Development of a real-time TaqMan® RT-PCR assay for the detection of infectious bronchitis virus in chickens, and comparison of RT-PCR and virus isolation
Development of a real-time TaqMan® RT-PCR assay for the detection of infectious bronchitis virus in chickens, and comparison of RT-PCR and virus isolation
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DOI：
10.1016/j.jviromet.2009.09.014
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发表时间：
2010-02-01
影响因子：
3.1
通讯作者：
Simanov, Lubov
中科院分区：
文献类型：
--
作者：
Meir, Rosie;Maharat, Ora;Simanov, Lubov
建立一种灵敏、特异的方法诊断传染性支气管炎病毒(IBV)具有重要意义。在这项研究中，描述了针对IBV高度保守的核衣壳(N)基因的实时定量TaqMan(R)RT-PCR的发展，并包括一个内参照。该检测是针对IBV的，没有检测到包括火鸡冠状病毒在内的其他禽类病原体。确定了胚胎适应菌株M41和胚胎适应不良变种IS/885/00的相对检测下限。对于M41，实时RT-PCR和病毒分离分别比针对N基因和S蛋白(S_1)基因的RT-PCR敏感一到两倍。对于IS/885/00，实时荧光定量RT-PCR的敏感性分别是病毒分离的10倍和N基因和SI基因的30倍和40倍。实时荧光RT-PCR和病毒分离比针对SI基因的RT-PCR直接检测实验感染雏鸡的气管拭子的敏感性高17-75%。直接检测临床标本的气管和泄殖腔拭子，荧光定量RT-PCR检测的阳性标本比S1基因RT-PCR多50%。针对N基因的实时RT-PCR比普通诊断方法更敏感，可以直接从临床标本中快速准确地检测到IBV，有助于鉴别诊断。(C)2009爱思唯尔B.V.保留所有权利。
A sensitive and specific method for the diagnosis of infectious bronchitis virus (IBV) is of great importance. In this study the development of a real-time TaqMan (R) RT-PCR targeting the highly conserved nucleocapsid (N) gene of IBV and including an internal PCR control is described. The assay was specific for IBV and did not detect other avian pathogens, including turkey coronaviruses. A comparative limit of detection was determined for M41, an embryo-adapted strain, and IS/885/00, a poorly embryo-adapted variant. For M41 real-time RT-PCR and virus isolation were one or two times more sensitive than RT-PCR targeting the N or spike glycoprotein (S1) genes, respectively. For IS/885/00, real-time RT-PCR was more sensitive by tenfold than virus isolation and 30- or 40-fold than by N gene or SI gene RT-PCR, respectively. Real-time RT-PCR and virus isolation were 17-75% more sensitive than RT-PCR targeting the SI gene for testing tracheal swabs directly from experimentally infected chicks. When tracheal and cloacal swabs from clinical specimens were tested directly, 50% more samples were positive by real-time RT-PCR than by the S1 gene RT-PCR. Real-time RT-PCR targeting the N gene is more sensitive than common diagnostic assays, allowing rapid and accurate IBV detection directly from clinical specimens, facilitating differential diagnosis. (C) 2009 Elsevier B.V. All rights reserved.