High-Throughput Procedure for Tick Surveys of Tick-Borne Encephalitis Virus and Its Application in a National Surveillance Study in Switzerland
High-Throughput Procedure for Tick Surveys of Tick-Borne Encephalitis Virus and Its Application in a National Surveillance Study in Switzerland
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DOI：
10.1128/aem.00391-10
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发表时间：
2010-07-01
影响因子：
4.4
通讯作者：
Beuret, Christian M.
中科院分区：
文献类型：
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作者：
Gaeumann, Rahel;Muehlemann, Kathrin;Beuret, Christian M.
森林脑炎(TBE)是一种中枢神经系统的病毒感染，在许多欧亚国家流行。在瑞士，临床病例的地理地图显示了TBE风险区域的特点。由于大规模接种疫苗应能显著减少TBE病例的数量，因此需要替代暴露风险评估方法。我们建立了一种新的基于聚合酶链式反应的检测方法，用于检测硬蜱中的TBE病毒(TBEV)。该方案包括一种自动化、高通量的核酸提取方法(QIAsymphone SP系统)和一步法检测欧洲亚型TBEV的双重实时逆转录-聚合酶链式反应(RT-PCR)，包括内部过程控制。在病毒浓度低至5×10(3)病毒基因组当量/亩的情况下，高可用性、重复性和同等性能L与改进的硫氰酸胍-苯酚-氯仿提取程序相比，更倾向于自动化程序。该实时RT-PCR方法可以快速、灵敏(最低检测限为10RNA拷贝/亩L)和特异性(对其他TBEV亚型、其他黄病毒或其他壁虱传播的病原体没有假阳性检测结果)检测欧洲TBEV亚型。新的检测方法被应用于一项国家监测研究，在该研究中，对62,343只蓖麻硬蜱进行了TBE病毒的筛查。共发现38个地方性疫源地，平均病毒流行率为0.46%。这些病灶与疾病图谱所定义的并不完全一致。因此，拟议的分子测试程序是进行适当的TBE监测的先决条件。我们的数据是瑞士人类TBE疾病病例图的独特补充。
Tick-borne encephalitis (TBE), a viral infection of the central nervous system, is endemic in many Eurasian countries. In Switzerland, TBE risk areas have been characterized by geographic mapping of clinical cases. Since mass vaccination should significantly decrease the number of TBE cases, alternative methods for exposure risk assessment are required. We established a new PCR-based test for the detection of TBE virus (TBEV) in ticks. The protocol involves an automated, high-throughput nucleic acid extraction method (QIAsymphony SP system) and a one-step duplex real-time reverse transcription-PCR (RT-PCR) assay for the detection of European subtype TBEV, including an internal process control. High usability, reproducibility, and equivalent performance for virus concentrations down to 5 x 10(3) viral genome equivalents/mu l favor the automated protocol compared to the modified guanidinium thiocyanate-phenol-chloroform extraction procedure. The real-time RT-PCR allows fast, sensitive (limit of detection, 10 RNA copies/mu l), and specific (no false-positive test results for other TBEV subtypes, other flaviviruses, or other tick-transmitted pathogens) detection of European subtype TBEV. The new detection method was applied in a national surveillance study, in which 62,343 Ixodes ricinus ticks were screened for the presence of TBE virus. A total of 38 foci of endemicity could be identified, with a mean virus prevalence of 0.46%. The foci do not fully agree with those defined by disease mapping. Therefore, the proposed molecular test procedure constitutes a prerequisite for an appropriate TBE surveillance. Our data are a unique complement of human TBE disease case mapping in Switzerland.