Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E)

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• 作者：Ayse G&uuml ; nes ^{ayse.guenes@vetmeduni.ac.at} ; Ana Marek ^{ana.marek@vetmeduni.ac.at} ; Beatrice Grafl ^{beatrice.grafl@vetmeduni.ac.at} ; Evelyn Berger ^{evelyn.schulz@vetmeduni.ac.at} ; Michael Hess ; ^{michael.hess@vetmeduni.ac.at}
• 关键词：BLAST ; basic local alignment search tool ; bp ; base pair ; CEL cells ; chicken embryo liver cells ; CELO ; chicken embryo lethal orphan ; CPE ; cytopathic effect ; CT ; threshold cycle ; d.p.i. ; days post-infection ; FAdV ; Fowl adenovirus ; FAdV-A to FAdV-E ; species
• 刊名：Journal of Virological Methods
• 出版年：2012
• 期刊代码：180_01660934
• 类别：med
• 出版时间：August, 2012
• 卷：183
• 期：2
• 页码：147-153
• 文件大小：725 K

摘要

The present study describes the development of a SYBR Green based real-time polymerase chain reaction (real-time PCR) for detection and quantitation of all fowl adenovirus (FAdV) species. Primers were designed based on conserved nucleotide sequences within the 52K gene. Ten-fold serial dilutions of a vector DNA were used as standard for quantitation. The real-time PCR had an efficiency of 98%, a regression squared value of 0.999 and showed a range of 6.73-6.73 脳 10⁸ copies of FAdV DNA per reaction. The assay was highly specific for FAdVs and an exact quantitation of all 5 FAdV species (FAdV-A to FAdV-E) could be demonstrated. It was shown, that twelve FAdV serotypes (FAdV-1 to 8a, and 8b to 11) were detectable and quantifiable. Other viral genomes as well as uninfected chicken embryo liver (CEL) cells did not produce positive signal. Cloacal swabs were taken during the animal experiment, which was performed with all FAdV species. Shedding of FAdVs was investigated in cell culture, by conventional PCR and by the developed real-time PCR. The real-time PCR was found more sensitive than cell culture and conventional PCR. Detection and quantitation of FAdVs in different type of samples was possible by the new real-time PCR.