title: Comparison of protocols for the analysis of type 1 porcine reproductive and respiratory syndrome virus by RT-PCR using oral fluids.
abstract: The detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OF) by quantitative real-time polymerase chain reaction (qRT-PCR) is gaining increasing popularity. However, the different steps leading to a result have not been extensively evaluated. The aim of the present study was to examine the effect on the performance of qRT-PCR with different sampling materials, conditions of storage of the OF, the need for centrifuging OF, as well as to compare RNA extraction methods and PCR mixes. For the assays, pen-based oral fluids were used, which were pooled and spiked in a serial dilution (up to genotype 100TCID50/mL) of type 1 PRRSV isolate 3267. Centrifugation at 15,000g for 15min resulted in an increase in sensitivity (1-2 PCR cycles) that was significant (P<0.05) at the lowest dilution tested. The TRIzol and MagMAX RNA extraction methods gave the maximum sensitivity, lowest threshold cycle (Ct), at equivalent virus concentrations. The AgPath-ID One-Step RT-PCR Kit PCR mix reagents were more sensitive for the detection of PRRSV using a purified plasmid as standard, but LSI VetMAX PRRSV EU/NA PRRSV reagents resulted in a slightly better sensitivity with OF (p<0.05). The present results may be useful to standardize protocols for optimizing detection of type 1 PRRSV in OF by qRT-PCR.
authors.affiliation: Centre de Recerca en Sanitat Animal (CReSA), IRTA-UAB, Edifici CReSA, Campus UAB, 08193, Bellaterra, Spain. Electronic address: elisa.gibert@irta.cat.
authors.forename: Elisa
authors.initials: E
authors.lastname: Gibert
authors.affiliation: Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona (UAB), Campus UAB, 08193, Bellaterra, Spain. Electronic address: gerard.martin@uab.cat.
authors.forename: Gerard
authors.initials: G
authors.lastname: Martín-Valls
authors.affiliation: Centre de Recerca en Sanitat Animal (CReSA), IRTA-UAB, Edifici CReSA, Campus UAB, 08193, Bellaterra, Spain; Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona (UAB), Campus UAB, 08193, Bellaterra, Spain. Electronic address: enric.mateu@uab.cat.
authors.forename: Enric
authors.initials: E
authors.lastname: Mateu
mesh_terms: D000818:Animals; D005838:Genotype; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009055:Mouth / Q000821:virology*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D013552:Swine
keywords: Diagnosis; Oral fluids; Porcine reproductive and respiratory syndrome virus; RNA extraction; qRT-PCR