Establishment of RT-RPA/RAA-based detection method for four henipa viruses
10.3760/cma.j.cn112866-20250629-00134
- VernacularTitle:四种亨尼帕病毒RT-RPA/RAA检测方法的建立
- Author:
Wenjun HE
1
;
Yuanyuan GUO
;
Sheng ZHANG
;
Mengjie YANG
;
Jing ZHANG
;
Wenwen LEI
;
Juan SONG
;
Guizhen WU
Author Information
1. 中国疾病预防控制中心病毒病预防控制所，传染病溯源预警与智能决策全国重点实验室，北京 102206
- Publication Type:Journal Article
- Keywords:
Nipah virus;
Langya virus;
Mojiang virus;
Cedar virus;
Reverse transcription recombinase polymerase amplification;
Reverse transcription recombinase aided
- From:
Chinese Journal of Experimental and Clinical Virology
2025;39(4):502-509
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a rapid and accurate duplex real-time fluorescent reverse transcription-recombinase polymerase amplification/recombinase-aided amplification（RT-RPA/RAA）detection method for identification and differentiation of Nipah virus（NiV），Langya virus（LayV），Mojiang virus（MoJV），and Cedar virus（CedV）.Methods:First，specific primers and probes were designed targeting the conserved L gene regions of NiV and LayV，as well as the conserved N gene regions of MoJV and CedV，respectively. The four viruses were divided into two groups for duplex detection. Subsequently，the optimal primer and probe combinations were screened by comparing the amplification efficiency of different primer pair combinations（F1/R1，F1/R2，F2/R1，F2/R2）. The reaction temperature was optimized through temperature gradient settings from 37 ℃ to 42 ℃，and the amounts of primers and probes were optimized to establish the duplex real-time fluorescent RT-RPA/RAA detection system. Finally，the detection performance was evaluated through specificity，sensitivity，and stability tests，as well as clinical sample validation.Results:The selected primer pairs（NiV primer pair F2/R1，LayV primer pair F1/R1，MoJV primer pair F2/R2，and CedV primer pair F2/R2）all demonstrated optimal amplification efficiency when combined with their corresponding probes. The optimal annealing temperature was 39 ℃，and the minimum detection limit was 101-102 copies/μl. The method could effectively distinguish target viruses from other non-target viruses，and repeated experiments showed good stability（ R2> 0.90）. Additionally，detection results for Malaysian NiV strains and various clinical samples were consistent with the Taqman multiplex qRT-PCR method. Conclusion:The duplex real-time fluorescent RT-RPA/RAA detection method successfully established in this study can rapidly and accurately identify and differentiate four important henipavirus-like viruses：NiV，LayV，MoJV，and CedV. It features simple operation，rapid reaction，high specificity，and good stability，providing an effective molecular detection tool for rapid field diagnosis，surveillance，and control of these zoonotic viruses.
