Establishment And Validation Of A Real-Time Fluorescent Pcr Freeze-Dried Type Laboratory Assay For 11 Sheep And Goats Disease Pathogens

In order to meet the growing market demand for sheep meat and milk,sheep and goats farming is expanding,however,problems such as the expansion of sheep disease epidemics come along with it.The precondition for strengthening the prevention and control of sheep diseases is to achieve early diagnosis of sheep diseases.Real-time fluorescent PCR assays are more efficient than other assays,shortening diagnosis time,reducing the possibility of contamination and non-specific reactions,and further improving the accuracy of results.However,fluorescent PCR assay technology also has problems such as low throughput of pathogens,insufficient sensitivity and high cost of product storage and transportation.To address these problems,this study combined 11 sheep diseases according to the sample types at the time of clinical sampling,and aimed to establish a real-time fluorescent PCR assay with high throughput,high sensitivity,no cold chain transportation,and easy-to-use freeze-dried reagent for Mycoplasma ovis(M.ovis),pathogenic Leptospira,Coxiella burnetii(C.burnetii),Bluetongue virus(BTV),Peste des pestis ruminants virus(PPRV),Brucella,exogenous jaagsiekte sheep retrovirus(ex JSRV),Chlamydia abortus(C.abortus),Orf virus(ORFV),Foot-andmouth disease(FMDV)and Capripox viruses(CPV).The 16 s rRNA of M.ovis,Lip L32 of Pathogenic Leptospira,IS1111 of C.burnetii,NS1 of BTV,M of PPRV,BCSP31 of Brucella,U3 of ex JSRV,omp A of C.abortus,B2 L of ORFV,ORF of FMDV,and RPO35 of CPV were used as target genes to design specific primers and probes.Constructing positive references based on the base sequences,in which RNA viruses were transcribed in vitro on the basis of the synthetic references.The positive reference was used as a template to screen the specific primers and probes.The 11 primers and probes were grouped according to the clinical sampling types and the primers and probe dosages were optimized.The ROC curves were evaluated the diagnostic value of the established method.The performance of the established method was evaluated in terms of specificity,limit of detection(LOD)and repetitiveness.The freeze-dried system was subjected to accelerated stability test and expiration date was predicted,and the performance of the established method was validated with actual samples.The results showed that a free-dried real-time fluorescence PCR method for the simultaneous detection of 11 sheep and goats pathogens was established,with no nonspecific amplification in systems containing different pathogen positive references,the areas under the ROC curves of the method were all in the range of 0.99 to 1.00.The LOD were 194 copies/m L for M.ovis,96 copies/m L for Pathogenic Leptospira,84copies/ml for C.burnetii,40 copies/ml for BTV,265 copies/m L for PPRV,68 copies/m L for Brucella,208 copies/m L for ex JSRV,236 copies/m L for C.abortus,118 copies/m L for ORFV,53 copies/m L for FMDV,723 copies/m L for CPV.The coefficient of variation of both intra-group and inter-group replicate tests was less than 5.00%.The accelerated thermostatic lyophilization test was used to predict the expiration date,and samples with the LOD of each pathogen were still detected after 6 days of acceleration at 55 ℃,suggesting an expiration date of at least 567 days at room temperature.140 clinical samples collected by the established method were tested,and 19 samples of M.ovis,65 samples of C.burnetii,12 samples of ex JSRV,26 samples of C.abortus were detected.The method in this work has the advantages of high throughput,high sensitivity,high stability,detection time within 2 h,room temperature storage,convenient transportation and easy storage,and can provide support for the clinical detection and epidemiological investigation of M.ovis,pathogenic Leptospira,C.burnetii,BTV,PPRV,Brucella,ex JSRV,C.abortus,ORFV,FMDV and CPV.