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目的建立嗜肺军团菌微滴式数字PCR(droplet digital PCR,ddPCR)检测方法,并与实时荧光定量PCR(realtime fluorescence quantitative PCR,qPCR)技术进行比较。方法针对嗜肺军团菌mip基因设计引物和探针,验证其特异性后,用于ddPCR和qPCR。优化ddPCR的退火温度、引物、探针浓度后,比较ddPCR和qPCR灵敏度和重复性,并对模拟样品进行检测。结果用5株非嗜肺军团菌测试特异性,结果表明具有良好的特异性。ddPCR与qPCR对DNA模板的检测限一致(42.6 fg),模拟样品检出限为300 cfu/ml,ddPCR的线性相关系数(0.999)略优于qPCR(0.982),ddPCR检测的RSD(0.78%~14.06%)均高于qPCR(0.30%~1.98%)。结论微滴式数字PCR方法灵敏度高、特异性强,可用于嗜肺军团菌检测,且可以对基因进行绝对定量。
Objective To establish a droplet digital PCR (ddPCR) assay for Legionella pneumophila and compare it with real-time fluorescence quantitative PCR (qPCR). Methods Primers and probes of Legionella pneumophila mip gene were designed and validated for their specificity and then used for ddPCR and qPCR. Optimize ddPCR annealing temperature, primer, probe concentration, compare ddPCR and qPCR sensitivity and repeatability, and the analog samples were tested. Results Five non-Legionella pneumophila strains were tested for specificity and the results showed good specificity. The detection limit of ddPCR and qPCR was the same as that of DNA template (42.6 fg), the detection limit was 300 cfu / ml, the linearity coefficient of ddPCR (0.999) was slightly better than that of qPCR (0.982), the RSD of ddPCR 14.06%) were higher than qPCR (0.30% ~ 1.98%). Conclusion The microdroplet digital PCR method has high sensitivity and specificity and can be used for the detection of Legionella pneumophila and can quantify the gene absolutely.