The Multiplex PCR Detection Method Of The Main Cause Of Bovine Respiratory Disease Complex Establishment And Application

In recent years,the breeding industry has developed rapidly.As one of the main industries of the breeding industry,the disease of cattle has attracted more and more attention.Among them,Bovine respiratory disease complex(BRDC),because of its complex and variable causes,is difficult to deal with,and has caused huge economic losses to the cattle industry all over the world.Although there are a large number of studies trying to treat bovine respiratory disease syndrome,these measures have limited impact on reducing bovine respiratory disease syndrome.Therefore,finding the cause of the disease from the root cause is more critical for symptomatic treatment.Many studies at home and abroad have shown that the causes of bovine respiratory syndrome include DNA viruses,RNA viruses,and even mycoplasma infections.Among them,Bovine herpes virus-1(BHV1)bovine respiratory syncytial virus(Bovine respiratory syncytial)virus,BRSV)Bovine corona viruses(BCo V),Bovine parinfluenza-3 virus(BPIV3),Bovine viral diarrhea virus(BVDV),Mycoplasma Bovis(Mycoplasma Bovis),MB)are several common clinical pathogens that cause bovine respiratory syndrome.If cattle carry these pathogens,they will lead to the occurrence of bovine respiratory syndrome,causing fever,runny nose or pneumonia,and even death.As for the rapid development of the aquaculture industry,it has caused huge obstacles.However,these pathogenic infections have very similar clinical symptoms,and simple clinical observations are difficult to distinguish,and these pathogenic infections often appear in the form of mixed infections.If mixed infections occur,the related pathogens should be tested separately.The operation is cumbersome,the time-consuming is too long,and there will be defects such as unobvious test results.In order to quickly differentiate and diagnose the multiple pathogenic infections or mixed infections that may occur in the bovine respiratory disease syndrome,this test attempts to construct a six-fold that can simultaneously identify the six pathogens of BVDV,BPIV3,BRSV,BCo V,MB and BHV1.The PCR detection and diagnosis method provides a scientific basis for the prevention and control of bovine respiratory diseases:1.Build and Test a Single PCR Method of BVDV,BPIV3,BRSV,BCo V,MB and BHV1 Bovine Respiratory Disease ComplexAccording to the gene sequences published in Genbank of BVDV 5’-UTR(MK059454),BPIV3 g N(JQ063064),BRSV g N(AF188553),BCo V g N(NC003045),MB uvr C(AF003959)and BHV1 g B(AJ004801),Six pairs of PCR primers were designed to amplify partial sequences of BVDV 5’-UTR,BPIV3 g N,BCo V g N,BRSV g N,MB uvr C and BHV1 g B,and the amplified target fragments were 139 bp,239 bp,304 bp,456 bp,527 bp and 798 bp.Through the sequencing and identification of the target gene and optimization of reaction conditions,a single PCR method for BVDV,BPIV3,BRSV,BCo V,MB and BHV1 was established,laying the foundation for the establishment of multiple PCR methods for pathogens of Bovine respiratory disease complex.2.Construction of Multiplex PCR Method for Six Common Pathogens of Bovine Respiratory Disease ComplexOn the basis of the above-constructed single PCR detection method for the six pathogenic genes of BVDV,BPIV3,BRSV,BCo V,MB and BHV1,the six-fold PCR reaction conditions of BVDV,BPIV3,BRSV,BCo V,MB and BHV1 were further optimized Adjust and change the controllable and adjustable conditions of template and primer concentration,dosage and annealing temperature.At the same time,the specificity,sensitivity and reproducibility of BVDV,BPIV3,BRSV,BCo V,MB and BHV1 six-fold PCR have been tested and studied.A multiplex PCR detection method for the simultaneous detection of six common bovine respiratory disease pathogens,BVDV,BPIV3,BRSV,BCo V,MB and BHV1,has been established.The results showed that:(1)The reaction system made up of 50μL with Gold Mix(green)is the optimal reaction system for six-fold PCR.The upstream and downstream primer concentrations of BVDV,BPIV3,BRSV,BCo V and BHV1 are all 1 μL;MB upstream primer Both the downstream primers and the downstream primers have a concentration of 1.5μL.(2)The multiple PCR amplified gene bands are most obvious when the annealing temperature is54.1℃,and each amplified gene has specific bands,that is,54.1℃ is a six-fold PCR Optimal annealing temperature;(3)The above can show that 94℃ 5 min(pre-denaturation);94℃45min(denaturation),54.1℃ 45 s(annealing),72℃ 30 s(extension),35 cycles;72℃ 15min(extended)is the optimal reaction conditions for BVDV,BPIV3,BRSV,BCo V,MB and BHV1 six-fold PCR.(3)The results of the sensitivity test showed that the established six-fold PCR of BVDV,BPIV3,BRSV,BCo V,MB and BHV1 had a minimum detection level of 44 pg for the nucleic acid template of the six pathogenic recombinant plasmids.The specific minimum detection levels for each pathogen were respectively BVDV 44 pg,BPIV3 32 pg,BRSV 43 pg,BCo V 76 pg,MB 75 pg and BHV1 125 pg.(4)The results of specificity and reproducibility test showed: BVDV,BPIV3,BRSV,BCo V,MB and BHV1 six-fold PCR can simultaneously amplify 139 bp,239 bp,304 bp,456 bp,527 bp and 798 bp.PBRSV,E.coli and normal tissues failed to be amplified,indicating that the amplification result was negative.And with BVDV,BPIV3,BRSV,BCo V,MB and BHV1 six-fold PCR in different time periods using the same procedure to test,the 5 results all amplified corresponding bands.The above test shows that: a successful re-PCR rapid detection method for detecting six major pathogens of bovine respiratory syndrome at the same time,the six-fold PCR established in the test can be used for rapid detection and diagnosis of clinical nasal swab samples and subsequent epidemiological investigations Prevention and control of and cattle.3.Preliminary Clinical Application of Multiplex PCR Diagnosis Method for Pathogen of Bovine Respiratory Disease ComplexThe results of testing 186 nasal swab samples by applying the established multiplex PCR diagnosis method for the pathogen of bovine respiratory disease syndrome showed that there were mixed infections of different degrees in different regions of Guizhou;there was no such phenomenon in 186 bovine nasal swab samples Five-fold infection and six-fold infection were detected;the mixed infection rate of 186 nasal swab samples from six cattle farms was9.14%(17/186),of which the positive rate of BVDV+MB double infection was 2.68%(5/186)The positive rate of BVDV+BHV1 double infection was 1.61%(3/186),the positive rate of MB+BHV1 double infection was 1.08%(2/186),and the positive rate of MB+BCo V double infection was 1.61%(3/186),the positive rate of BRSV+BPIV3 double infection was 1.08%(2/186),the positive rate of BVDV+MB+BHV1 triple infection was 0.54 %(1/186),and the triple infection of BVDV+MB+BCo V was positive The rate was 0.54 %(1/186),and the positive rate of quadruple infection of BVDV+MB+BRSV+BPIV3 was 0.54 %(1/186).By comparing with the results of the single PCR detection method constructed in the experiment,it is found that the similarity rate of the detection result of the six-fold PCR detection method and the detection result of the single PCR detection method.By comparing the test results of clinical nasal swab samples,it is determined that a six-fold PCR detection method for bovine respiratory disease syndromes BVDV,BPIV3,BRSV,BCo V,MB and BHV1 has been successfully established in this test and can complete the above six tests.Simultaneous detection of various pathogens can also detect and diagnose clinical samples of nasal swabs with single or mixed infections.