Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi
Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi
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DOI：
10.1128/jcm.42.7.3164-3168.2004
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发表时间：
2004-07-01
影响因子：
9.4
通讯作者：
Massung, RF
中科院分区：
文献类型：
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作者：
Courtney, JW;Kostelnik, LM;Massung, RF
建立了同时检测嗜吞噬细胞无形体和伯氏疏螺旋体的多重荧光定量PCR方法。对各种无形体属、疏螺旋体属、埃里希体属和立克次体属物种以及汉赛巴尔通体和大肠杆菌进行了检测，发现该检测对A.嗜吞噬细胞菌和所测试的疏螺旋体属物种（B. burgdorferi，B. parkeri，B. andersonii和B. bissettii）。该测定的分析灵敏度与先前描述的巢式PCR测定的分析灵敏度相当（A.嗜吞噬细胞菌，16 S rRNA; B. burgdorferi，弗拉基因），扩增相当于八分之一A.嗜吞噬细胞菌感染的细胞和50个疏螺旋体螺旋体。A.嗜吞噬细胞菌和B. burgdorferi大于或等于4个对数级。从A.嗜吞噬细胞菌和B.将伯氏螺旋体掺入从未感染的蜱和阴性对照小鼠和人血液提取的DNA中，这些背景DNA显示对测定的灵敏度或特异性没有显著影响。在现场收集的肩突硬蜱蜱上测试了该测定法，与先前描述的基于非探针的PCR测定法相比，该测定法具有100%的一致性。据我们所知，这是第一个报告的实时多重PCR检测，可用于同时和快速筛选样品的A。嗜吞噬细胞菌和疏螺旋体属，这是美国最常见的两种蜱传播的传染性病原体。
A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi,fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was greater than or equal to4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.