A Single-Copy Sensitive and Field-Deployable One-Pot RT-RPA CRISPR/Cas12a Assay for the Specific Visual Detection of the Nipah Virus

IF 3.5 2区农林科学 Q2 INFECTIOUS DISEASES Transboundary and Emerging Diseases Pub Date : 2024-11-20 DOI:10.1155/2024/4118007

Kaikai Jin, Pei Huang, Boyi Li, Zengguo Cao, Zanheng Huang, Zimo Zhang, Meihui Liu, Hao Li, Lijuan Niu, Tianyi Zhang, Yuanyuan Li, Xuemeng Li, Hualei Wang, Haili Zhang

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Abstract

Nipah virus (NiV) is an emerging bat-borne zoonotic virus that can be transmitted to humans and other animals through infected bats or contaminated foods. The disease is highly lethal in humans (40%–75%) and has the potential for human-to-human transmission. Currently, there are no approved treatments or vaccines for NiV infection in humans or animals. Consequently, there is a pressing need for a highly sensitive, precise, and visually detectable assay to enable early intervention and mitigate the transmission of NiV infection. Here, we report a single-copy sensitive, field-deployable, one-pot visual reverse transcription-recombinase polymerase amplification (RT-RPA)-clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associate system (Cas)12 for the detection of NiV. The assay works by targeting the N gene of NiV, and the results are directly visible to the naked eye. The assay has demonstrated the ability to detect as few as 5.5 copies/μl of positive plasmids or 5.5 × 10¹ copies/μl of RNA transcripts when reacted at constant temperature for 40 min. It showed high specificity for NiV and had no cross-reaction with other pathogens, including rabies virus (RABV), Japanese encephalitis virus (JEV), herpes simplex virus type 1 (HSV-1), Hendra virus (HeV), and Streptococcus suis (S. suis), that can cause clinical symptoms similar to those of NiV infection. Moreover, this assay had a 100% coincidence rate with the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method recommended by the World Organization for Animal Health (WOAH) for the detection of simulated clinical samples, indicating that it has great potential as an ultrasensitive, simple, and portable novel assay for the onsite diagnosis of NiV infection.

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用于特异性肉眼检测尼帕病毒的单拷贝灵敏且可现场部署的一锅式 RT-RPA CRISPR/Cas12a 检测方法

尼帕病毒（NiV）是一种新出现的蝙蝠传播人畜共患病病毒，可通过受感染的蝙蝠或受污染的食物传播给人类和其他动物。这种疾病对人类的致死率很高（40%-75%），并有可能在人与人之间传播。目前，针对人类或动物感染 NiV 的治疗方法或疫苗尚未获得批准。因此，我们迫切需要一种高灵敏度、精确和可目测的检测方法，以实现早期干预并减少 NiV 感染的传播。在这里，我们报告了一种单拷贝灵敏、可现场部署、一次可视的反转录-重组聚合酶扩增（RT-RPA）-簇状规则间隔短回文重复（CRISPR）/CRISPR 关联系统（Cas）12 检测 NiV 的方法。该检测方法以 NiV 的 N 基因为靶标，检测结果肉眼直接可见。该检测方法在恒温反应 40 分钟后，可检测到低至 5.5 个拷贝/μl 的阳性质粒或 5.5 × 101 个拷贝/μl 的 RNA 转录本。它对 NiV 的特异性很高，与其他病原体无交叉反应，包括狂犬病毒 (RABV)、日本脑炎病毒 (JEV)、1 型单纯疱疹病毒 (HSV-1)、亨德拉病毒 (HeV) 和猪链球菌 (S.suis)，这些病原体可引起与 NiV 感染类似的临床症状。此外，这种检测方法与世界动物卫生组织（WOAH）推荐的用于检测模拟临床样本的反转录定量聚合酶链反应（RT-qPCR）方法的吻合率为 100%，表明它作为一种超灵敏、简单、便携的新型检测方法，在现场诊断 NiV 感染方面具有巨大潜力。

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来源期刊

Transboundary and Emerging Diseases 农林科学-传染病学

CiteScore

8.90

自引率

9.30%

发文量

350

审稿时长

1 months

期刊介绍： Transboundary and Emerging Diseases brings together in one place the latest research on infectious diseases considered to hold the greatest economic threat to animals and humans worldwide. The journal provides a venue for global research on their diagnosis, prevention and management, and for papers on public health, pathogenesis, epidemiology, statistical modeling, diagnostics, biosecurity issues, genomics, vaccine development and rapid communication of new outbreaks. Papers should include timely research approaches using state-of-the-art technologies. The editors encourage papers adopting a science-based approach on socio-economic and environmental factors influencing the management of the bio-security threat posed by these diseases, including risk analysis and disease spread modeling. Preference will be given to communications focusing on novel science-based approaches to controlling transboundary and emerging diseases. The following topics are generally considered out-of-scope, but decisions are made on a case-by-case basis (for example, studies on cryptic wildlife populations, and those on potential species extinctions): Pathogen discovery: a common pathogen newly recognised in a specific country, or a new pathogen or genetic sequence for which there is little context about — or insights regarding — its emergence or spread. Prevalence estimation surveys and risk factor studies based on survey (rather than longitudinal) methodology, except when such studies are unique. Surveys of knowledge, attitudes and practices are within scope. Diagnostic test development if not accompanied by robust sensitivity and specificity estimation from field studies. Studies focused only on laboratory methods in which relevance to disease emergence and spread is not obvious or can not be inferred (“pure research” type studies). Narrative literature reviews which do not generate new knowledge. Systematic and scoping reviews, and meta-analyses are within scope.