建立基于小沟结合剂探针的定量实时PCR以检测伯氏疏螺旋体狭义种，并通过ospA特异性常规PCR鉴别斯氏疏螺旋体。
Establishment of a minor groove binder-probe based quantitative real time PCR to detect Borrelia burgdorferi sensu lato and differentiation of Borrelia spielmanii by ospA-specific conventional PCR.
作者信息
Strube Christina, Montenegro Victor M, Epe Christian, Eckelt Elke, Schnieder Thomas
机构信息
Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany.
出版信息
Parasit Vectors. 2010 Aug 10;3:69. doi: 10.1186/1756-3305-3-69.
BACKGROUND
Borrelia burgdorferi sensu lato (sl), the causative agent of Lyme borreliosis, is transmitted by ticks of the genus Ixodes as vector. For identification of Borrelia infections in ticks a TaqMan minor groove binder (MGB) probe-based quantitative real time PCR (qPCR) was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an Ixodes spp. positive control to verify successful DNA isolation. Besides qPCR, an ospA-specific conventional PCR for species-specific identification of B. spielmanii was established. Afterwards 1000 I. ricinus flagged in the city of Hanover, Germany, were investigated for B. burgdorferi sl infections followed by species identification. Furthermore, I. hexagonus ticks were investigated to proof applicability of the PCRs.
RESULTS
Quantitative real time PCR (qPCR) identifying B. burgdorferi sl in ticks was able to detect 1-10 copies per reaction. B. spielmanii ospA-specific conventional PCR was also highly specific and showed no cross reactions with the other tested Borrelia species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was B. garinii, followed by B. afzelii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). 70.6% of I. ricinus were mono-infected, whereas 28.0% and 1.4% were infected with two and three Borrelia species, respectively. From 232 I. hexagonus collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with B. burgdorferi sl, which were identified as B. afzelii, B. garinii and B. spielmanii.
CONCLUSIONS
The evaluated qPCR to detect B. burgdorferi sl in Ixodes spp. is highly specific and sensitive. As a duplex qPCR including detection of Ixodes spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from the vector tick. Establishment of a B. spielmanii specific conventional PCR filled the gap in PCR identification of principal European Borrelia genospecies. Practical application showed that all European pathogenic Borrelia spp. were present in I. ricinus flagged in recreational areas of the city of Hanover and confirmed I. hexagonus as reservoir for pathogenic Borrelia spp.
背景
莱姆病疏螺旋体（Borrelia burgdorferi sensu lato，sl）是莱姆病的病原体，通过硬蜱属（Ixodes）蜱作为媒介传播。为了鉴定蜱中的疏螺旋体感染情况，建立了一种基于TaqMan小沟结合剂（MGB）探针的定量实时PCR（qPCR）方法，该方法靶向5S - 23S基因间隔区。扩展为双链qPCR后，加入了硬蜱属物种的阳性对照以验证DNA成功提取。除了qPCR外，还建立了一种用于特异性鉴定斯氏疏螺旋体（B. spielmanii）的ospA特异性常规PCR方法。之后，对德国汉诺威市标记的1000只蓖麻硬蜱（I. ricinus）进行了莱姆病疏螺旋体sl感染情况调查，随后进行物种鉴定。此外，还对六角硬蜱（I. hexagonus）进行了调查，以验证PCR方法的适用性。
结果
用于鉴定蜱中莱姆病疏螺旋体sl的定量实时PCR（qPCR）能够检测到每个反应1 - 10个拷贝。斯氏疏螺旋体ospA特异性常规PCR也具有高度特异性，与其他测试的疏螺旋体物种没有交叉反应。在1000只汉诺威蜱中，24.3%呈阳性，而暗视野显微镜检测的阳性率仅为7.4%。与蜱的发育阶段相关，幼虫的阳性率为1.7%，若虫为18.1%，成虫为34.6%。最常见的物种是伽氏疏螺旋体（B. garinii），其次是阿氏疏螺旋体（B. afzelii）、斯氏疏螺旋体、瓦氏疏螺旋体（B. valaisiana）和狭义疏螺旋体（B. burgdorferi sensu stricto，ss）。70.6%的蓖麻硬蜱为单一感染，而28.0%和1.4%分别感染了两种和三种疏螺旋体物种。从德国不同地点刺猬身上采集的232只六角硬蜱中，qPCR检测到5.7%感染了莱姆病疏螺旋体sl，鉴定为阿氏疏螺旋体、伽氏疏螺旋体和斯氏疏螺旋体。
结论
评估用于检测硬蜱属中莱姆病疏螺旋体sl的qPCR具有高度特异性和敏感性。作为一种包括检测硬蜱属物种DNA的双链qPCR，它是第一种基于DNA的技术，包含了从媒介蜱成功提取DNA的对照。建立斯氏疏螺旋体特异性常规PCR填补了欧洲主要疏螺旋体基因种PCR鉴定的空白。实际应用表明，汉诺威市休闲区标记的蓖麻硬蜱中存在所有欧洲致病性疏螺旋体物种，并证实六角硬蜱是致病性疏螺旋体物种的储存宿主。
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