TaqMan MGB探针实时荧光定量-聚合酶链反应检测人粒细胞无形体Msp2基因方法的建立  

Establishment of real-time PCR assay to detect Anaplasma phagocytophilum Msp2 gene with TaqMan MGB probe

作　　者：付秀萍[1] 王景泉[1] 贺金荣[1] 张景山[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《疾病监测》2012年第2期141-144,共4页Disease Surveillance

基　　金：国家基础研究项目-973计划(No.2010CB530200)~~

摘　　要：目的建立敏感、特异、实时荧光定量-聚合酶链反应(real-time fluorescence quantitative,FQ-PCR)方法,用于人粒细胞无形体病的检测。方法根据无形体特异外膜蛋白Msp2基因为靶基因设计引物以及TaqMan MGB探针,建立FQ-PCR方法,并对湖北省随州和河北省张家口地区的蜱标本进行了检测。结果本研究建立的TaqMan MGB探针具有良好的特异性,建立的FQ-PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.99),灵敏性评估发现每个20μl PCR反应管中只要有35个拷贝的目的基因即可被检测到,即最低检出浓度为2拷贝/μl,并且具有较好的重复性。共检测蜱标本426只,其中豪猪血蜱253只,共49组,阳性7份。结论本研究建立的FQ-PCR方法具有很高的特异性和敏感性,可用于人粒细胞无形体感染的快速检测。进一步证实了豪猪血蜱可能是粒细胞无形体的媒介宿主。Objective To establish a highly specific and sensitive real-time PCR assay to detect Anaplasma phagocytophilum. Methods A pair of primers and a Taq Man MGB probe were designed according to the Msp2 outer membrane gene sequence. By using real-time PCR assay, Anaplasma phagocytophilum was detected in ticks samples collected from Hubei and Hebei provinces. Results A linear relationship between threshold cycle（Ct） of the quantitative real-time PCR and the DNA copy number could be demonstrated（ r = 0.99）. The standard curve showed that 35 copies target genes per reaction can be detected by this assay. The lowest detection limit of this assay was 2 CFU per txl. The assay showed high species specificity and good reproducibility. A total of 426 tick samples were detected by this assay, including 253 haemaphysalis hystricis in 49 groups, 7 samples were positive. Conclusion The results suggested that the real-time PCR assay with TaqMan MGB is highly specific and sensitive for the detection of Anaplasrna phagocytophilurn and may be used in the diagnosis of this infection.

关 键 词：人粒细胞无形体 REAL-TIME PCR Taq-Man MGB探针 豪猪血蜱 

分 类 号：R440[医药卫生—诊断学]