title: Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E).
abstract: 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 6.73-6.73×10(8) 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.
authors.affiliation: Clinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria. ayse.guenes@vetmeduni.ac.at
authors.forename: Ayse
authors.initials: A
authors.lastname: Günes
authors.forename: Ana
authors.initials: A
authors.lastname: Marek
authors.forename: Beatrice
authors.initials: B
authors.lastname: Grafl
authors.forename: Evelyn
authors.initials: E
authors.lastname: Berger
authors.forename: Michael
authors.initials: M
authors.lastname: Hess
mesh_terms: D000257:Adenoviridae Infections / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002460:Cell Line; D002642:Chick Embryo; D002988:Cloaca / Q000821:virology; D025681:Fowl adenovirus A / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D016679:Genome, Viral; D025202:Molecular Diagnostic Techniques; D058889:Molecular Typing; D011201:Poultry Diseases / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D017201:Virus Shedding
chemical_list: D000914:Antibodies, Viral