China (1501015B), the Earmarked Fund For China Agriculture Research System (CARS-40), and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Compliance with ethical standards
Conflict of interest
None of the authors has competing interests to declare.
This study was performed in strict concordance with the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The protocols for animal experiments were approved by the Jiangsu Administrative Committee for Laboratory Animals (approval number: SYXK-SU-2007-0005), and complied with the guidelines of Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals.
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