title: Development of SYBR green-based real-time PCR and duplex nested PCR assays for quantitation and differential detection of species- or type-specific porcine Torque teno viruses.
abstract: Porcine Torque teno virus (TTV), a single-stranded circular DNA virus, has been incriminated in swine diseases recently. Multiple infection with porcine TTV species 1 (PTTV1) and species 2 (PTTV2), each consisting of two types (PTTV1a and 1b) or subtypes (PTTV2b and 2c), in a single pig had been reported by our group previously. The present study described three novel assays for quantitation and differential detection of porcine TTV. First, we developed two SYBR green-based real-time PCR assays to quantify viral loads of two porcine TTV species, respectively. The PTTV1- and PTTV2-specific real-time PCR primer sequences were selected to target conserved regions identified by multiple alignments of ten available porcine TTV full-length genomes. Furthermore, by coupling the two singleplex PCR assays, a duplex real-time PCR assay followed by melting curve analysis was established for simultaneous detection and differentiation of PTTV1 and PTTV2. In addition, a type-specific duplex nested PCR was also developed to simultaneously detect and distinguish between the two types, PTTV1a and 1b, in PTTV1 species. These assays provide rapid and practical tools for molecular diagnosis of species- or type-specific porcine TTV.
authors.affiliation: Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0913, USA.
authors.forename: Y W
authors.initials: YW
authors.lastname: Huang
authors.forename: B A
authors.initials: BA
authors.lastname: Dryman
authors.forename: K K
authors.initials: KK
authors.lastname: Harrall
authors.forename: E M
authors.initials: EM
authors.lastname: Vaughn
authors.forename: M B
authors.initials: MB
authors.lastname: Roof
authors.forename: X J
authors.initials: XJ
authors.lastname: Meng
mesh_terms: D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers; D004266:DNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D003959:Diamines; D025202:Molecular Diagnostic Techniques; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D011804:Quinolines; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013194:Staining and Labeling; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D022783:Torque teno virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I