Romanova Nadya, Corredor Juan Carlos, Nagy Eva
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
J Virol Methods. 2009 Jul;159(1):58-63. doi: 10.1016/j.jviromet.2009.02.026. Epub 2009 Mar 9.
The purpose of the study was to develop a highly sensitive real-time polymerase chain reaction (PCR) assay to detect and quantitate fowl adenovirus (FAdV) DNA in chicken tissues, using FAdV-9 as a model. The assay had a dynamic range of 7 logs and minimum detection limit of 9.4 viral genome copies. It was shown to be highly specific, as tissues from uninfected chickens and other viral genomes, such as those of Marek's disease virus, fowlpox virus and infectious laryngotracheitis virus did not produce positive signal. The sensitivity of the real-time PCR was comparable with nested PCR and it was 100 times more sensitive than the conventional PCR. The assay was validated by testing DNA from tissues of chickens infected with FAdV-9 collected at different days post-infection. FAdV-9 DNA was detected in liver, bursa of Fabricius and cecal tonsil tissues in a range of 10(2)-10(7) copies per 100 ng of total DNA. High amounts of viral DNA were present in the cecal tonsils for a week after inoculation making this tissue an ideal sample source for the diagnosis of FAdV infection. This assay is an excellent research and diagnostic tool that provides high sensitivity, specificity and rapid post-PCR analyses.
本研究的目的是以禽腺病毒9型（FAdV-9）为模型，开发一种高灵敏度的实时聚合酶链反应（PCR）检测方法，用于检测和定量鸡组织中的禽腺病毒（FAdV）DNA。该检测方法的动态范围为7个对数，最低检测限为9.4个病毒基因组拷贝。结果表明，该方法具有高度特异性，未感染鸡的组织以及其他病毒基因组，如马立克氏病病毒、禽痘病毒和传染性喉气管炎病毒的组织均未产生阳性信号。实时PCR的灵敏度与巢式PCR相当，比传统PCR高100倍。通过检测感染FAdV-9的鸡在感染后不同天数采集的组织DNA，对该检测方法进行了验证。在肝脏、法氏囊和盲肠扁桃体组织中检测到FAdV-9 DNA，每100 ng总DNA中的拷贝数范围为10²-10⁷。接种后一周内，盲肠扁桃体中存在大量病毒DNA，使该组织成为诊断FAdV感染的理想样本来源。该检测方法是一种优秀的研究和诊断工具，具有高灵敏度、特异性和快速的PCR后分析能力。
