猪胸膜肺炎放线杆菌微滴数字PCR定量检测方法的建立

Development of Droplet Digital PCR for the Quantitative Detection of Actinobacillus pleuropneumoniae

摘要:

为实现对猪胸膜肺炎的致病菌猪胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）的量化研究，本实验根据猪胸膜肺炎放线杆菌特异性基因ApxIV的保守序列，设计引物探针，通过优化反应条件，建立可对其准确定量的微滴数字PCR（droplet digital PCR，ddPCR）检测方法，并对该方法的特异性、灵敏度、稳定性以及适用性进行验证，与实时荧光定量PCR（Quantitative real-time PCR，qPCR）检测方法进行比较分析。结果表明：ddPCR最佳退火温度为52 ℃；线性相关系数（R2）为0.9957，线性关系良好；灵敏度达153.8 copies/μL，是qPCR的两倍；特异性良好，与其它常见猪病原无交叉反应。在实际样品检测中，110份样品的ddPCR与qPCR检测结果的Kappa系数为0.9529，说明两种检测方法结果高度一致，并且ddPCR可对qPCR无法判断阴阳性的可疑样本进行定量分析。本实验建立的ddPCR方法敏感性高、特异性强、可用于猪胸膜肺炎放线杆菌的定量检测。

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.