Department of Biotechnology, University of Johannesburg, PO Box 17011, Doornfontein 2028, South Africa.
J Virol Methods. 2010 Oct;169(1):34-8. doi: 10.1016/j.jviromet.2010.06.010. Epub 2010 Jun 25.
Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis distributed widely in Africa, Asia, Russia and the Balkans. Occurrence of segment reassortment has been established and may be associated with pathogenicity. The ability to distinguish between an infection with a reassortant and a non-reassortant variant may have prognostic value. In this study the use of Simple-Probe technology and real-time PCR was investigated to detect rapidly CCHF viral nucleic acid and to distinguish between reassorted and non-reassorted variants of southern African CCHF virus (CCHFV) isolates. A Simple-Probe was designed based on multiple alignments of 19 CCHFV partial M segment sequences that included reassorted and non-reassorted isolates from two phylogenies. Real-time PCR followed by probe-specific melting-curve analysis allowed differentiation of three selected isolates representative of two phylogenetically distinct groups, SPU 431/85 (group IV), 383/87 (group IV) and 103/87 (group III) based on melting temperatures. Within group IV there are two distinct lineages which were represented in the optimization. A further 16 clinical isolates could be genotyped into groups III and IV using the Simple-Probe real-time PCR assay developed in this study. This assay provides a reliable and sensitive tool for the differentiation between reassorted and non-reassorted variants of CCHFV which finds application for diagnosis and epidemiologic and surveillance studies.
克里米亚-刚果出血热（CCHF）是一种广泛分布于非洲、亚洲、俄罗斯和巴尔干地区的蜱传病毒性人畜共患病。已经确定了片段重排的发生，并且可能与致病性有关。区分重组和非重组变体感染的能力可能具有预后价值。在这项研究中，使用 Simple-Probe 技术和实时 PCR 来快速检测 CCHF 病毒核酸，并区分南非 CCHF 病毒（CCHFV）分离株的重组和非重组变体。根据包含来自两个系统发育的重组和非重组分离株的 19 个 CCHFV 部分 M 片段序列的多重比对，设计了一种 Simple-Probe。实时 PCR 后进行探针特异性熔解曲线分析，允许根据融解温度区分三个选定的代表两个系统发育上不同组的分离株，SPU 431/85（组 IV）、383/87（组 IV）和 103/87（组 III）。在组 IV 中，有两个不同的谱系在优化中得到了代表。使用本研究中开发的 Simple-Probe 实时 PCR 检测方法，可以将另外 16 个临床分离株分为 III 组和 IV 组。该检测方法为区分 CCHFV 的重组和非重组变体提供了一种可靠且敏感的工具，可用于诊断以及流行病学和监测研究。
