副猪嗜血杆菌微滴数字PCR定量检测方法的建立

Development of a Droplet Digital PCR for the Detection of Haemophilus parasuis

根据副猪嗜血杆菌(Haemophilus parasuis,HPS)OMP P2基因的保守序列设计特异性引物和Taq Man探针,通过反应条件优化和特异性、敏感性、重复性试验以及临床样品的检测,建立可对其准确定量的微滴数字PCR(droplet digital PCR,ddPCR)检测方法,并与实时荧光定量PCR(Quantitative real-time PCR,qPCR)检测方法进行比较分析。结果表明,该方法与猪传染性胸膜肺炎放线杆菌、猪链球菌、枯草芽孢杆菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌以及猪圆环病毒2型、猪瘟病毒无交叉反应;其灵敏性优于qPCR,线性相关系数(R2)为0.996,呈良好线性关系,最低可检测到2.647 copies/μL的阳性质粒,变异系数为3.29%。临床样品定量检测结果表明,ddPCR具有敏感性高、特异性好等优点,其检出率稍高于qPCR。本研究建立的ddPCR能够准确定量检测HPS,将为HPS相关研究提供有益参考。

Specific primers and one Taq Man probes were designed according to the conserved sequence of the OMP P2 gene of Haemophilus parasuis(HPS) in this study. An accurate quantitative droplet digital PCR(ddPCR) method was developed through the optimization of reaction conditions, specificity, sensitivity and reproducibility tests, analysis of clinical samples, and comparison with the real-time quantitative PCR(qPCR). Under the optimized reaction conditions, the results showed that ddPCR exhibited no cross-reactions with Actinobacillus pleuropneumoniae, Streptococcus suis, Bacillus subtilis, Escherichia coli, Salmonella, Staphylococcus aureu, Porcine circovirus 2, and swine fever virus. The developed method had a higher sensitivity than qPCR with a correlation coefficient(R2) of 0.996, a good linearity, detection limit of positive plasmid as 2.647 copies/μL, and coefficient of variation as 3.29%. The quantitative analysis results of clinical samples showed that ddPCR possessed advantages of high sensitivity and good specificity with a detection rate slightly higher than that for qPCR. The ddPCR developed in this study can accurately and quantitatively detect HPS, which will provide a useful reference for HPS related research.