title: Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green-based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples.
abstract: Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 10(7) DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green-based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected (n = 23) or non-PMWS-affected pigs (n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥ 10(2.5)). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 10(7.0 ± 1.5) copies/100 ng of total DNA sample, while the cPCR detected up to 10(4.8 ± 1.5). A mean difference of 10(1.8) was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.
authors.affiliation: Laboratório de Virologia, FEPAGRO Saúde Animal, Instituto de Pesquisas Veterinárias Desidério Finamor, Eldorado do Sul, Rio Grande do Sul, Brazil. Brazil.ddezen@gmail.com
authors.forename: Diogenes
authors.initials: D
authors.lastname: Dezen
authors.forename: Franciscus A M
authors.initials: FA
authors.lastname: Rijsewijk
authors.forename: Thais F
authors.initials: TF
authors.lastname: Teixeira
authors.forename: Carine L
authors.initials: CL
authors.lastname: Holz
authors.forename: Ana P
authors.initials: AP
authors.lastname: Varela
authors.forename: Samuel P
authors.initials: SP
authors.lastname: Cibulski
authors.forename: Tatiane Shäffer
authors.initials: TS
authors.lastname: Gregianini
authors.forename: Helena B C R
authors.initials: HB
authors.lastname: Batista
authors.forename: Ana C
authors.initials: AC
authors.lastname: Franco
authors.forename: Paulo M
authors.initials: PM
authors.lastname: Roehe
mesh_terms: D000818:Animals; D017925:Circovirus / Q000145:classification* / Q000235:genetics*; D004279:DNA, Viral / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D053570:Porcine Postweaning Multisystemic Wasting Syndrome / Q000175:diagnosis / Q000821:virology*; D012680:Sensitivity and Specificity; D013552:Swine; D019562:Viral Load
chemical_list: D004279:DNA, Viral; D005456:Fluorescent Dyes