Abstract:In order to achieve the quantitative study of Actinobacillus pleuropneumonia (APP), a pathogenic bacterium for porcine pleuropneumonia, a primer was designed according to the conserved sequence of the specific ApxIV gene of APP in this study. An accurate and quantitative droplet digital PCR (ddPCR) detection method was developed through optimizing reaction conditions, and the specificity, sensitivity, stability and applicability of this method were verified and compared with those of the quantitative real-time PCR (qPCR) detection method. The results showed that the optimal annealing temperature of the ddPCR method was 52 ℃, and the linear correlation coefficient (R2) value was 0.9957, indicating good linearity; the sensitivity was 153.8 copies/μL, which was twice that of qPCR; a the specificity was good, and there were no cross-reactions with other common swine pathogens. In the actual tests, the Kappa coefficient of ddPCR and qPCR detection of the 110 samples was 0.9529, indicating that the results obtained by the two methods were highly consistent, and the ddPCR method was also capable of analyzing quantitatively suspicious samples that could not be differentiated by the qPCR method. The ddPCR method established in this experiment has high sensitivity and specificity, thus can be used for quantitative detection of APP.
