title: Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus.
abstract: The accuracy and rapid diagnosis of PRRSV infection is a major prerequisite for every control and/or eradication strategy. In this study two real time RT-PCR based on different chemistry analysis (TaqMan Probes and SYBR Green) have been developed and validated before comparison to an end point two-step RT-PCR validated previously. All assays were aimed at discrimination between PRRSV genotypes. Furthermore, an exogenous internal control (IC) system had also been implemented in qRT-PCR. A rigorous analytical validation, executed on infected cell cultures and serum, demonstrated good sensitivity, specificity and repeatability. In particular RT-PCR was exceptionally sensitive and could detect a viral titre in the order of a magnitude of 1 copies/μL, 10-fold lower than other qRT-PCR described in this study. Optimal diagnostic performances have been demonstrated analyzing samples retrieved from an experimental infection, with RT-PCR again outperforming real time RT-PCR assays. All tests, showing substantial agreement between them, were able to detect early stages of viraemia (1 DPI) and some animals were classified as positive until the end of the study (76 DPI). Therefore, this supports the assays usefulness in animals with different clinical conditions and in a broad range of epidemiological scenarios. The benefits and disadvantages of different assays were also considered and discussed.
authors.affiliation: Department of Animal Medicine, Production and Health (MAPS), University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy. Electronic address: michele.drigo@unipd.it.
authors.forename: Michele
authors.initials: M
authors.lastname: Drigo
authors.affiliation: Department of Animal Medicine, Production and Health (MAPS), University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Franzo
authors.affiliation: Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020 Legnaro, PD, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Belfanti
authors.affiliation: Department of Animal Medicine, Production and Health (MAPS), University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Martini
authors.affiliation: Department of Animal Medicine, Production and Health (MAPS), University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mondin
authors.affiliation: Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020 Legnaro, PD, Italy.
authors.forename: Letizia
authors.initials: L
authors.lastname: Ceglie
mesh_terms: D000818:Animals; D060005:Genotyping Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis; D019316:Porcine respiratory and reproductive syndrome virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine
keywords: Genotyping; PRRSV; RT-PCR; qRT-PCR