嗜肺巴斯德菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

摘要

Objective To develop a TaqMan MGB probe real-time fluorescence quantitative PCR assay for rapid detection of Pasteurella pneumotropica. Methods Primers and probes specific to Pasteurella pneumotropica 16S rRNA gene were designed. A TaqMan MGB probe real-time fluorescence quantitative PCR was established and its specificity,sensitivity and stability were assessed. Pasteurella pneumotropica were detected in 1 680 specimens during 2008 ~ 2011 by using TaqMan MGB probe real-time fluorescence quantitative PCR. Results The specificity of TaqMan MGB probe real-time fluorescence quantitative PCR was high and did not crossreact with Pasteurella multocida, Pasteurella aerogenes, Bordetella bronchisep-tica,Klebsiella pneumoniae, Escherichia coli and Pseudomonasaeruginosa. It could detect as few as 22 copies of Pasteurella pneumotropica per well. The standard curve showed every concentration range had good linear relationship,correlation coefficient was 0. 999,and the slope was -3. 488,PCR rate of 100%. To detect Pasteurella pneumotropica in 1 680 specimens, a total of 137 specimens showed positive by TaqMan MGB probe real-time fluorescence quantitative PCR. The TaqMan MGB probe real-time fluorescence quantitative PCR assay method described here could be used for specific detecting Pasteurella pneumotropica from specimens directly. Conclusions TaqMan MGB probe real-time fluorescence quantitative PCR have peculiar characteristics of specific,sensitive and stability,suitable for faster detection of Pasteurella pneumotropica.%目的 建立嗜肺巴斯德菌的TaqMan MGB探针实时荧光定量PCR快速检测方法.方法 针对嗜肺巴斯德菌16SrRNA基因设计特异性引物和探针,建立嗜肺巴斯德菌TaqMan MGB探针实时荧光定量PCR检测方法,并验证该方法的特异度、敏感度和稳定性.对2008～2011年采集的1 680份样本进行检测.结果 嗜肺巴斯德菌TaqMan MGB探针实时荧光定量PCR检测方法具有高度特异性,对多杀巴斯德菌、产气巴斯德菌、支气管鲍特杆菌、肺炎克雷伯杆菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测灵敏度达22拷贝.标准曲线显示备浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.488,PCR效率为100％.荧光定量PCR检测1 680份样本,检出137份嗜肺巴斯德菌阳性.该方法可直接从样本中特异性地检出嗜肺巴斯德菌.结论 TaqMan MGB探针实时荧光定量PCR方法具有灵敏、特异、稳定的特性,适用于嗜肺巴斯德菌的快速检测.