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
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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。Abstract: 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.
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