犊牛腹泻主要病原菌四重实时荧光定量PCR检测方法的建立OA

To enable rapid diagnosis and epidemiological prevention and control of bacterial diarrhea in calves,this study selected conserved sequences of specific target genes from four major pathogens—Escherichia coli,Salmonella,Clostridium perfringens,and Mycobacterium paratuberculosis—as detection targets.Four pairs of specific primers and minor groove binder(MGB)-TaqMan probes were designed for each pathogen,and recombinant positive plasmids were constructed.Singleplex real-time fluorescence quantitative polymerase chain reaction(qPCR)assays capable of detecting each of the four pathogens individually were established,and the reaction conditions were optimized.Based on the optimized singleplex qPCR assays,the optimal reaction conditions for a quadruplex qPCR assay were determined.Standard curves were generated using recombinant positive plasmids.The sensitivity,specificity,and repeatability of the method were further evaluated.Clinical samples were tested to assess its feasibility.The results showed that recombinant positive plasmids for all four pathogens were successfully constructed.The optimal annealing temperature and time for the singleplex qPCR assays were 55.0℃and 60 s,respectively.The optimal primer concentrations for the singleplex qPCR assays targeting E.coli,Salmonella,C.perfringens,and M.paratuberculosis were 200,300,200,and 200 nmol/L,respectively,while the optimal probe concentrations were 300,300,200,and 200 nmol/L.For the quadruplex qPCR assay,the optimal primer concentrations were 200,100,100,and 200 nmol/L,and the optimal probe concentrations were 300,100,100,and 200 nmol/L,respectively.The detection limits of the quadruplex qPCR assay for recombinant positive plasmids of E.coli,Salmonella,C.perfringens,and M.paratuberculosis were 100,100,101,and 100 copies/μL,respectively,indicating high sensitivity.The assay specifically detected only the target pathogens and showed no cross-reactivity with other pathogens,demonstrating good specificity.The intra-assay and inter-assay coefficients of variation were both less than 10%,indicating good repeatability.Clinical sample testing showed that the detection rates of E.coli and M.paratuberculosis using the established quadruplex qPCR assay were higher than those obtained using PCR.These results indicate that the quadruplex qPCR assay developed in this study,which simultaneously detects four bacterial pathogens,exhibits high sensitivity,specificity,and repeatability.It can be applied to the differential diagnosis of E.coli,Salmonella,C.perfringens,and M.paratuberculosis,providing robust technical support for the early diagnosis and prevention of calf diarrhea.