Establishment Of Multiple PCR And Multiple Fluorescent Quantitative PCR For Detection Of Common Diseases Of Cattle And Sheep

In recent years,the state strive to develop the herbivorous animal husbandry,increasing the number of cattle and sheep breeding and large-scale degree arise.With the rapid development of cattle and sheep breeding,the epidemic form of cattle and sheep epidemic disease tends to be complex,and often show the increased incidence of disease,mixed infection increased trend,through a single disease laboratory testing is difficult to make a diagnosis.Therefore,the establishment of multiplex PCR and multiplex fluorescent quantitative PCR can simultaneously and respectively on a variety of diseases for rapid diagnosis,and which is particularly important for laboratory rapid detection of cattle and sheep epidemic disease and has strong practical application value for the primary prevention of animal epidemic disease.In this study,Peste des petits ruminants,Capripox,Bluetongue,Bovine viral diarrhea,Infectious bovine rhinotracheitis and other cattle and sheep often epidemic disease as the object of the study.Through a large number of comparing the N gene of PPRV,the P32 gene of CPV and the NS3 gene of BTV in GenBank.,and selected the highly conserved gene sequence as the target sequence,which established a single PCR method of PPRV,BTV and CPV,at the same time,established a multiplex PCR method for simultaneous amplification of PPRV,BTV and CPV.Through a large number of comparing the 5'UTR gene of BVDV,the gB gene of IBRV in GenBank.,and selected the highly conserved gene sequence as the target sequence,which established a single PCR method of BVDV,IBRV,at the same time,established a multiplex PCR method for simultaneous amplification of BVDV,IBRV.The results show that the single PCR method has better sensitivity and specificity,the detection limit of PPRV is 60pg,the detection limit of BTV is 1ng,the detection limit of CPV is 8pg,the detection limit of BVDV is 0.12ng,the detection limit of IBRV 0.6pg.The establishment of PPRV,BTV,CPV multiple PCR method has better sensitivity and specificity,the detection limit of PPRV 4ng,BTV 10ng,CPV 0.8pg,BVDV,IBRV multiplex PCR for detection limit of BVDV was 0.12pg,IBRV was 6pg.On the basis of conventional PCR,Through designing premers and probes,which established a single fluorescence quantitative PCR method of PPRV,BTV and CPV,at the same time,established a multiplex fluorescence quantitative method for simultaneous amplification of PPRV,BTV and CPV.And which established a single fluorescence quantitative PCR method of BVDV,IBRV,at the same time,established a duplex fluorescence quantitative method for simultaneous amplification of BVDV,IBRV.The results show that the single fluorescence quantitative method has better sensitivity and specificity,the detection limit of PPRV is 0.06pg,the detection limit of BTV is 0.1 pg,the detection limit of CPV is 0.04pg,the detection limit of BVDV is 0.1 pg,the detection limit of IBRV 0.06pg.The establishment of PPRV,BTV,CPV multiple fluorescence quantitative PCR method has better sensitivity and specificity,the detection limit of PPRV 0.06pg,BTV 0.12pg,CPV 0.04pg,BVDV,IBRV multiplex fluorescent quantitative PCR for detection limit of BVDV was 1.2pg,IBRV was 0.8pg.In conclusion,this study established PPRV,BTV,CPV single and multiple PCR method single and multiple fluorescence quantitative PCR method,BVDV,IBRV single and multiple PCR method single and multiple fluorescence quantitative PCR method,which can fast and accurately detect these common diseases of cattle and sheep,and has a strong application value.