title: Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-based multiplex PCR assay.
abstract: A new, rapid, and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR method was developed for simultaneous detection and differentiation of nine avian respiratory pathogens. The respiratory pathogens included in this study were avian influenza subtypes H5, H7, and H9, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Haemophilus paragallinarum (HPG). Ten pairs of primers were designed using conserved and specific sequence genes of AIV subtypes and respiratory pathogens from GenBank. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The specific DNA product amplification peaks of nine respiratory pathogens were observed on the GeXP analyzer. Non-respiratory avian pathogens, including chicken infectious anemia virus, fowl adenovirus, avian reovirus and infectious bursal disease virus, did not produce DNA products. The detection limit for the GeXP-multiplex assay was determined to be 100 copies/μl using various pre-mixed plasmids/ssRNAs containing known target genes of the respiratory pathogens. Further, GeXP-multiplex PCR assay was 100% specific when 24 clinical samples with respiratory infections were tested in comparison with conventional PCR method. The GeXP-multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of nine avian respiratory pathogens.
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China. Electronic address: xiezhixun@126.com.
authors.forename: Zhixun
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China.
authors.forename: Sisi
authors.initials: S
authors.lastname: Luo
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China.
authors.forename: Liji
authors.initials: L
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China.
authors.forename: Jiabo
authors.initials: J
authors.lastname: Liu
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China.
authors.forename: Yaoshan
authors.initials: Y
authors.lastname: Pang
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China.
authors.forename: Xianwen
authors.initials: X
authors.lastname: Deng
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China.
authors.forename: Zhiqin
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning 530001, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Fan
authors.affiliation: Department of Pathobiology and Veterinary Science, University of Connecticut, 61 North Eagleville Road, Storrs, CT 06269-3089, USA. Electronic address: mazhar.khan@uconn.edu.
authors.forename: Mazhar I
authors.initials: MI
authors.lastname: Khan
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D001715:Bird Diseases / Q000175:diagnosis* / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods*; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D014780:Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
keywords: Avian respiratory pathogens; GenomeLab Gene Expression Profiler (GeXP); Multiplex PCR