title: Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues.
abstract: The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time polymerase chain reaction (PCR) assay targeting the highly conserved rep gene (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally infected conventional pigs, experimentally infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2x10(3) to 2.2x10(10) copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes; thymus; tonsil; ileal Peyer's patches; and spleen), and non-lymphoid (myocardium; lung; kidney; liver; and gluteal muscle) tissues from naturally infected pigs. Across all tissues and sera of naturally infected pigs, the mean PCV2 concentration was 3.0logs higher in wasting versus non-wasting pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in quantification of PCV2 in a variety of tissues from naturally and experimentally infected pigs.
authors.affiliation: Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4, Canada.
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: McIntosh
authors.forename: Anju
authors.initials: A
authors.lastname: Tumber
authors.forename: John C S
authors.initials: JC
authors.lastname: Harding
authors.forename: Steven
authors.initials: S
authors.lastname: Krakowka
authors.forename: John A
authors.initials: JA
authors.lastname: Ellis
authors.forename: Janet E
authors.initials: JE
authors.lastname: Hill
mesh_terms: D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D017925:Circovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis*; D003959:Diamines; D005243:Feces / Q000821:virology; D005456:Fluorescent Dyes; D005838:Genotype; D005856:Germ-Free Life; D007150:Immunohistochemistry / Q000662:veterinary; D008221:Lymphoid Tissue / Q000821:virology; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary*; D053570:Porcine Postweaning Multisystemic Wasting Syndrome / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D011804:Quinolines; D012680:Sensitivity and Specificity; D044967:Serum / Q000821:virology; D013552:Swine; D019562:Viral Load / Q000662:veterinary; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D014764:Viral Proteins; C098022:SYBR Green I