猪丹毒丝菌SYBR Green Ⅰ荧光定量PCR检测方法的建立OA北大核心CSTPCD

In order to establish a rapid and sensitive detection method for E.rhusiopathiae,grol gene was connected with TA/Blunt Zero vector to construct a recombinant plasmid.Using the recombinant plasmid DNA as the template,the optimal reaction conditions were screened,and a SYBR Green Ⅰ fluores-cence quantitative PCR detection method was established.The sensitivity,specificity and repeatabil-ity of the method were performed.The results showed that this method has a good linear relationship with plasmid standards within the concentration range of 2 X 102～2 X 109 copies/μL,with a linear cor-relation coefficient of R2=0.999 5.The standard curve is y=-3.231x+41.834,no nonspecific amplifi-cation was observed.The minimum detection limit for plasmid standards is 20 copies/μL,and the sensi-tivity is 1 × 103 times higher than that of common PCR methods.The test results of Bordetella bronchisepti-ca,Streptococcus suis 3(Ss3),Streptococcus suis 2(Ss2),Escherichia coli(E.coli),Salmonella and Pas-teurella multocida(Pm),indicating that the method has good specificity.The coefficient of variation within and between groups in the repeated experiment were less than 2％,indicating that the method had good repeatability.The method established in this experiment was used to detect 20 suspected E.rhu-siopathiae clinical samples,and the positive detection rate was 60％,higher than the ordinary PCR de-tection method(50％).The method established in this experiment has high sensitivity,high specifici-ty,and good reproducibility,which is suitable for rapid clinical diagnosis of E.rhusiopathiae infec-tion.