Simbu 血清群正布尼亚病毒属的群特异性 RT-qPCR 检测方法的建立和分析验证。
Development and analytical validation of a group-specific RT-qPCR assay for the detection of the Simbu serogroup orthobunyaviruses.
机构信息
Faculty of Veterinary Medicine, University of Lisbon, Av. da Universidade Técnica, 1300-477, Lisbon, Portugal.
Vector and Vector-borne Diseases Programme, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa.
出版信息
J Virol Methods. 2019 Sep;271:113685. doi: 10.1016/j.jviromet.2019.113685. Epub 2019 Jun 17.
The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses, with a cosmopolitan distribution. This group of arthropod-borne viruses includes important pathogens of humans and domestic animals e.g. Oropouche orthobunyavirus and Schmallenberg virus. Sensitive and specific diagnostic tools are required for recognition and control of outbreaks. A novel TaqMan® RT-qPCR assay was developed, optimised and analytically validated for the broad detection of the Simbu serogroup orthobunyaviruses. A region in the S segment, which encodes the nucleocapsid protein, was used to design a group primer set and a pair of differently labelled TaqMan® minor groove binder probes to distinguish phylogenetic clade A and B of the serogroup. Efficiencies determined for seven members of the group were 99% for Akabane orthobunyavirus (AKAV), 96% for Simbu orthobunyavirus (SIMV), 96% for Shuni orthobunyavirus (SHUV), 97% for Sathuperi orthobunyavirus (SATV), 84% for Shamonda orthobunyavirus (SHAV), 93% for Ingwavuma virus (INGV, now classified as Manzanilla orthobunyavirus) and 110% for Sabo virus (SABOV, now classified as AKAV). The 95% limit of detection (TCID50/reaction) was 10-3.61 for AKAV, 10-2.38 for SIMV, 10-3.42 for SHUV, 10-3.32 for SATV, 10-1.67 for SHAV, 100.39 for INGV and 10-2.70 for SABOV.
辛布血清群属于正布尼亚病毒科，包含 32 种已确认的具有三段负链单链 RNA 的病毒，其分布具有世界性。这组节肢动物传播的病毒包括人类和家畜的重要病原体，例如奥罗普切正布尼亚病毒和沙姆林贝格病毒。需要敏感和特异的诊断工具来识别和控制暴发。开发了一种新的 TaqMan® RT-qPCR 检测方法，用于广泛检测辛布血清群正布尼亚病毒。使用 S 片段中编码核衣壳蛋白的区域来设计一组引物和一对不同标记的 TaqMan®小沟结合探针，以区分血清群的进化枝 A 和 B。对该组的 7 个成员进行的效率测定结果为：阿卡班正布尼亚病毒（AKAV）为 99%，辛布正布尼亚病毒（SIMV）为 96%，舒尼正布尼亚病毒（SHUV）为 96%，萨图佩里正布尼亚病毒（SATV）为 97%，沙蒙德正布尼亚病毒（SHAV）为 84%，因格瓦武马病毒（INGV，现归类为曼萨尼拉正布尼亚病毒）为 93%，萨博病毒（SABOV，现归类为 AKAV）为 110%。95%的检测限（TCID50/反应）为：AKAV 为 10-3.61，SIMV 为 10-2.38，SHUV 为 10-3.42，SATV 为 10-3.32，SHAV 为 10-1.67，INGV 为 100.39，SABOV 为 10-2.70。
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