Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Insel Riems, Greifswald, Germany.
Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, Germany.
Biomed Res Int. 2017;2017:4248756. doi: 10.1155/2017/4248756. Epub 2017 May 24.
The genus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most species are available, there has been also a demand for a broad-range assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known species. This assay has been used as a screening and confirmation tool for presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented.
该属包括节肢动物传播的病毒，这些病毒可导致人类和重要经济动物的大量感染。虽然已有针对大多数 种的特定检测的 RT-PCR 方案，但也需要一种涵盖该属所有成员的广谱检测方法。在属水平上保持特异性与对每个目标种具有同等敏感性之间的平衡特别具有挑战性。在本研究中，开发了一种结合基于 SYBR Green 的 RT-qPCR 和低密度 DNA 微阵列的新型检测方法。验证实验证实，该 RT-qPCR 对所有测试的黄病毒的检测和定量均具有大致相等的灵敏度。将这些 PCR 产物进行杂交，在携带代表所有已知 种的 84 个不同寡核苷酸探针的微阵列上进行杂交。该方法已用作实验室和现场样本中 存在的筛选和确认工具，并在国际 NAT 研究的外部质量评估中成功进行。用该检测方法对 26 株进行了测试，与原始方法甚至与种特异性 RT-PCR 相比，其具有同等或更高的特征。例如，展示了对来自希腊的 340 个蚊子样本池的西尼罗河病毒检测的测试结果。
