A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2
A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2
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DOI：
10.1016/j.jviromet.2016.03.011
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发表时间：
2016-08-01
影响因子：
3.1
通讯作者：
Decaro, Nicola
中科院分区：
文献类型：
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作者：
Dowgier, Giulia;Mari, Viviana;Decaro, Nicola
犬腺病毒是引起狗、土狼、红狐和狼以及其他食肉动物和海洋哺乳动物疾病的主要原因。犬腺病毒1型（CAdV-1）和犬腺病毒2型（CAdV-2）分别引起犬传染性肝炎（ICH）和传染性气管支气管炎（ITB）。在这项研究中，双重实时PCR检测CADV-1和CADV-2的同时检测和表征，通过使用一个单一的引物对和病毒特异性探针。通过检测专门制备的标准DNA和已知对CAdV-1、CAdV-2或两种病毒均呈阳性的各种基质的临床样品，验证了该检测方法。由于未观察到与不同CAdV类型的交叉反应，因此证明该测定具有高度特异性，并且灵敏度高，能够检测不到10个拷贝的CAdV-1/CAdV-2 DNA。低的批内和批间变异系数证明了高的重复性，从而证实了该检测方法用于定量检测CAdV-1和CAdV-2的快速诊断和流行病学调查的潜在用途。(C)© 2016 Elsevier B. V.版权所有。
Canine adenoviruses are a major cause of disease in dogs, coyotes, red foxes and wolves, as well as in other carnivores and marine mammals. Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB), respectively. In this study, a duplex real-time PCR assay for simultaneous detection and characterisation of CAdV-1 and CAdV-2 was developed by using a single primer pair and virus-specific probes. The assay was validated testing standard DNAs produced on purpose and clinical samples of various matrices known to be positive for CAdV-1, CAdV-2 or both viruses.Precise calculation of DNA loads in samples containing a wide range of viral amounts was allowed by generating a standard curve for absolute quantification. The assay was proven to be highly specific, since no cross-reactions with the different CAdV type was observed, and sensitive, being able to detect less than 10 copies of CAdV-1/CAdV-2 DNA. The low intra-assay and interassay coefficient of variations demonstrated a high repeatability, thus confirming the potential use of this assay for quantitative detection of CAdV-1 and CAdV-2 for rapid diagnosis and epidemiological investigations. (C) 2016 Elsevier B.V. All rights reserved.