【摘要】 目的建立一种敏感、特异的实时荧光定量PCR方法,用于检测临床标本中的恙虫病东方体。方法根据恙虫病东方体GroEL蛋白基因序列设计引物和探针,建立实时荧光定量PCR检测方法。并从灵敏度、特异度、重复性及临床标本的检测能力等方面对本方法进行综合评价。结果建立的实时荧光定量PCR方法具有良好的特异性,可检测出恙虫病东方体Gilliam、Karp、Kato和Kawasaki株。建立的标准曲线循环阈值(Ct)与模板拷贝数呈现良好的线性关系(r=0.99),灵敏度分析显示样品最低检出浓度为21.8拷贝/μL。批内及批间重复性实验变异系数均小于1.5%,显示本方法具有良好的重复性。对本实验室保存的82份临床标本进行检测,并与常规巢式PCR方法比较,本方法检测出26份阳性标本中的25份,检出率为96.15%。结论本研究建立的实时荧光定量PCR方法具有较高的敏感性、特异性和稳定性,可用于临床病人及暴发疫情的实验室快速检测。更多还原

【Abstract】 Based on the GroEL gene of Orientia tsutsugamushi,we designed a pair of primers and TaqMan probe for real-time PCR assay in order to detect O.tsutsugamushi clinical specimens.The sensitivity,specificity,reproducibility and veracity of diagnoses with clinical specimens of this method were evaluated.A linear relationship between threshold cycle(CT)of the real-time PCR and the DNA copy number was demonstrated(r=0.99).The standard curve showed that 21.8copies/reaction could be detected by this method.The coefficients of variation of intra-and inter-assay reproducibility were both less than1.5%.Compared with that of the nested PCR,the positive detection rate of real-time PCR assay was 96.15%(25/26)in 82 clinical specimens.Results showed that this real-time PCR is a highly specific,sensitive and timesaving assay for detection of O.tsutsugamushi,which can be applied for the rapid diagnosis of clinical specimens.更多还原