title: Comparison of commercial real-time reverse transcription-PCR assays for reliable, early, and rapid detection of heterologous strains of porcine reproductive and respiratory syndrome virus in experimentally infected or noninfected boars by use of different sample types.
abstract: The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days -2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays (VetMax from Applied Biosystems, Foster City, CA [abbreviated AB]; VetAlert from Tetracore, Rockville, MD [TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold, <30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa, USA.
authors.forename: Priscilla F
authors.initials: PF
authors.lastname: Gerber
authors.forename: Kevin
authors.initials: K
authors.lastname: O'Neill
authors.forename: Olajide
authors.initials: O
authors.lastname: Owolodun
authors.forename: Chong
authors.initials: C
authors.lastname: Wang
authors.forename: Karen
authors.initials: K
authors.lastname: Harmon
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Zhang
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Halbur
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
authors.forename: Xiang-Jin
authors.initials: XJ
authors.lastname: Meng
authors.forename: Tanja
authors.initials: T
authors.lastname: Opriessnig
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D001826:Body Fluids / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: D000914:Antibodies, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D014759:Viral Envelope Proteins; C114789:glycoprotein 5, PRRSV