TaqMan-探针实时荧光定量PCR同时检测立克次体目中7种重要病原菌

Simultaneous detection of 7 important Rickettsiales pathogens by TaqMan-probe quantitative real-time PCR

目的建立及优化检测立克次体目中7种重要病原菌的TaqMan-探针实时荧光定量PCR(qPCR)方法,可同步检测并明确感染类型。方法根据普氏立克次体、莫氏立克次体和斑点热群立克次体ompB基因、恙虫病东方体groEL基因、查菲埃立克体16S rRNA基因、嗜吞噬细胞无形体gltA基因和贝氏柯克斯体com1基因序列合成引物和探针,优化反应体系及反应程序至同一方案,对该方法灵敏度、特异性和重复性等进行评价,并使用该方法对模拟样本和实际样本进行检测。结果7种病原菌标准曲线的Ct值与模板拷贝数均呈良好的线性关系(均R^(2)>0.9900),所建立方法最低检测限均为1×10拷贝数/μl且具有良好的特异性。96份蜱虫核酸提取物样本中,1份样本检出贝氏柯克斯体,3份样本检出斑点热群立克次体;80份不明原因发热患者血标本DNA中,1份样本检出恙虫病东方体,2份样本检出斑点热群立克次体。结论本研究基于TaqMan-探针qPCR方法,将7种病原菌反应体系及反应程序优化至同一方案,克服目前不同立克次体目病原菌采用不同的反应体系和反应程序的缺点,可将临床样本中立克次体目的7种重要病原菌精确定位检测至种,明确感染病原菌类型并缩短检测时间,有助于对患者的精准诊治。

Objective To establish and optimize a TaqMan-probe quantitative real-time PCR(qPCR)assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types.Methods Based on the ompB gene of Rickettsia prowazekii,Rickettsia mooseri and spotted fever group rickettsiae,the groEL gene of Orientia tsutsugamushi,the 16S rRNA of Ehrlichia chaffeensis,the gltA gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii,we synthesized primers and TaqMan-probes and optimized the reaction system and reaction process to same solution.The sensitivity,specificity and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples.Results The Ct value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies(all R^(2)>0.9900),the minimum detection limit was 10 copies/μl,showing good specificity.In the 96 tick nucleic acid extracts,Coxiella burnetii was detected in 1 sampleand spotted fever group Rickettsiae was detected in 3 samples.In the 80 blood samples from patients with undefined febrile illness,Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples.Conclusions In this study,based on the established TaqMan-probe qPCR assay,the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution.This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens,which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.