Establishment And Primary Application Molecular Detection Methods For Infectious Bovine Rhinotracheitis And Akabane Disease

Infectious bovine rhinotracheitis （IBR） and Akabane disease are two important harmfulviral transmitted diseases in cattle industry.Import-export cattle and their products should beperformed quarantine for these two diseases. The main detection methods to IBRV and AKAVinclude immunofluorescent antibody test,Neutralization test,Enzyme-linked immunosorbentassay,Electron microscopy,Colloidal gold technique,and so on.Although these methods playedan important role in detecting diseases, they have some difficulty such as lowersensitivy,operation complex and cost a long time,and so on. It is urgent to establish a fast,accurate early cattle disease diagnosis to prevent and clear such infectious recessive diseases forthe healthy and rapid development of China’s livestock. The thesis has established the rapiddetection system of IBR and AKA firstly,including Multiplex Polymerase Chain Reaction, Genechip and realtime fluores-cence quantitative PCR. A series of reaction conditions areoptimized,and they form detection reagent kits which increase detection speed and improvedetection limits. The research abstract was summarized below：（1）Establishment and primary application of Multiplex Polymerase Chain Reaction fordetection of IBRV and AKAV. A multiplex polymerase chain reaction（M-PCR） was establishedand optimized to detect bovine herpesvirus1（BHV1） and akabane virus （AKAV）simultaneously．The genes were amplified by polymerase chain reaction（PCR） and cloned to thePGM-T vector.Two sets of specific primers were designed according to the conservative gBsequence of IBRV and the S sequence of AKAV in the GenBank．The optimal annealingtemperature is59℃and the optimal Mg²⁺concentration is1.5nmol/μL. The detection limit ofthe M-PCR assay was0.13pg/25μL for IBRV and1.04pg/25μL for AKAV.（2）Establishment and primary application of oligonucleotide microarray for detection ofIBRV and AKAV. Based on the multiplex PCR, oligonucleotide probes were designed to capturethe Sense strands of the PCR product, and the specific Sense primers were labeled withfluorescein （Cy3） modification at5，-terminal. Through the experiment,the suitable hybridisationtemperature was38℃. The detection limit of the oligonucleotide microarray was0.013pg/25μLfor IBRV and0.104pg/25μL for AKAV.（3）Establishment and primary application of realtime fluorescence quantitative PCR fordetection of IBRV and AKAV. Based on the two positive standard plasmid builded in M-PCR, two sets of specific primers were designed. The reaction parameters were optimized to developSYBR Green I fluorescence quantitative PCR assay for detecting the two viruses respectively.The detection limit of the realtime fluores-cence quantitative PCR was1.3fg （360copies）/25μLfor IBRV and1.04fg （926copies）/25μL for AKAV.Out of these three above-mentioned methods,the realtime fluorescence quantitative PCR hasthe highest sensitivity，oligonucleotide microarray takes second place,and the M-PCR has thelowest sensitivity. The operation is relatively simple and the instrument is cheaper in theM-PCR. Oligonucleotide microarray has complicated operation and expensive instrument,but ithas the advantage of high throughput.The realtime fluorescence quantitative PCR can’t detect thetwo viruses simultaneously. In conclusion,each method has its own merits and demerits andshould be used according to different situations.