Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcas
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RESEARCH ARTICLE
Open Access
Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus Xiyu Zhang1,2, Ming Yao1, Zhihui Tang1, Daning Xu3, Yan Luo4, Yunfei Gao5 and Liping Yan1,2*
Abstract Background: Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results: The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion: The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks. Keywords: Real-time PCR, AIV, NDV, DTMUV, Ducks, Clinical detection
Background China is the largest duck producer of the world, accounting for more than half of the world’s total duck meat stock. During the breeding of ducks, virus infection is a serious problem and has caused huge economic loss * Correspondence: [email protected] 1 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China 2 MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing 210095, China Full list of author information is available at the end of the article
to the waterfowl industry, among which, duck-derived avian influenza virus (AIV), duck-derived Newcastle disease virus (NDV), and duck Tembusu virus (DTMUV) are the most common virus. AIV belongs to influenza A virus of Orthomyxoviridae and has many serotypes, most of which have been detected in domestic ducks and wild ducks. The high-pathogenicity subtypes such as H5 and H7 are highly virulent to ducks of various ages and breeds [1–3]. Although the low-pathogenicity subtypes such as H3, H6, H9 and H10 are often detected in duck
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permit
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