嗜肺军团菌微滴式数字PCR检测方法的建立

Establishment of droplet digital PCR assay for the detection of Legionella pneumophila

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） method for the detection of Legionella pneumophila and comparison with real - time fluorescence quantitative PCR （ qPCR）. Methods The primers and probe were designed for ddPCR and qPCR according to mip gene after verifying its specificity. The annealing temperature, primer, probe concentration of ddPCR were optimized simultaneously. The sensitivity and repeatability of the 2 methods were compared, and the simulated samples were tested. Results The highly specificity of thisprimers and probe was evaluated using 5 non - targeted bacteria. The detection limits of ddPCR and qPCR for DNA were 42.6 fg and 300 cfu/ml for artificially samples. The ddPCR（0. 999） showed slightly higher degree of linearity than the qPCR （0. 982 ）. The relative standard deviation （RSD） of ddPCR （0. 78 % - 14.06% ） was higher than that of qPCR（0.30% - 1. 98% ）. Conclusion The ddPCR is of high sensitivity and specificity, and can be used for the detection of Legionella pneumophila. And it can perform full quantitativity to gene.