Specific Detection of RHDV GI.1 and GI.2 by RT-LAMP-CRISPR/Cas12a Platform

IF 3.5 2区农林科学 Q2 INFECTIOUS DISEASES Transboundary and Emerging Diseases Pub Date : 2024-11-19 DOI:10.1155/tbed/3881457

Mengting Wu, Mengmeng Chen, Rulong Qiu, Lei Ge, Zhiyu Fan, Bo Hu, Houjun Wei, Yiming Li, Fang Wang, Yanhua Song

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Abstract

Rabbit hemorrhagic disease is a highly contagious and acute fatal disease caused by rabbit hemorrhagic disease virus (RHDV). The first outbreak of RHDV2 in 2020 has posed a serious threat to the rabbit breeding industry in China. An effective and specific detection strategy for RHDV GI.1 (RHDV1) and GI.2 (RHDV2) is urgently needed. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-CRISPR/Cas12a-based dual readout portable detection platform. The platform showed excellent specificity to identify RHDV1 and RHDV2 strains and no cross-reaction with other prevalent pathogens of rabbit. The detection limit for RHDV1 and RHDV2 by RT-LAMP-CRISPR/Cas12a could reach 10 copies/μl of the VP60 gene per reaction. Furthermore, 74 clinical samples were detected for both RHDV1 and RHDV2. RT-LAMP-CRISPR/Cas12a-based dual readout portable detection platform showed 25.68% (19/74) RHDV1-positive samples, 43.24% (32/74) RHDV2-positive samples, and 8.11% (6/74) RHDV1/RHDV2 double positive samples, respectively. The coincidence rates of detection RHDV1 and RHDV2 between RT-LAMP-CRISPR/Cas12a and quantitative real-time-polymerase chain reaction (qPCR) were both 97.30%. RT-LAMP-CRISPR/Cas12a showed higher sensitivity and detection rate compared with qPCR. Moreover, the results were visible to the naked eye within 1.5 h combined with lateral flow strips (LFSs) and visual fluorescence. The RT-LAMP-CRISPR/Cas12a portable platform has the advantages of high sensitivity, specificity, fast, low equipment requirements, which can be used in clinical practice in rural areas and resource-limited settings.

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利用 RT-LAMP-CRISPR/Cas12a 平台特异性检测 RHDV GI.1 和 GI.2

兔出血性疾病是由兔出血性疾病病毒（RHDV）引起的一种高度传染性的急性致死性疾病。2020 年首次暴发的 RHDV2 对我国养兔业构成了严重威胁。目前亟需一种有效、特异的RHDV GI.1（RHDV1）和GI.2（RHDV2）检测策略。本研究建立了基于反转录环介导等温扩增（RT-LAMP）-CRISPR/Cas12a双读出便携式检测平台。该平台在鉴定 RHDV1 和 RHDV2 株系方面表现出极好的特异性，并且与兔的其他流行病原体没有交叉反应。RT-LAMP-CRISPR/Cas12a对RHDV1和RHDV2的检测限可达到每次反应10个VP60基因拷贝/μl。此外，74 份临床样本都检测到了 RHDV1 和 RHDV2。基于RT-LAMP-CRISPR/Cas12a的双读出便携式检测平台显示，RHDV1阳性样本占25.68%（19/74），RHDV2阳性样本占43.24%（32/74），RHDV1/RHDV2双阳性样本占8.11%（6/74）。RT-LAMP-CRISPR/Cas12a和实时聚合酶链反应（qPCR）检测RHDV1和RHDV2的重合率均为97.30%。与 qPCR 相比，RT-LAMP-CRISPR/Cas12a 的灵敏度和检出率更高。此外，结合横向流动条（LFS）和可视荧光，肉眼可在 1.5 小时内看到检测结果。RT-LAMP-CRISPR/Cas12a便携式平台具有灵敏度高、特异性强、快速、设备要求低等优点，可用于农村地区和资源有限环境下的临床实践。

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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.