title: Immuno-PCR for one step detection of H5N1 avian influenza virus and Newcastle disease virus using magnetic gold particles as carriers.
abstract: Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 10(2) to 10(-5)EID(50)/ml. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10(-4)EID(50)/ml AIV and NDV. To further evaluate the specificity and the clinical application of this IPCR assay for AIV H5N1 and NDV, the tracheal swab specimens, taken from chickens which were infected with H5N1/AIV, H9N2/AIV, H7N2/AIV, NDV, IBDV, IBV/H(120), were detected by IPCR. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step.
authors.affiliation: Northwest Agriculture and Forestry University, College of Veterinary Medicine, Yangling, Shaanxi 712100, China.
authors.forename: MingJun
authors.initials: M
authors.lastname: Deng
authors.forename: Ling
authors.initials: L
authors.lastname: Long
authors.forename: XiZhi
authors.initials: X
authors.lastname: Xiao
authors.forename: ZhenXing
authors.initials: Z
authors.lastname: Wu
authors.forename: FengJuan
authors.initials: F
authors.lastname: Zhang
authors.forename: YanMing
authors.initials: Y
authors.lastname: Zhang
authors.forename: XiaoLong
authors.initials: X
authors.lastname: Zheng
authors.forename: XueQian
authors.initials: X
authors.lastname: Xin
authors.forename: Qun
authors.initials: Q
authors.lastname: Wang
authors.forename: DongLai
authors.initials: D
authors.lastname: Wu
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry; D000956:Antigens, Viral / Q000737:chemistry; D019921:Biotinylation; D002645:Chickens; D006046:Gold / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology; D009521:Newcastle Disease / Q000175:diagnosis / Q000821:virology; D009522:Newcastle disease virus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014132:Trachea / Q000821:virology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D006046:Gold