Tan Fuli, Li Yuchang, Kang Xiaoping, Chen Yuehong, Zhang Sen, Li Jing, Feng Ye, Li Xiaokun, Liang Runxin, Wang Fei, Li Xiangdong, Jiang Tao
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China.
Infect Med (Beijing). 2025 Jul 19;4(3):100196. doi: 10.1016/j.imj.2025.100196. eCollection 2025 Sep.
In recent years, frequent outbreaks of infectious diseases caused by hemorrhagic fever viruses have posed serious threats to global public health. The pathogens are variable and highly infectious, such as Sudan (SEBOV), Zaire (ZEBOV), Marburg (MARV), (LASV), Rift Valley fever (RVFV), Sin Nombre (SNV), etc. To improve the efficiency of pathogen detection, a method for simultaneous screening multiplex targets is in a great demand.
Utilizing dual-probe hybridization and melting curve analysis, a multiplex nucleic acid polymerase assay for eight hemorrhagic fever viruses test (named the MPA-eight-virus assay) was developed in this study. The sensitivity for each target was improved by optimizing primer and probe selection as well as amplification conditions; the usability of MPA-eight-virus assay was validated by simulated samples preparation and test.
The MPA-eight-virus assay achieved high sensitivity and specificity for the targets, with a limit of detection (LOD) of 1.83-691.00 copies/µL for all eight targets; Notably, the LOD for MARV was 1.83 copies/µL and that for SNV was 9.32 copies/µL.
The MPA-eight-virus assay is high throughput, time-saving, accurate, and cost-effective, making it potentially useful for prevention and control of severe viral hemorrhagic fever.
近年来，由出血热病毒引起的传染病频繁爆发，对全球公共卫生构成了严重威胁。病原体种类多样且传染性极强，如苏丹埃博拉病毒（SEBOV）、扎伊尔埃博拉病毒（ZEBOV）、马尔堡病毒（MARV）、拉沙病毒（LASV）、裂谷热病毒（RVFV）、汉坦病毒（SNV）等。为提高病原体检测效率，一种同时筛查多个靶标的方法亟待开发。
本研究利用双探针杂交和熔解曲线分析技术，开发了一种用于检测8种出血热病毒的多重核酸聚合酶检测方法（命名为MPA - 八病毒检测法）。通过优化引物和探针的选择以及扩增条件，提高了每个靶标的检测灵敏度；通过模拟样本制备和检测，验证了MPA - 八病毒检测法的实用性。
MPA - 八病毒检测法对靶标具有高灵敏度和特异性，所有8个靶标的检测限（LOD）为1.83 - 691.00拷贝/微升；值得注意的是，MARV的LOD为1.83拷贝/微升，SNV的LOD为9.32拷贝/微升。
MPA - 八病毒检测法具有高通量、省时、准确且经济高效的特点，对严重病毒性出血热的防控具有潜在应用价值。
