Mengting Wu, Mengmeng Chen, Rulong Qiu, Lei Ge, Zhiyu Fan, Bo Hu, Houjun Wei, Yiming Li, Fang Wang, Yanhua Song
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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.</p>\n </div>","PeriodicalId":234,"journal":{"name":"Transboundary and Emerging Diseases","volume":"2024 1","pages":""},"PeriodicalIF":3.5000,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1155/tbed/3881457","citationCount":"0","resultStr":"{\"title\":\"Specific Detection of RHDV GI.1 and GI.2 by RT-LAMP-CRISPR/Cas12a Platform\",\"authors\":\"Mengting Wu, Mengmeng Chen, Rulong Qiu, Lei Ge, Zhiyu Fan, Bo Hu, Houjun Wei, Yiming Li, Fang Wang, Yanhua Song\",\"doi\":\"10.1155/tbed/3881457\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n <p>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.</p>\\n </div>\",\"PeriodicalId\":234,\"journal\":{\"name\":\"Transboundary and Emerging Diseases\",\"volume\":\"2024 1\",\"pages\":\"\"},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-11-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1155/tbed/3881457\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Transboundary and Emerging Diseases\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1155/tbed/3881457\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"INFECTIOUS DISEASES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Transboundary and Emerging Diseases","FirstCategoryId":"97","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1155/tbed/3881457","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"INFECTIOUS DISEASES","Score":null,"Total":0}
Specific Detection of RHDV GI.1 and GI.2 by RT-LAMP-CRISPR/Cas12a Platform
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.
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.
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