title: A duplex real-time PCR assay for the detection and quantification of avian reovirus and Mycoplasma synoviae.
abstract: BACKGROUND
  Infectious arthritis in broilers represents an economic and health problem, resulting in severe losses due to retarded growth and downgrading at the slaughterhouse. The most common agents associated with cases of infectious arthritis in poultry are avian reovirus (ARV) and Mycoplasma synoviae (MS). The accurate differentiation and rapid diagnosis of ARV and MS are essential prerequisites for the effective control and prevention of these avian pathogens in poultry flocks. This study thus aimed to develop and validate a duplex real-time PCR assay for the simultaneous detection and quantification of ARV and MS.
  
  
  METHODS
  Specific primers and probes for each pathogen were designed to target the special sequence of the ARV σC gene or the MS phase-variable surface lipoprotein hemagglutinin (vlhA) gene. A duplex real-time PCR assay was developed, and the reaction conditions were optimized for the rapid detection and quantification of ARV and MS.
  
  
  RESULTS
  The duplex real-time PCR assay was capable of ARV- and MS-specific detection without cross-reaction with other non-targeted avian pathogens. The sensitivity of this assay was 2 × 10(1) copies for a recombinant plasmid containing ARV σC or MS vlhA gene, and 100 times higher than that of conventional PCR. This newly developed PCR assay was also reproducible and stable. All tested field samples of ARV and/or MS were detectable with this duplex real-time PCR assay compared with pathogen isolation and identification as well as serological tests.
  
  
  CONCLUSIONS
  This duplex real-time PCR assay is highly specific, sensitive and reproducible and thus could provide a rapid, specific and sensitive diagnostic tool for the simultaneous detection of ARV and MS in poultry flocks. The assay will be useful not only for clinical diagnostics and disease surveillance but also for the efficient control and prevention of ARV and MS infections.
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. lhuang405@126.com.|College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China. lhuang405@126.com.
authors.forename: Li
authors.initials: L
authors.lastname: Huang
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. xiezhixun@126.com.
authors.forename: Zhixun
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. xie3120371@163.com.
authors.forename: Liji
authors.initials: L
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. dengxianwen@126.com.
authors.forename: Xianwen
authors.initials: X
authors.lastname: Deng
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. xzqman2002@sina.com.
authors.forename: Zhiqin
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. 2004-luosisi@163.com.
authors.forename: Sisi
authors.initials: S
authors.lastname: Luo
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. huangjiaoling728@126.com.
authors.forename: Jiaoling
authors.initials: J
authors.lastname: Huang
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China. tingtingzeng1986@163.com.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zeng
authors.affiliation: College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China. feng@public.nn.gx.cn.
authors.forename: Jiaxun
authors.initials: J
authors.lastname: Feng
mesh_terms: D000818:Animals; D001170:Arthritis, Infectious / Q000175:diagnosis / Q000662:veterinary*; D058491:Bacterial Load / Q000379:methods; D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006388:Hemagglutinins / Q000235:genetics; D008074:Lipoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D045805:Mycoplasma synoviae / Q000302:isolation & purification*; D030101:Orthoreovirus, Avian / Q000302:isolation & purification*; D011200:Poultry; D011201:Poultry Diseases / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods; D014764:Viral Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D006388:Hemagglutinins; D008074:Lipoproteins; D008565:Membrane Proteins; D014764:Viral Proteins