Yin Jingqi, Cui Jin, Zheng Hui, Guo Tingting, Wei Rong, Sha Zhou, Gu Shaopeng, Ni Bo
College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030031, China.
China Animal Health and Epidemiology Center, Qingdao 266011, China.
Viruses. 2025 Mar 27;17(4):483. doi: 10.3390/v17040483.
Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription-recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks.
尼帕病毒（NiV）是一种严重的人畜共患病原体，对公众健康构成重大威胁。猪是NiV的天然宿主，有可能将这种疾病传播给人类。建立一种快速、灵敏且准确的即时检测方法对于及时识别受感染猪群至关重要。在本研究中，我们开发了一种基于逆转录重组酶聚合酶扩增（RT-RAA）和规律成簇间隔短回文重复序列（CRISPR）相关蛋白13a（Cas13a）系统的NiV检测方法，用于精确检测NiV。选择NiV基因的高度保守区域作为检测靶点。我们首先设计了11对RT-RAA引物，并根据RT-RAA效率确定了最佳引物组合和反应温度。此外，根据荧光信号强度选择了最有效的crRNA序列。结果表明，所开发方法的最佳反应温度为37℃。检测限低至1.565拷贝/μL。特异性测试显示，该方法与六种常见猪病毒的核酸无交叉反应，这六种病毒包括塞内卡病毒A（SVA）、口蹄疫病毒（FMDV）、经典猪瘟病毒（CSFV）、猪流行性腹泻病毒（PEDV）、非洲猪瘟病毒（ASFV）和伪狂犬病病毒（PRV）。使用模拟临床样本进行的验证测试显示符合率为100%。检测结果可通过荧光读数仪或侧向流动试纸条（LFS）进行可视化。与传统检测方法相比，这种基于RT-RAA-CRISPR/Cas13a的方法快速、简单，不需要专业仪器。此外，试剂可冻干保存，无需冷链运输。该检测技术为NiV的即时诊断以及预防和控制疫情提供了一种方便高效的新工具。
