Establishment And Adhibition Of LAMP&SYBR Green Ⅰ Real-time Quantitative PCR Detection Methods For Ornithobacetrium Rhinotracheale

Ornithobacterium rhinotracheale (ORT) is a Gram-negative pleomorphic, rod-shapedbacterium that has been identified as an emerging respiratory pathogen in chickens andturkeys. There are18serotypes of ORT, designated by the letters A through R. It growsslowly, producing only pinpoint colonies on blood agar at37℃in the presence of5%CO2at24h. Optimal growth occurs when plates have been incubated for48h. Colonies then appeargray to grayish white, opaque, convex and circular with a diameter of1-3mm. Infection withthis organism has been associated with respiratory disease, increased mortality, and growthretardation in poultry and other avian species throughout the world. The organism has beenreported worldwide and has also been isolated from wild birds. ORT was first discovered in1986, followed by the Netherlands, Belgium, United States, Germany, France, Canada andJapan have reported that the ORT was isolated. The disease caused by ORT is characterizedby lacrimation, dyspnea, tracheitis, nasal exudates, sinusitis, coughing, and oedema in facialsubcutis, growth retardation airsacculitis, and fibrinous pneumonia in severely affected birds.Postmortem lesions associated with Ornithobacterium rhinotracheale infection have beenidentified as fibrinopurulent pneumonia, air sacculitis, peritonitis with foamy exudates andarthritis. For further comprehension of the ORT epidemic and theory basis of prevention andcontrol, the research focuses on the establishment of the diagnosis method. This researchcontent was divided into two parts:1．Establishment and adhibition of LAMP system for rapid detection of OrnithobacteriumrhinotrachealeTo establish a rapid diagnostic method for Ornithobacterium rhinotracheale (ORT) basedon the loop-mediated isothermal amplification (LAMP). A suit of special primers weredesigned for ORT with Primer Explorer V4.The reaction system and conditions wereestablished and optimized, meanwhile its specificity and sensitivity were assessed. TheOrnithobacterium rhinotracheale (ORT) DNA could be amplified by incubation at63℃within1h and the amplification products could be observed by naked-eye.Specificity testshowed that the target bacterium could be specially detected by the method, while theamplification results of Salmonella pullorum, Escherichia coli, Staphylococcusaureus, Pasteurella multocida and Haemophilus paragallinarum were negative.Sensitivity testshowed that the detection limit of the system was found to be1×101DNA sample, which was104-fold higher than that of the traditional PCR. The detection rate was100%by using thisnewly developed LAMP methed to test positive for the separation of the strains. Theestablished LAMP diagnostic method appeared to be fast accurate, highly sensitive andspecific for the purpose.2．Development and application of SYBR GreenⅠreal-time quantitative PCR technique forOrnithobacterium rhinotrachealeThe objective of the study is to establish a method for detecting Ornithobacteriumrhinotracheale by SYBR Green I fluorescence quantitative PCR detection method and toapplication in clinical samples detection. A pair of specific primers was designed accordingto the Ornithobacterium rhinotracheale（GenBank login number: JF810495.1）gene sequence.The target214bp fragment was amplified in PCR gene amplification, and inserted intopMD18-T vector after being purified. Then the restructured vectors were transformed toEscherichia coli DH5α. After PCR identifying, enzyme cutting and sequencing, positiveplasmid as template was extracted to establish SYBR-Green Ⅰ real-time fluorescencequantitative PCR standard curve. Sensitivity, reproducibility and specificity of the methodwere determined. Results the standard curve indicated the linear relationship between cyclethreshold (CT) and template concentration. The standard curve linear relationship was morethan0.99. The Tm was81.3-83.2and the sensitive degree is1.44×102copies per all, Specificresults showed that only amplification curve of Ornithobacterium rhinotracheale can bedetected, repeatability test indicated the variation coefficient is less than0.7%; clinicalsample detection rate was100%. The newly established SYBR Green Ⅰ real-timefluorescent quantitative PCR assay might be a useful diagnostic method forepidemiologically investigating and closely observing the newly emerged Ornithobacteriumrhinotracheale.