多重实时 PCR 检测在出血热综合征病毒中的应用。

Application of multiplex realtime PCR detection for hemorrhagic fever syndrome viruses.

机构信息

Department of Convergence Engineering, Graduate School of Venture, Hoseo University, Seoul, 06724, South Korea; MDx Center, Diagnosis Division, iNtRON Biotechnology, South Korea.

Department of Convergence Engineering, Graduate School of Venture, Hoseo University, Seoul, 06724, South Korea.

出版信息

J Infect Public Health. 2023 Dec;16(12):1933-1941. doi: 10.1016/j.jiph.2023.10.012. Epub 2023 Oct 6.

DOI:10.1016/j.jiph.2023.10.012

PMID:37866271

Abstract

BACKGROUND

Multiplex real-time PCR is a quick and cost effective method for detection of various gene simultaneously. HFSV (Hemorrhagic Fever Syndrome Virus) is a newly emerging infectious disease because of globalization and climate change. We tried to develop a molecular diagnostic technique for various causative viruses and evaluate its usefulness for improving public health.

METHODS

Molecular diagnostic test method that qualitatively detects viruses causing viral hemorrhagic fevers hired Taq-Man Real-time RT-PCR technique. The Ct value was experimentally observed three or more times at the RNA concentration before and after the detection limit. After designing a multiplex real-time RT-PCR test for target gene of selected 17 viruses, the detection limit for each target and the presence or absence of cross-reaction and interference reaction were evaluated to determine its availability.

RESULTS

Six kinds of viruses, including Crimean-Congo hemorrhagic fever virus, Omsk hemorrhagic fever virus, Sabia virus, Chapare virus, Yellow fever virus, and Variola virus (A4L gene, B12R gene), were able to confirm the detection limit of 0.5 copies/μl, and other Ebola virus, Marburg virus, Rift Valley fever virus, Kyasanur Forest disease virus, Junin virus, Guanarito virus, Machupo virus, Chikungunya virus, Hantavirus, Dengue virus types 1-4, and Lassa virus (L gene, GPC gene), and 11 kinds of viruses, the detection limit was confirmed at 5 copies/μl. No cross-reaction or interference between detected genes was observed.

CONCLUSION

The virus test method developed through this study using multiplex is expected to be used for public health and quarantine as a test method that can be used when a hemorrhagic fever virus of unknown cause is introduced.

摘要

背景

多重实时 PCR 是一种快速且具有成本效益的方法，可同时检测多种基因。HFVS（出血热综合征病毒）是一种由于全球化和气候变化而新出现的传染病。我们试图开发一种用于检测各种病原体病毒的分子诊断技术，并评估其在改善公共卫生方面的有用性。

方法

采用 Taq-Man 实时 RT-PCR 技术对导致病毒性出血热的病毒进行定性检测的分子诊断测试方法。在检测限前后，将 RNA 浓度重复实验观察 Ct 值三次或更多次。在为选定的 17 种病毒的目标基因设计多重实时 RT-PCR 检测后，评估每种靶基因的检测限以及是否存在交叉反应和干扰反应，以确定其可用性。

结果

六种病毒，包括克里米亚-刚果出血热病毒、鄂木斯克出血热病毒、沙贝病毒、查帕雷病毒、黄热病病毒和天花病毒（A4L 基因、B12R 基因），能够确认检测限为 0.5 拷贝/μl，其他埃博拉病毒、马尔堡病毒、裂谷热病毒、基孔肯雅热病毒、Junin 病毒、瓜纳里托病毒、马丘波病毒、基孔肯雅热病毒、汉坦病毒、登革热病毒 1-4 型和拉萨病毒（L 基因、GPC 基因），检测限在 5 拷贝/μl 时得到确认。未观察到检测基因之间的交叉反应或干扰。