title: The use of endogenous and exogenous reference RNAs for qualitative and quantitative detection of PRRSV in porcine semen.
abstract: Semen is known to be a route of porcine reproductive and respiratory syndrome virus (PRRSV) transmission. A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = 36). However, the amount of UBE2D2 mRNA in porcine semen varied (up to 106-fold), thus its use is limited to qualitative detection of PRRSV RNA. For quantitation, a synthetic, non-metazoan RNA was added to the RNA isolation reaction at an exact copy number. The photosynthesis gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from Arabidopsis thaliana was used as an exogenous spike. Unexpectedly, PRRSV RNA was detected in a herd of specific pathogen-free (SPF) boars which were tested ELISA-negative for anti-PRRSV antibodies. Therefore, RT-PCR for seminal cell-associated PRRSV is a powerful tool for managing the SPF status during quarantine programs and for routine outbreak investigations.
authors.affiliation: Institute of Animal Breeding and Genetics, Department for Animal Breeding and Reproduction, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.
authors.forename: Sandra
authors.initials: S
authors.lastname: Revilla-Fernández
authors.forename: Barbara
authors.initials: B
authors.lastname: Wallner
authors.forename: Klaus
authors.initials: K
authors.lastname: Truschner
authors.forename: Alexandra
authors.initials: A
authors.lastname: Benczak
authors.forename: Gottfried
authors.initials: G
authors.lastname: Brem
authors.forename: Friedrich
authors.initials: F
authors.lastname: Schmoll
authors.forename: Mathias
authors.initials: M
authors.lastname: Mueller
authors.forename: Ralf
authors.initials: R
authors.lastname: Steinborn
mesh_terms: D000818:Animals; D008969:Molecular Sequence Data; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D012661:Semen / Q000821:virology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013552:Swine; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D044763:Ubiquitin-Conjugating Enzymes; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase