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

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20 μl 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。

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

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 μl.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 Anaplasma phagocytophilum and may be used in the diagnosis of this infection.