The use of endogenous and exogenous reference RNAs for qualitative and quantitative detection of PRRSV in porcine semen.
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.
Revilla-Fernández
Sandra
S
Institute of Animal Breeding and Genetics, Department for Animal Breeding and Reproduction, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.
Wallner
Barbara
B
Truschner
Klaus
K
Benczak
Alexandra
A
Brem
Gottfried
G
Schmoll
Friedrich
F
Mueller
Mathias
M
Steinborn
Ralf
R
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
D012333:RNA, Messenger; D012367:RNA, Viral; D044763:Ubiquitin-Conjugating Enzymes; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase