布鲁氏菌微滴式数字PCR方法的建立及应用OA

In order to detect Brucella timely,rapidly and accurately,primers and probe were designed targeting its virB10 gene,and a droplet digital PCR(ddPCR)assay was established,then used to detect clinical samples after evaluation on its sensitivity,specificity and repeatability,as well as generation of a standard curve.Subsequently,the antigen nucleic acid quantified by this method was used as a standard to evaluate the limit of detection(LOD),low-concentration sensitivity and specificity of three common fluorescence quantitative PCR kits.The results showed that the amplification efficiency reached the highest when the concentrations of primers and probe were 900 and 300 nmol/L,respectively,at the annealing temperature of 60℃;the LOD was 1.01 copies/μL,without specific amplification for several common pathogens,and the coefficients of variation in intra-assay and inter-assay repeatability tests ranged from 2.80%to 5.08%;and the results for clinical samples were consistent with those obtained by a common real-time fluorescence PCR assay.Three kits(A,B and C)were compared using a nucleic acid standard quantified at 6 240 copies/μL by ddPCR,it was found that the LOD of all the kits was 97.50 copies/μL,their sensitivities were 96%,92%and 80%,respectively,and specificities were 100%for all the kits,indicating the best overall performance of kit A.In conclusion,the established ddPCR assay was highly sensitive,specific and reproducible,and applicable for detecting Brucella in clinical samples and for selecting appropriate kits,thereby supporting future control and eradication of the disease as a key tool.