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Article(id=1241036335667016152, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241036327177744706, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202503292, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1742140800000, receivedDateStr=2025-03-17, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773815719763, onlineDateStr=2026-03-18, pubDate=1757433600000, pubDateStr=2025-09-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773815719763, onlineIssueDateStr=2026-03-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773815719763, creator=13701087609, updateTime=1773815719763, updator=13701087609, issue=Issue{id=1241036327177744706, tenantId=1146029695717560320, journalId=1227665162245664772, year='2025', volume='52', issue='17', pageStart='3073', pageEnd='3264', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773815717738, creator=13701087609, updateTime=1773840080282, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241138511152206262, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241036327177744706, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241138511152206263, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241036327177744706, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=3227, endPage=3234, ext={EN=ArticleExt(id=1241036336082252276, articleId=1241036335667016152, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Establishment of a rapid detection method for arthropod-borne viruses based on RT-RAA and CRISPR/Cas13a, columnId=1228016572065837304, journalTitle=Modern Preventive Medicine, columnName=Experimental Technology and Applications, runingTitle=null, highlight=null, articleAbstract=
Objective

To combine reverse transcription recombinase-aided amplification (RT-RAA) technology with the CRISPR/Cas13a detection system to establish a rapid, sensitive, and specific detection method for arthropod-borne viruses (RT-RAA-CRISPR/Cas13a).

Methods

We designed and synthesized RT-RAA isothermal amplification primers and specific crRNAs for four arthropod-borne viruses, namely, Zika (ZIKV), dengue (DENV), Japanese encephalitis (JEV), and chikungunya (CHIKV), and screened for the optimal combinations of amplification primers and crRNAs to establish the detection system.Viral nucleic acids were detected by fluorescence and lateral flow assay to determine the sensitivity and specificity. A TCEP/EDTA-based heat lysis protocol was optimized to enable virus detection without nucleic acid extraction.

Results

Highly efficient amplification primers and crRNAs for ZIKV, DENV, JEV and CHIKV were screened, and an RT-RAA-CRISPR/Cas13a fluorescence and lateral flow assay was established for detection of arthropod-borne viruses. The method can complete the detection in 40 min, and the lowest detection limit was 102 copies/μl (ZIKV, DENV and CHIKV) to 101 copies/μl (JEV), and the sensitivity was comparable to that of RT-qPCR. There was no cross-reactivity among the four pathogens, and the specificity was high. Meanwhile, this method combined with nucleic acid extraction-free method can directly detect virus particles in liquid samples, which has the potential for field site application.

Conclusion

The CRISPR/Cas13a-assisted RT-RAA method established in this study demonstrates notable advantages, including simple operation, rapid reaction, high specificity, superior sensitivity, and low cost, and does not rely on specialized nucleic acid detection equipment, but only requires a thermostatic heating instrument to complete the detection. The method is suitable for the immediate detection of arthropod-borne viruses, offering a novel technical platform for arbovirus identification.

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目的

将逆转录酶重组酶辅助扩增（reverse transcriptase recombinase-aided amplification，RT-RAA）技术与CRISPR/Cas13a检测系统相结合，建立一种快速、灵敏、特异的虫媒病毒检测方法（RT-RAA-CRISPR/Cas13a）。

方法

针对寨卡病毒、登革病毒、乙型脑炎病毒和基孔肯雅病毒四种虫媒病毒基因序列设计合成RT-RAA等温扩增引物及特异性crRNA并筛选最佳扩增引物和crRNA组合建立检测体系；通过荧光法和试纸条法检测虫媒病毒核酸，确定检测方法的灵敏度及特异性；优化一种基于TCEP/EDTA的加热裂解法，用于免核酸提取的病毒检测。

结果

筛选出针对寨卡病毒、登革病毒、乙型脑炎病毒和基孔肯雅病毒的高效扩增引物和crRNA，建立了用于虫媒病毒检测的RT-RAA-CRISPR/Cas13a荧光及试纸条检测方法。该方法可在40 min内完成检测，最低检测下限为102 copies/μl（寨卡病毒、登革病毒和基孔肯雅病毒）至101 copies/μl（乙型脑炎病毒），灵敏度与RT-qPCR相当。四种病原体之间无交叉反应，特异性强。同时该方法结合核酸免提取方法可直接检测液体样本中病毒颗粒，具有野外现场应用潜力。

结论

本研究建立的CRISPR/Cas13a辅助RT-RAA方法具有操作简便、反应快速、特异性强、灵敏度高以及低成本等显著优势，且不依赖专业的核酸检测设备，只需一个恒温加热仪器即可完成检测。该方法适用于虫媒病毒的即时检测，为虫媒病毒的检测提供了新的技术手段。

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吴家红，E-mail:，
姜玉庭，E-mail:。
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吴家红与姜玉庭为共同通信作者

熊丽（1998—），女，硕士在读，研究方向：媒介生物及虫媒病防控

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authorNames=Power GM, Vaughan AM, Qiao LX, journalName=BMJ Glob Health, refType=null, unstructuredReference=Power GM, Vaughan AM, Qiao LX, et al. Socioeconomic risk markers of arthropod-borne virus (arbovirus) infections: a systematic literature review and meta-analysis[J]. BMJ Glob Health, 2022, 7(4): e007735., articleTitle=Socioeconomic risk markers of arthropod-borne virus (arbovirus) infections: a systematic literature review and meta-analysis, refAbstract=null), Reference(id=1241139387958874416, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2016, volume=72, issue=1, pageStart=80, pageEnd=90, url=null, language=null, rfNumber=[2], rfOrder=1, authorNames=Sun JF, Wu D, Zhou HQ, journalName=Journal of Infection, refType=null, unstructuredReference=Sun JF, Wu D, Zhou HQ, et al. The epidemiological characteristics and genetic diversity of dengue virus during the third largest historical outbreak of dengue in Guangdong, China, in 2014[J]. Journal of Infection, 2016, 72(1): 80-90., articleTitle=The epidemiological characteristics and genetic diversity of dengue virus during the third largest historical outbreak of dengue in Guangdong, China, in 2014, refAbstract=null), Reference(id=1241139388025983284, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2021, volume=107, issue=6, pageStart=15, pageEnd=17, url=null, language=null, rfNumber=[3], rfOrder=2, authorNames=Zhang J, Shu Y, Shan XY, journalName=International Journal of Infectious Diseases, refType=null, unstructuredReference=Zhang J, Shu Y, Shan XY, et al. Co-circulation of three dengue virus serotypes led to a severe dengue outbreak in Xishuangbanna, a border area of China, Myanmar, and Laos, in 2019[J]. International Journal of Infectious Diseases, 2021, 107(6): 15-17., articleTitle=Co-circulation of three dengue virus serotypes led to a severe dengue outbreak in Xishuangbanna, a border area of China, Myanmar, and Laos, in 2019, refAbstract=null), Reference(id=1241139388097286456, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2021, volume=27, issue=3, pageStart=254, pageEnd=257, url=null, language=null, rfNumber=[4], rfOrder=3, authorNames=胡学锋, 李勃, 韩辉, journalName=中华卫生杀虫药械, refType=null, unstructuredReference=胡学锋，李勃，韩辉，等.黄热病和寨卡病毒病输入性病例流行病学特征比较[J].中华卫生杀虫药械，2021，27（3）：254-257., articleTitle=黄热病和寨卡病毒病输入性病例流行病学特征比较, refAbstract=null), Reference(id=1241139388202144062, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2021, volume=27, issue=3, pageStart=254, pageEnd=257, url=null, language=null, rfNumber=[4], rfOrder=4, authorNames=Hu XF, Li B, Han H, journalName=Chinese Journal of Hygienic Insecticides & Equipments, refType=null, unstructuredReference=Hu XF, Li B, Han H, et al. Comparison of epidemiological features of imported cases between yellow fever and Zika virus disease[J]. Chinese Journal of Hygienic Insecticides & Equipments, 2021, 27(3): 254-257.(In Chinese), articleTitle=Comparison of epidemiological features of imported cases between yellow fever and Zika virus disease, refAbstract=null), Reference(id=1241139388319584578, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2021, volume=22, issue=1, pageStart=66, pageEnd=69, url=null, language=null, rfNumber=[5], rfOrder=5, authorNames=刘克娜, 马杨岭, 王钦, journalName=中国预防医学杂志, refType=null, unstructuredReference=刘克娜，马杨岭，王钦，等.2010-2019年云南省流行性乙型脑炎流行特征分析[J].中国预防医学杂志，2021，22（1）：66-69., articleTitle=2010-2019年云南省流行性乙型脑炎流行特征分析, refAbstract=null), Reference(id=1241139388416053574, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2021, volume=22, issue=1, pageStart=66, pageEnd=69, url=null, language=null, rfNumber=[5], rfOrder=6, authorNames=Liu KN, Ma YL, Wang Q, journalName=China Preventive Medicine, refType=null, unstructuredReference=Liu KN, Ma YL, Wang Q, et al. Epidemiological characteristics of Epidemic encephalitis B in Yunnan in 2010-2019[J]. China Preventive Medicine, 2021, 22(1): 66-69.(In Chinese), articleTitle=Epidemiological characteristics of Epidemic encephalitis B in Yunnan in 2010-2019, refAbstract=null), Reference(id=1241139388508328266, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2020, volume=26, issue=3, pageStart=280, pageEnd=282, url=null, language=null, rfNumber=[6], rfOrder=7, authorNames=蒋凤, 梁禄祥, 芮莉萍, journalName=中国疫苗和免疫, refType=null, unstructuredReference=蒋凤，梁禄祥，芮莉萍，等.2007-2019年贵州省流行性乙型脑炎流行病学特征[J].中国疫苗和免疫，2020，26（3）：280-282., articleTitle=2007-2019年贵州省流行性乙型脑炎流行病学特征, refAbstract=null), Reference(id=1241139388588020045, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2020, volume=26, issue=3, pageStart=280, pageEnd=282, url=null, language=null, rfNumber=[6], rfOrder=8, authorNames=Jiang F, Liang LX, Rui LP, journalName=Chinese Journal of Vaccines and Immunization, refType=null, unstructuredReference=Jiang F, Liang LX, Rui LP, et al. Epidemiological characteristics of Japanese encephalitis in Guizhou province, 2007-2019[J]. Chinese Journal of Vaccines and Immunization, 2020, 26(3): 280-282.(In Chinese), articleTitle=Epidemiological characteristics of Japanese encephalitis in Guizhou province, 2007-2019, refAbstract=null), Reference(id=1241139388680294739, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2024, volume=47, issue=1, pageStart=79, pageEnd=83, url=null, language=null, rfNumber=[7], rfOrder=9, authorNames=金安松, 钟莲梅, journalName=中国国境卫生检疫杂志, refType=null, unstructuredReference=金安松， 钟莲梅.2016-2022年云南省流行性乙型脑炎分子流行病学、实验室及影像学检查[J].中国国境卫生检疫杂志，2024，47（1）：79-83., articleTitle=2016-2022年云南省流行性乙型脑炎分子流行病学、实验室及影像学检查, refAbstract=null), Reference(id=1241139388751597908, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2024, volume=47, issue=1, pageStart=79, pageEnd=83, url=null, language=null, rfNumber=[7], rfOrder=10, authorNames=Jin AS, Zhong LM, journalName=Chinese Journal of Frontier Health and Quarantine, refType=null, unstructuredReference=Jin AS, Zhong LM. Molecular epidemiology, laboratory and imaging of Japanese encephalitis in Yunnan Province from 2016 to 2022[J]. Chinese Journal of Frontier Health and Quarantine, 2024, 47(1): 79-83.(In Chinese), articleTitle=Molecular epidemiology, laboratory and imaging of Japanese encephalitis in Yunnan Province from 2016 to 2022, refAbstract=null), Reference(id=1241139388818706774, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2020, volume=47, issue=8, pageStart=1345, pageEnd=1348, url=null, language=null, rfNumber=[8], rfOrder=11, authorNames=刘丽珺, 漆琪, 刘宇, journalName=现代预防医学, refType=null, unstructuredReference=刘丽珺，漆琪，刘宇，等.2015-2018年四川省542例流行性乙型脑炎病例流行病学特征分析[J].现代预防医学，2020，47（8）：1345-1348., articleTitle=2015-2018年四川省542例流行性乙型脑炎病例流行病学特征分析, refAbstract=null), Reference(id=1241139388961313113, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2020, volume=47, issue=8, pageStart=1345, pageEnd=1348, url=null, language=null, rfNumber=[8], rfOrder=12, authorNames=Liu LJ, Qi Q, Liu Y, journalName=Modern Preventive Medicine, refType=null, unstructuredReference=Liu LJ, Qi Q, Liu Y, et al. Characteristics of 542 Japanese encephalitis cases in Sichuan province from 2015 to 2018[J]. Modern Preventive Medicine, 2020, 47(8): 1345-1348.(In Chinese), articleTitle=Characteristics of 542 Japanese encephalitis cases in Sichuan province from 2015 to 2018, refAbstract=null), Reference(id=1241139389049393499, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2011, volume=null, issue=null, pageStart=100, pageEnd=103, url=null, language=null, rfNumber=[9], rfOrder=13, authorNames=袁达康, 黄勇, 陈平华, 中华医学会医学病毒学分会, journalName=null, refType=null, unstructuredReference=袁达康， 黄勇， 陈平华.我国首起基孔肯雅热社区暴发疫情的流行病学调查分析[C]//中华医学会医学病毒学分会.新发和再发传染病防治热点研讨会论文集.珠海，2011，东莞：东莞市疾病预防控制中心，2011：100-103., articleTitle=我国首起基孔肯雅热社区暴发疫情的流行病学调查分析, refAbstract=null), Reference(id=1241139390462873949, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2011, volume=null, issue=null, pageStart=100, pageEnd=103, url=null, language=null, rfNumber=[9], rfOrder=14, authorNames=Yuan DK, Huang Y, Chen PH, Medical Virology Branch of Chinese Medical Association, journalName=null, refType=null, unstructuredReference=Yuan DK, Huang Y, Chen PH. Epidemiological investigation and analysis of the first community outbreak of chikungunya fever in our country[C]//Medical Virology Branch of Chinese Medical Association. Proceedings of the Symposium on the Prevention and Treatment of Emerging and Re-emerging Infectious Diseases. Zhuhai, 2011, Dongguan: Dongguan Center for Disease Control and Prevention, 2011:100-103.(In Chinese), articleTitle=Epidemiological investigation and analysis of the first community outbreak of chikungunya fever in our country, refAbstract=null), Reference(id=1241139390525788510, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2018, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[10], rfOrder=15, authorNames=曹国平, 钟建跃, 郑灿杰, 浙江省医学会热带病与寄生虫病分会, journalName=null, refType=null, unstructuredReference=曹国平，钟建跃，郑灿杰，等.中国第二起基孔肯雅热暴发疫情调查结果分析[C]//浙江省医学会热带病与寄生虫病分会.2018年浙江省医学会热带病与寄生虫病学术年会论文汇编.绍兴，2018，衢州：衢州市疾病预防控制中心，2018., articleTitle=中国第二起基孔肯雅热暴发疫情调查结果分析, refAbstract=null), Reference(id=1241139390609674593, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2018, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[10], rfOrder=16, authorNames=Cao GP, Zhong JY, Zheng CJ, Tropical Diseases and Parasitic Diseases Branch of Zhejiang Medical Association, journalName=null, refType=null, unstructuredReference=Cao GP, Zhong JY, Zheng CJ, et al. Analysis of the results of the investigation of the second chikungunya outbreak in China[C]//Tropical Diseases and Parasitic Diseases Branch of Zhejiang Medical Association.Proceedings of the 2018 Annual Meeting of Tropical Diseases and Parasitic Diseases of Zhejiang Medical Association. Shaoxing, 2018, Quzhou:Quzhou Center for Disease Control and Prevention, 2018.(In Chinese), articleTitle=Analysis of the results of the investigation of the second chikungunya outbreak in China, refAbstract=null), Reference(id=1241139390697754980, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2023, volume=46, issue=2, pageStart=113, pageEnd=118, url=null, language=null, rfNumber=[11], rfOrder=17, authorNames=陈禹希, 余方友, journalName=中华检验医学杂志, refType=null, unstructuredReference=陈禹希， 余方友.即时检测与虫媒传染病的实验诊断：应用与挑战[J].中华检验医学杂志，2023，46（2）：113-118., articleTitle=即时检测与虫媒传染病的实验诊断：应用与挑战, refAbstract=null), Reference(id=1241139390769058149, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2023, volume=46, issue=2, pageStart=113, pageEnd=118, url=null, language=null, rfNumber=[11], rfOrder=18, authorNames=Chen YX, Yu FY, journalName=Chinese Journal of Laboratory Medicine, refType=null, unstructuredReference=Chen YX, Yu FY. Point-of-care testing technology and experimental diagnosis of insect-borne diseases: applications and challenges[J]. Chinese Journal of Laboratory Medicine, 2023, 46(2): 113-118.(In Chinese), articleTitle=Point-of-care testing technology and experimental diagnosis of insect-borne diseases: applications and challenges, refAbstract=null), Reference(id=1241139390827778407, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2021, volume=20, issue=5, pageStart=593, pageEnd=605, url=null, language=null, rfNumber=[12], rfOrder=19, authorNames=Kevadiya BD, Machhi J, Herskovitz J, journalName=Nature Materials, refType=null, unstructuredReference=Kevadiya BD, Machhi J, Herskovitz J, et al. Diagnostics for SARS-CoV-2 infections[J]. Nature Materials, 2021, 20(5): 593-605., articleTitle=Diagnostics for SARS-CoV-2 infections, refAbstract=null), Reference(id=1241139390903275882, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2020, volume=14, issue=4, pageStart=3822, pageEnd=3835, url=null, language=null, rfNumber=[13], rfOrder=20, authorNames=Udugama B, Kadhiresan P, Kozlowski HN, journalName=ACS Nano, refType=null, unstructuredReference=Udugama B, Kadhiresan P, Kozlowski HN, et al. Diagnosing COVID-19: the disease and tools for detection[J]. ACS Nano, 2020,14(4): 3822-3835., articleTitle=Diagnosing COVID-19: the disease and tools for detection, refAbstract=null), Reference(id=1241139390970384749, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2022, volume=12, issue=9, pageStart=677, pageEnd=null, url=null, language=null, rfNumber=[14], rfOrder=21, authorNames=Boonbanjong P, Treerattrakoon K, Waiwinya W, journalName=Biosensors-Basel, refType=null, unstructuredReference=Boonbanjong P, Treerattrakoon K, Waiwinya W, et al.Isothermal amplification technology for disease diagnosis[J].Biosensors-Basel, 2022, 12(9): 677., articleTitle=Isothermal amplification technology for disease diagnosis, refAbstract=null), Reference(id=1241139391020716399, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2022, volume=40, issue=11, pageStart=1326, pageEnd=1345, url=null, language=null, rfNumber=[15], rfOrder=22, authorNames=Li LX, Shen GW, Wu M, journalName=Trends in Biotechnology, refType=null, unstructuredReference=Li LX, Shen GW, Wu M, et al. CRISPR-Cas-mediated diagnostics[J]. Trends in Biotechnology, 2022, 40(11): 1326-1345., articleTitle=CRISPR-Cas-mediated diagnostics, refAbstract=null), Reference(id=1241139391092019570, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2022, volume=203, issue=7, pageStart=431, pageEnd=446, url=null, language=null, rfNumber=[16], rfOrder=23, authorNames=Kostyusheva A, Brezgin S, Babin Y, journalName=Methods, refType=null, unstructuredReference=Kostyusheva A, Brezgin S, Babin Y, et al. CRISPR-Cas systems for diagnosing infectious diseases[J]. Methods, 2022, 203(7): 431-446., articleTitle=CRISPR-Cas systems for diagnosing infectious diseases, refAbstract=null), Reference(id=1241139391146545525, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2017, volume=60, issue=12, pageStart=1364, pageEnd=1371, url=null, language=null, rfNumber=[17], rfOrder=24, authorNames=Fu SH, Song S, Liu H, journalName=Science China.Life Sciences, refType=null, unstructuredReference=Fu SH, Song S, Liu H, et al. ZIKA virus isolated from mosquitoes: a field and laboratory investigation in China, 2016[J]. Science China.Life Sciences, 2017, 60(12): 1364-1371., articleTitle=ZIKA virus isolated from mosquitoes: a field and laboratory investigation in China, 2016, refAbstract=null), Reference(id=1241139391209460088, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2024, volume=31, issue=3, pageStart=153, pageEnd=162, url=null, language=null, rfNumber=[18], rfOrder=25, authorNames=丁康慧, 黄俊, 范娇, journalName=寄生虫与医学昆虫学报, refType=null, unstructuredReference=丁康慧，黄俊，范娇，等.基于CRISPR/Cas13a系统的发热伴血小板减少综合征病毒快速检测方法的建立[J].寄生虫与医学昆虫学报，2024，31（3）：153-162., articleTitle=基于CRISPR/Cas13a系统的发热伴血小板减少综合征病毒快速检测方法的建立, refAbstract=null), Reference(id=1241139391284957562, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2024, volume=31, issue=3, pageStart=153, pageEnd=162, url=null, language=null, rfNumber=[18], rfOrder=26, authorNames=Ding KH, Huang J, Fan J, journalName=Acta Parasitology et Medica Entomologica Sinica, refType=null, unstructuredReference=Ding KH, Huang J, Fan J, et al. A rapid CRISPR/Cas13a system-based method for detecting severe fever with thrombocytopenia syndrome virus[J].Acta Parasitology et Medica Entomologica Sinica, 2024, 31(3): 153-162.(In Chinese), articleTitle=A rapid CRISPR/Cas13a system-based method for detecting severe fever with thrombocytopenia syndrome virus, refAbstract=null), Reference(id=1241139391347872124, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2023, volume=32, issue=3, pageStart=1, pageEnd=9, url=null, language=null, rfNumber=[19], rfOrder=27, authorNames=蒋静, 林颖峥, 蔡一村, journalName=中国动物传染病学报, refType=null, unstructuredReference=蒋静，林颖峥，蔡一村，等.基于RAA-CRISPR/Cas13a的口蹄疫病毒检测方法的建立[J].中国动物传染病学报，2023，32（3）：1-9., articleTitle=基于RAA-CRISPR/Cas13a的口蹄疫病毒检测方法的建立, refAbstract=null), Reference(id=1241139391410786686, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2023, volume=32, issue=3, pageStart=1, pageEnd=9, url=null, language=null, rfNumber=[19], rfOrder=28, authorNames=Jiang J, Lin YZ, Cai YC, journalName=Chinese Journal of Animal Infectious Diseases, refType=null, unstructuredReference=Jiang J, Lin YZ, Cai YC, et al. Establishment of a RAA-CRISPR/Cas13a-based detection method for foot-and-mouth disease virus[J]. Chinese Journal of Animal Infectious Diseases, 2023, 32(3): 1-9.(In Chinese), articleTitle=Establishment of a RAA-CRISPR/Cas13a-based detection method for foot-and-mouth disease virus, refAbstract=null), Reference(id=1241139391469506944, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2022, volume=13, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[20], rfOrder=29, authorNames=Zhao LX, Qiu MY, Li XJ, journalName=Frontiers in Microbiology, refType=null, unstructuredReference=Zhao LX, Qiu MY, Li XJ, et al. CRISPR-Cas13a system: A novel tool for molecular diagnostics[J]. Frontiers in Microbiology, 2022, 13:1060947., articleTitle=CRISPR-Cas13a system: A novel tool for molecular diagnostics, refAbstract=null), Reference(id=1241139391524032898, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2022, volume=10, issue=4, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[21], rfOrder=30, authorNames=Qiu XT, Liu XP, Ma X, journalName=Microbiol Spectr, refType=null, unstructuredReference=Qiu XT, Liu XP, Ma X, et al. One-Pot isothermal LAMP-CRISPR-Based assay for klebsiella pneumoniae detection[J]. Microbiol Spectr,2022, 10(4): e0154522., articleTitle=One-Pot isothermal LAMP-CRISPR-Based assay for klebsiella pneumoniae detection, refAbstract=null), Reference(id=1241139391591141764, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2023, volume=14, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[22], rfOrder=31, authorNames=Wu Y, Zhan J, Shan Z, journalName=Frontiers in Microbiology, refType=null, unstructuredReference=Wu Y, Zhan J, Shan Z, et al. CRISPR-Cas13a-based detection method for avian influenza virus[J]. Frontiers in Microbiology, 2023, 14: 1288951., articleTitle=CRISPR-Cas13a-based detection method for avian influenza virus, refAbstract=null), Reference(id=1241139391649862022, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2023, volume=11, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[23], rfOrder=32, authorNames=Mao X, Xu M, Luo S, journalName=Front Bioeng Biotechnol, refType=null, unstructuredReference=Mao X, Xu M, Luo S, et al.Advancements in the synergy of isothermal amplification and CRISPR-cas technologies for pathogen detection[J]. Front Bioeng Biotechnol, 2023, 11: 1273988., articleTitle=Advancements in the synergy of isothermal amplification and CRISPR-cas technologies for pathogen detection, refAbstract=null), Reference(id=1241139391805051272, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2024, volume=275, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[24], rfOrder=33, authorNames=Zhang H, Wang YN, Chen CG, journalName=International Journal of Biological Macromolecules, refType=null, unstructuredReference=Zhang H, Wang YN, Chen CG, et al.A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick[J]. International Journal of Biological Macromolecules, 2024, 275(Pt 2): 133720., articleTitle=A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick, refAbstract=null), Reference(id=1241139391880548745, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, doi=null, pmid=null, pmcid=null, year=2018, volume=360, issue=6387, pageStart=444, pageEnd=448, url=null, language=null, rfNumber=[25], rfOrder=34, authorNames=Myhrvold C, Freije CA, Gootenberg JS, journalName=Science, refType=null, unstructuredReference=Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science, 2018, 360(6387):444-448., articleTitle=Field-deployable viral diagnostics using CRISPR-Cas13, refAbstract=null)], funds=[Fund(id=1241139387686244645, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, awardId=2023YFF0724601, language=CN, fundingSource=国家重点研发计划(2023YFF0724601), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241139377838019427, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, xref=1., ext=[AuthorCompanyExt(id=1241139377842213732, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, companyId=1241139377838019427, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=School of Public Health, Guizhou Medical University, Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry Of Education, Guiyang, Guizhou 561113, China), AuthorCompanyExt(id=1241139377850602341, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, companyId=1241139377838019427, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.贵州医科大学公共卫生与健康学院，环境污染与疾病监控教育部重点实验室，贵州 贵阳 561113)]), AuthorCompany(id=1241139377968042861, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, xref=2., ext=[AuthorCompanyExt(id=1241139377976431469, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, companyId=1241139377968042861, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.病原微生物生物安全全国重点实验室，北京 100071)]), AuthorCompany(id=1241139378077094772, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, xref=3., ext=[AuthorCompanyExt(id=1241139378093871990, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, companyId=1241139378077094772, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.贵州医科大学基础医学院，贵州 贵阳 561113)])], figs=[ArticleFig(id=1241139386402787555, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=EN, label=Figure 1, caption=Primer screening for arbovirus RT-RAA detection, figureFileSmall=cIBe4p4JijVNQQBj3Hx+yg==, figureFileBig=QIosvH+F+RZ7dpiaTta6fg==, tableContent=null), ArticleFig(id=1241139386524422376, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=CN, label=图1, caption=虫媒病毒RT-RAA引物筛选

注：图A为虫媒病毒不同检测引物的筛选；图B为四种虫媒病毒RT-RAA检测电泳图；NC为阴性对照。

, figureFileSmall=cIBe4p4JijVNQQBj3Hx+yg==, figureFileBig=QIosvH+F+RZ7dpiaTta6fg==, tableContent=null), ArticleFig(id=1241139386650251504, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=EN, label=Figure 2, caption=Establishment of CRISPR/Cas13a detection method and specificity test, figureFileSmall=2kkCrw86GoiH4FBIh6AMAA==, figureFileBig=/8U3WNlTPmd/DMvrKHqmSg==, tableContent=null), ArticleFig(id=1241139386729943288, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=CN, label=图2, caption=CRISPR/Cas13a检测方法的建立及特异性检验

注：图A为四种虫媒病毒的CRISPR检测荧光曲线；图B为反应孔的最终荧光亮度；图C为四种虫媒病毒的CRISPR核酸检测试纸检测结果；Z为ZIKV；D为DENV；J为JEV；C为CHIKV。

, figureFileSmall=2kkCrw86GoiH4FBIh6AMAA==, figureFileBig=/8U3WNlTPmd/DMvrKHqmSg==, tableContent=null), ArticleFig(id=1241139386830606590, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=EN, label=Figure 3, caption=Sensitivity test of RT-RAA-CRISPR/Cas13a method for ZIKV、DENV、JEV、CHIKV detection, figureFileSmall=gDxQRGvR0OFLHr/7Uq966g==, figureFileBig=m1yU393hPCp9keId5MWgOA==, tableContent=null), ArticleFig(id=1241139386931269891, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=CN, label=图3, caption=RT-RAA-CRISPR/Cas13a法检测ZIKV、DENV、JEV、CHIKV的灵敏度

注：图A、D、G和J分别为ZIKV、DENV、JEV和CHIKV的CRISPR/Cas13a荧光法灵敏度检测；图B、E、H和K分别为ZIKV、DENV、JEV和CHIKV的CRISPR/Cas13a核酸检测试纸法灵敏度检测；图C、F、I和L分别为ZIKV、DENV、JEV和CHIKV的RT-qPCR法灵敏度检测。

, figureFileSmall=gDxQRGvR0OFLHr/7Uq966g==, figureFileBig=m1yU393hPCp9keId5MWgOA==, tableContent=null), ArticleFig(id=1241139387010961673, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=EN, label=Figure 4, caption=Detection sensitivity of the viral nucleic acid extraction-free method, figureFileSmall=aYxBfjFQaNRGVCr9QLOGOA==, figureFileBig=PIeQa9PP0fl/pCxBq8bZMg==, tableContent=null), ArticleFig(id=1241139387111624972, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=CN, label=图4, caption=病毒免提核酸方法的检测灵敏度

注：图A、D、G分别为ZIKV、DENV和JEV倍比稀释后的RT-RAA法灵敏度检测；图B、E和H分别为ZIKV、DENV和JEV倍比稀释后免提核酸的CRISPR/Cas13a荧光法灵敏度检测；图C、F和I分别为ZIKV、DENV和JEV倍比稀释后提取核酸的RT-qPCR法灵敏度检测。

, figureFileSmall=aYxBfjFQaNRGVCr9QLOGOA==, figureFileBig=PIeQa9PP0fl/pCxBq8bZMg==, tableContent=null), ArticleFig(id=1241139387208093967, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=EN, label=Table 1, caption=

ZIKV, DENV, JEV, CHIKV RT-RAA primers, RT-qPCR primers and crRNA sequences

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称序列（5'-3'）
D-RT-RAA-F1GAAATTAATACGACTCACTATAGGGCAACACAACTACAGACCAGGCTACCATAC
D- RT-RAA-F2GAAATTAATACGACTCACTATAGGGCAACTACAGACCAGGCTACCATACACAAAC
D- RT-RAA-R1GTCCTCTATTTCCACAGTCCTCAGTCACC
D- RT-RAA-R2AGATCGGCAGCACCATTCTGTTATGAGTT
Z- RT-RAA-F1GAAATTAATACGACTCACTATAGGGCAAAGACAAATAACAGCTTTGTCGTGGATGG
Z-RT-RAA-F2GAAATTAATACGACTCACTATAGGGTACTTCGTCAGAGCAGCAAAGACAAATAAC
Z- RT-RAA-F3GAAATTAATACGACTCACTATAGGGGGAAATCGTACTTCGTCAGAGCAGCAAAGA
Z- RT-RAA-R1ACCTTGAGCCAGACACTAGTGTGAAATACC
Z- RT-RAA-R2AATCTTCTCTAACCTTGAGCCAGACACTAG
Z- RT-RAA-R3GATCACACTCTAATGAATAATCTTCTCTAA
J- RT-RAA-F1GAAATTAATACGACTCACTATAGGGTTTACAGTAACACCCAATGCTCCTTCGGTAG
J-RT-RAA-F1GAAATTAATACGACTCACTATAGGGAAGTTTACAGTAACACCCAATGCTCCTTCGG
J- RT-RAA-R1CCACTCCCTATGGACCAGAAATGACTTTGAC
J- RT-RAA-R2AGTTCTCTGTTTCTCCACGCTGTGCTCGAA
C- RT-RAA-F1GAAATTAATACGACTCACTATAGGGCTGGATGAAGCAAAGGAGCAACTATTACT
C-RT-RAA-F1GAAATTAATACGACTCACTATAGGGGCAACTATTACTTAAGAAACTCCAGGAGAA
C- RT-RAA-R1GTATAGTCCACAGCCTCTCTTTAGTCTCT
C- RT-RAA-R2TCTGACATTAAGTATAGTCCACAGCCTCTC
Z-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCUGUUCCAUGCUCUAUGUUUGAGUGGG
D-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCACUGUGGUUCCUUCGCAGAAAUCAAAG
J-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCGCUUCAGUGUUCAGUCCACUCCUUGGC
C-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUGCGCGACUGAUACCUGCUUCUGUUGG
Z-RT-qPCR-FAAGTTTGCATGCTCCAAGAAAAT
Z-RT-qPCR-RCAGCATTATCCGGTACTCCAGAT
Z-RT-qPCR-PACCGGGAAGAGCATCCAGCCAGA
D-RT-qPCR-FAAACCGTGCTGCCTGTAGCT
D-RT-qPCR-RTCCTCTAACCGCTAGTCCAATACG
D-RT-qPCR-PCTTCCATGGTTTGTGGCCTCCCAG
J-RT-qPCR-FGGCTCTTATCACGTTCTTCAAGTTT
J-RT-qPCR-RTGCTTTCCATCGGCCYGAAA
J-RT-qPCR-PAGCATTAGCCCCGACCAAGGCG
C-RT-qPCR-FCATGGCCAACAGAAGCAGGT
C-RT-qPCR-RACAGCCTCTCTTTAGTCTCTGGA
FAM-RNA-BHQFAM-UUUUUU- BHQ
FAM-RNA-BiotinFAM-UUUUUUUUUUUUUUUUUUUU- Biotin
), ArticleFig(id=1241139387308757268, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=CN, label=表1, caption=

ZIKV、DENV、JEV、CHIKV RT-RAA引物、RT-qPCR引物及crRNA序列

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称序列（5'-3'）
D-RT-RAA-F1GAAATTAATACGACTCACTATAGGGCAACACAACTACAGACCAGGCTACCATAC
D- RT-RAA-F2GAAATTAATACGACTCACTATAGGGCAACTACAGACCAGGCTACCATACACAAAC
D- RT-RAA-R1GTCCTCTATTTCCACAGTCCTCAGTCACC
D- RT-RAA-R2AGATCGGCAGCACCATTCTGTTATGAGTT
Z- RT-RAA-F1GAAATTAATACGACTCACTATAGGGCAAAGACAAATAACAGCTTTGTCGTGGATGG
Z-RT-RAA-F2GAAATTAATACGACTCACTATAGGGTACTTCGTCAGAGCAGCAAAGACAAATAAC
Z- RT-RAA-F3GAAATTAATACGACTCACTATAGGGGGAAATCGTACTTCGTCAGAGCAGCAAAGA
Z- RT-RAA-R1ACCTTGAGCCAGACACTAGTGTGAAATACC
Z- RT-RAA-R2AATCTTCTCTAACCTTGAGCCAGACACTAG
Z- RT-RAA-R3GATCACACTCTAATGAATAATCTTCTCTAA
J- RT-RAA-F1GAAATTAATACGACTCACTATAGGGTTTACAGTAACACCCAATGCTCCTTCGGTAG
J-RT-RAA-F1GAAATTAATACGACTCACTATAGGGAAGTTTACAGTAACACCCAATGCTCCTTCGG
J- RT-RAA-R1CCACTCCCTATGGACCAGAAATGACTTTGAC
J- RT-RAA-R2AGTTCTCTGTTTCTCCACGCTGTGCTCGAA
C- RT-RAA-F1GAAATTAATACGACTCACTATAGGGCTGGATGAAGCAAAGGAGCAACTATTACT
C-RT-RAA-F1GAAATTAATACGACTCACTATAGGGGCAACTATTACTTAAGAAACTCCAGGAGAA
C- RT-RAA-R1GTATAGTCCACAGCCTCTCTTTAGTCTCT
C- RT-RAA-R2TCTGACATTAAGTATAGTCCACAGCCTCTC
Z-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCUGUUCCAUGCUCUAUGUUUGAGUGGG
D-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCACUGUGGUUCCUUCGCAGAAAUCAAAG
J-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCGCUUCAGUGUUCAGUCCACUCCUUGGC
C-crRNAGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUGCGCGACUGAUACCUGCUUCUGUUGG
Z-RT-qPCR-FAAGTTTGCATGCTCCAAGAAAAT
Z-RT-qPCR-RCAGCATTATCCGGTACTCCAGAT
Z-RT-qPCR-PACCGGGAAGAGCATCCAGCCAGA
D-RT-qPCR-FAAACCGTGCTGCCTGTAGCT
D-RT-qPCR-RTCCTCTAACCGCTAGTCCAATACG
D-RT-qPCR-PCTTCCATGGTTTGTGGCCTCCCAG
J-RT-qPCR-FGGCTCTTATCACGTTCTTCAAGTTT
J-RT-qPCR-RTGCTTTCCATCGGCCYGAAA
J-RT-qPCR-PAGCATTAGCCCCGACCAAGGCG
C-RT-qPCR-FCATGGCCAACAGAAGCAGGT
C-RT-qPCR-RACAGCCTCTCTTTAGTCTCTGGA
FAM-RNA-BHQFAM-UUUUUU- BHQ
FAM-RNA-BiotinFAM-UUUUUUUUUUUUUUUUUUUU- Biotin
), ArticleFig(id=1241139387401031960, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=EN, label=Table 2, caption=

Detection systems using CRISPR-based fluorescence and lateral flow assay

, figureFileSmall=null, figureFileBig=null, tableContent=
成分ZIKV、CHIKVDENV、JEV
荧光体系体积（μl）试纸体系体积（μl）荧光体系体积（μl）试纸体系体积（μl）
Cas13a(1 μmol/L)2525
crRNA(1 μmol/L)2525
RNA Reporter(5/2 μmol/L)2525
RNase Inhibitor（10 U）12.512.5
ATP12.50.51.25
GTP12.50.51.25
CTP12.50.51.25
UTP12.50.51.25
TranscriptAid Enzyme Mix12.50.51.25
5×TranscriptAid Reaction Buffer2512.5
5×Cleavage Buffer2525
DNase/RNase free water255.513.75
扩增产物2525
总体积20502050
), ArticleFig(id=1241139387510083870, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036335667016152, language=CN, label=表2, caption=

CRISPR荧光法与试纸条法检测体系

, figureFileSmall=null, figureFileBig=null, tableContent=
成分ZIKV、CHIKVDENV、JEV
荧光体系体积（μl）试纸体系体积（μl）荧光体系体积（μl）试纸体系体积（μl）
Cas13a(1 μmol/L)2525
crRNA(1 μmol/L)2525
RNA Reporter(5/2 μmol/L)2525
RNase Inhibitor（10 U）12.512.5
ATP12.50.51.25
GTP12.50.51.25
CTP12.50.51.25
UTP12.50.51.25
TranscriptAid Enzyme Mix12.50.51.25
5×TranscriptAid Reaction Buffer2512.5
5×Cleavage Buffer2525
DNase/RNase free water255.513.75
扩增产物2525
总体积20502050
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基于RT-RAA和CRISPR/Cas13a的虫媒病毒快速检测方法的建立
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熊丽 1, 2 , 郭晓霞 2 , 李冬琴 1, 2 , 刘露 2 , 李明强 2 , 蒲希 2 , 张恒端 2 , 邢丹 2 , 赵彤言 2 , 吴家红 1, 3 , 姜玉庭 2
现代预防医学 | 实验技术及其应用 2025,52(17): 3227-3234
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现代预防医学 | 实验技术及其应用 2025, 52(17): 3227-3234
基于RT-RAA和CRISPR/Cas13a的虫媒病毒快速检测方法的建立
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熊丽1, 2, 郭晓霞2, 李冬琴1, 2, 刘露2, 李明强2, 蒲希2, 张恒端2, 邢丹2, 赵彤言2, 吴家红1, 3 , 姜玉庭2
作者信息
  • 1.贵州医科大学公共卫生与健康学院，环境污染与疾病监控教育部重点实验室，贵州 贵阳 561113
  • 2.病原微生物生物安全全国重点实验室，北京 100071
  • 3.贵州医科大学基础医学院，贵州 贵阳 561113

    • 熊丽（1998—），女，硕士在读，研究方向：媒介生物及虫媒病防控

通讯作者:

吴家红，E-mail:，
姜玉庭，E-mail:。
Establishment of a rapid detection method for arthropod-borne viruses based on RT-RAA and CRISPR/Cas13a
Li XIONG1, 2, Xiao-xia GUO2, Dong-qin LI1, 2, Lu LIU2, Ming-qiang LI2, Xi PU2, Heng-duan ZHANG2, Dan XING2, Tong-yan ZHAO2, Jia-hong WU1, 3 , Yu-ting JIANG2
Affiliations
  • School of Public Health, Guizhou Medical University, Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry Of Education, Guiyang, Guizhou 561113, China
出版时间: 2025-09-10 doi: 10.20043/j.cnki.MPM.202503292
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摘要
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目的

将逆转录酶重组酶辅助扩增（reverse transcriptase recombinase-aided amplification，RT-RAA）技术与CRISPR/Cas13a检测系统相结合，建立一种快速、灵敏、特异的虫媒病毒检测方法（RT-RAA-CRISPR/Cas13a）。

方法

针对寨卡病毒、登革病毒、乙型脑炎病毒和基孔肯雅病毒四种虫媒病毒基因序列设计合成RT-RAA等温扩增引物及特异性crRNA并筛选最佳扩增引物和crRNA组合建立检测体系；通过荧光法和试纸条法检测虫媒病毒核酸，确定检测方法的灵敏度及特异性；优化一种基于TCEP/EDTA的加热裂解法，用于免核酸提取的病毒检测。

结果

筛选出针对寨卡病毒、登革病毒、乙型脑炎病毒和基孔肯雅病毒的高效扩增引物和crRNA，建立了用于虫媒病毒检测的RT-RAA-CRISPR/Cas13a荧光及试纸条检测方法。该方法可在40 min内完成检测，最低检测下限为102 copies/μl（寨卡病毒、登革病毒和基孔肯雅病毒）至101 copies/μl（乙型脑炎病毒），灵敏度与RT-qPCR相当。四种病原体之间无交叉反应，特异性强。同时该方法结合核酸免提取方法可直接检测液体样本中病毒颗粒，具有野外现场应用潜力。

结论

本研究建立的CRISPR/Cas13a辅助RT-RAA方法具有操作简便、反应快速、特异性强、灵敏度高以及低成本等显著优势，且不依赖专业的核酸检测设备，只需一个恒温加热仪器即可完成检测。该方法适用于虫媒病毒的即时检测，为虫媒病毒的检测提供了新的技术手段。

虫媒病毒  /  逆转录酶重组酶辅助扩增（RT-RAA）  /  CRISPR/Cas13a  /  核酸检测
Objective

To combine reverse transcription recombinase-aided amplification (RT-RAA) technology with the CRISPR/Cas13a detection system to establish a rapid, sensitive, and specific detection method for arthropod-borne viruses (RT-RAA-CRISPR/Cas13a).

Methods

We designed and synthesized RT-RAA isothermal amplification primers and specific crRNAs for four arthropod-borne viruses, namely, Zika (ZIKV), dengue (DENV), Japanese encephalitis (JEV), and chikungunya (CHIKV), and screened for the optimal combinations of amplification primers and crRNAs to establish the detection system.Viral nucleic acids were detected by fluorescence and lateral flow assay to determine the sensitivity and specificity. A TCEP/EDTA-based heat lysis protocol was optimized to enable virus detection without nucleic acid extraction.

Results

Highly efficient amplification primers and crRNAs for ZIKV, DENV, JEV and CHIKV were screened, and an RT-RAA-CRISPR/Cas13a fluorescence and lateral flow assay was established for detection of arthropod-borne viruses. The method can complete the detection in 40 min, and the lowest detection limit was 102 copies/μl (ZIKV, DENV and CHIKV) to 101 copies/μl (JEV), and the sensitivity was comparable to that of RT-qPCR. There was no cross-reactivity among the four pathogens, and the specificity was high. Meanwhile, this method combined with nucleic acid extraction-free method can directly detect virus particles in liquid samples, which has the potential for field site application.

Conclusion

The CRISPR/Cas13a-assisted RT-RAA method established in this study demonstrates notable advantages, including simple operation, rapid reaction, high specificity, superior sensitivity, and low cost, and does not rely on specialized nucleic acid detection equipment, but only requires a thermostatic heating instrument to complete the detection. The method is suitable for the immediate detection of arthropod-borne viruses, offering a novel technical platform for arbovirus identification.

Arthropod-borne virus  /  Reverse transcription recombinase-aided amplification (RT-RAA)  /  CRISPR/Cas13a  /  Nucleic acid detection
熊丽, 郭晓霞, 李冬琴, 刘露, 李明强, 蒲希, 张恒端, 邢丹, 赵彤言, 吴家红, 姜玉庭. 基于RT-RAA和CRISPR/Cas13a的虫媒病毒快速检测方法的建立. 现代预防医学, 2025 , 52 (17) : 3227 -3234 . DOI: 10.20043/j.cnki.MPM.202503292
Li XIONG, Xiao-xia GUO, Dong-qin LI, Lu LIU, Ming-qiang LI, Xi PU, Heng-duan ZHANG, Dan XING, Tong-yan ZHAO, Jia-hong WU, Yu-ting JIANG. Establishment of a rapid detection method for arthropod-borne viruses based on RT-RAA and CRISPR/Cas13a[J]. Modern Preventive Medicine, 2025 , 52 (17) : 3227 -3234 . DOI: 10.20043/j.cnki.MPM.202503292
正文
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虫媒病毒（arbovirus）是一类通过蚊、蜱等病媒昆虫传播的病原体[1]，包括登革病毒（Dengue virus，DENV）、寨卡病毒（Zika virus，ZIKV）、乙型脑炎病毒（Japanese encephalitis virus，JEV）和基孔肯雅病毒（Chikungun virus，CHIKV）在内的虫媒病毒每年导致全球数百万人感染。我国近年来也多次发生因输入性病例引发的虫媒传染病疫情。2014年广东省因东南亚输入病例导致登革热大规模暴发，感染人数超4.5万例[2]。2019年位于中缅边境的云南省西双版纳傣族自治州爆发严重的登革热疫情，共报告病例3 900例[3]；2016年全国确诊24例从南美洲和大洋洲输入性寨卡病毒病病例，其中包括4起聚集性疫情[4]；2007—2022年，云贵川地区持续存在的乙型脑炎流行，感染患者造成不同程度病症，甚至死亡[5-8]；2010年广东东莞和2017年浙江衢州先后发生输入性基孔肯雅热并引发本土疫情，两次共确诊病例69人[9-10]。随着全球气候变化、全球贸易、森林破坏等因素的影响，媒介分布范围以及虫媒传染病流行范围在持续扩大。
蚊虫等病媒昆虫通常栖息于野外、森林等远离实验室的环境，使传统的检测方法在媒介携带病原体监测中面临诸多挑战。血清学检测和逆转录定量聚合酶链反应虽准确灵敏，但依赖实验室条件和专业人员，无法满足野外快速检测需求[11-13]。以重组酶辅助扩增（recombinase-aid amplification，RAA）为代表的等温扩增技术能够在恒温条件下快速扩增靶标核酸序列，具有快速和便携性等优势，适用于现场检测[14]。等温扩增技术灵敏度高，但特异性不足，限制其在复杂样本中的应用。成簇的规律间隔短回文重复序列及其相关蛋白（clustered regularly interspaced short palindromic repeats/CRISPR associated protein, CRISPR/Cas）系统以其高特异性和精准的靶向识别能力，有效弥补了等温扩增技术特异性不足的缺陷[15]，等温扩增技术能在短时间内将靶标核酸序列扩增至可检测水平，而CRISPR系统则通过其特异性识别功能，确保检测结果的可靠性[16]，两者结合的优势为虫媒病毒的现场快速检测提供助力，为应对突发公共卫生事件提供了重要的技术支持。
我国与东南亚、拉丁美洲、非洲等地区的贸易以及人员交流日益频繁，虫媒病病例或者携带虫媒病毒的媒介输入风险也随之升高。并且我国野外还存在携带ZIKV的蚊虫[17]，可能发生从森林循环溢出至人间传播的风险。目前已有将多酶恒温扩增结合CRISPR/Cas13a系统用于蜱传发热伴血小板减少综合征病毒（SFTSV）的检测[18]，但针对蚊媒病毒的CRISPR检测方法研究较少，因此，本研究针对DENV、ZIKV、JEV和CHIKV四种蚊媒病毒，开发一种高效、快速、便携的虫媒病毒检测方法，并应用于病人血清和野外蚊虫携带虫媒病毒检测，该方法的建立将有助于虫媒传染病快速诊断和提升野外虫媒病毒监测预警能力。
1　材料与方法
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1.1　检测方法概述
根据检测的虫媒病毒序列，设计并筛选等温扩增引物，通过等温扩增提升检测靶标核酸浓度，从而提高检测灵敏度；在引物5'端引入T7启动子，加入T7转录酶获取大量病毒单链RNA片段；针对各病毒设计特异性crRNA，利用CRISPR/Cas13a的反式切割活性切割RNA探针，根据RNA探针两端引入的基团类型，可通过荧光监测装置或试纸条读取结果；通过病毒核酸与crRNA间的交叉反应评价建立方法的特异性；通过将靶标核酸进行梯度系列稀释评价建立方法的灵敏度。
1.2　病毒核酸获取
ZIKV SZ01株、DENV-2 NGC株和JEV SA14-14-2株由病原微生物生物安全全国重点实验室保存。使用总RNA提取试剂盒（Qiagen）根据说明书进行病毒核酸提取。由于CHIKV活毒需在生物安全三级实验室操作，本研究采用体外合成方法获取病毒RNA片段。选取CHIKV中国株（GenBank编号MH400249）基因组5751～5948（198 nt）片段，在其5'端加T7启动子序列（GAAATTAATACGACTCACTATAGGG），3'端加EcoR I酶切位点（GAATTC）后，将序列提交北京擎科生物科技股份有限公司合成并克隆入pUC57载体。将重组质粒使用EcoR I限制性内切酶（Thermo Fisher Scientific）酶切线性化，进行1.5%琼脂糖凝胶电泳，微柱浓缩DNA凝胶回收试剂盒（ZOMANBIO/庄盟生物）进行回收，再使用T7体外转录试剂盒（TranscriptAid T7 High Yield Transcription Kit，Thermo Scientific）转录CHIKV RNA片段并纯化。纯化后的RNA进行2%琼脂糖凝胶电泳验证条带大小与预期相符后进行下游实验。
1.3　引物和crRNA的设计与合成
利用Primer Premier 6.0软件，针对ZIKV（GenBank编号KU866423.2）、DENV-2（GenBank编号AF038403.1）、JEV（GenBank编号JN604986.1）、CHIKV（GenBank编号MH400249.1）设计逆转录酶重组酶辅助扩增（reverse transcriptase recombinase-aided amplification，RT-RAA）与逆转录实时定量聚合酶链反应（reverse transcription quantitative real-time polymerase chain reaction, RT-qPCR）的引物，并由生工生物工程(上海)股份有限公司合成。在RT-RAA扩增引物之间按碱基反向互补的原则设计crRNA，crRNA序列由广州艾迪基因科技有限责任公司合成。见表1。
1.4　RT-RAA
使用RT-基础型核酸扩增试剂（杭州众测生物科技有限公司）依据说明书配制扩增反应体系(50 μl)，盖紧管盖后颠倒6～8次充分混合溶液，将反应体系置于金属浴中，42℃孵育20 min。反应结束后使用2%琼脂糖凝胶电泳分析扩增条带。
1.5　CRISPR荧光法与试纸条法检测
本研究综合使用Thermo Scientific公司的T7转录试剂盒和广州博徕斯生物科技有限公司的Cas13a反应体系，实现单管转录和CRISPR/Cas13a检测。参考试剂盒说明书以及其他文献报道，不断优化各成分比例后，获得适用于本研究针对的四种虫媒病毒核酸的反应体系，见表2。荧光法检测使用Light Cycler 96RFqPCR仪进行，检测条件：37℃孵育30～60 min，每30 s采集一次荧光信号，实时监测荧光强度变化。试纸条检测体系在金属浴上37℃恒温孵育20 min，孵育结束后，用移液枪将整个反应体系转移到核酸检测试纸条上，等待3～5 min即可肉眼读取结果。试纸条检测结果的判定标准：试纸条上出现T线及C线或只出现T线，均判定为阳性；出现C线且T线未出现，判定为阴性。
1.6　RT-qPCR法检测
为比较建立的CRISPR检测方法与传统RT-qPCR方法的优劣，使用一步法RT-qPCR探针法检测试剂盒（Promega）对ZIKV、DENV、JEV模板进行检测。RT-qPCR体系(20 μl)：GoTaq ® Probe qPCR Master Mix 10 μl，GoScriptTM RT Mix for 1-Step RT-qPCR 0.4 μl，上、下游引物及探针(10 μmol/L)各1 μl，模板2 μl，Nuclease-Free Water 4.6 μl。充分混合溶液并离心后，将其置于实时荧光定量PCR仪中，45℃逆转录15 min；95℃预变性2 min；40个循环（95℃变性15 s，60℃退火/延伸1 min），每个循环结束时采集荧光信号。使用两步法RT-qPCR对CHIKV模板进行检测，将8 μl RNA样本与2 μl PrimeScriptTM RT Master Mix（TaKaRa）混匀后37℃反转录反应15 min，85℃灭活5 min，合成的cDNA使用qPCR试剂盒（Qiagen）进行检测，反应体系(20 μl)：2× Quanti Nova SYBR Green PCR Master Mix 10 μL，QN ROX Reference Dye 2 μl，上、下游引物(10 μmol/L)各1 μl，模板cDNA 1 μl，Nuclease-Free Water 5 μl。充分混合溶液并离心后，将其置于实时荧光定量PCR仪中，95℃ PCR热启动2 min；40个循环（95℃变性5 s，60℃退火/延伸10 s），进行熔解曲线分析。
1.7　免提取核酸检测
本研究探索了裂解液中不同浓度三羧乙基膦(TCEP）和乙二胺四乙酸（EDTA），以及不同温度和持续时间获取病毒核酸效果后，最终获得如下反应条件：将TCEP和EDTA分别以100 mmol/L和1 mmol/L的终浓度加入病毒液中，采用两步法进行病毒裂解和核酸释放，ZIKV：50℃ 20 min，64℃ 5 min；DENV：37℃ 20 min，64℃ 5 min；JEV：50℃20 min，72℃ 5 min。
2　结果
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2.1　虫媒病毒RT-RAA引物筛选
针对DENV、ZIKV、JEV、CHIKV四种病毒分别设计上下游引物进行等温扩增，ZIKV设计上下游引物各3条，其他三种病毒设计上下游引物各2条，每种病毒的上下游引物进行两两配对组合后进行RT-RAA与琼脂糖凝胶电泳，根据条带的亮度和特异性，最终筛选出以下引物用于后续检测：DENV引物组合D-F1/R1、ZIKV引物组合Z-F2/R1、JEV引物组合J-F1/R2、CHIKV引物组合为C-F1/R1，见图1A。将筛选的四对引物重新RT-RAA扩增并电泳，条带清晰、单一，大小符合预期，见图1B。
2.2　CRISPR/Cas13a荧光检测
利用2.1筛选到的等温扩增产物，经T7转录生成病毒单链RNA（ssRNA），再与CRISPR/Cas13a、crRNA和荧光底物进行混合，在37℃条件下反应，实时监测荧光强度变化，结果显示四种病毒反应孔均有明显的荧光增强，见图2A；在反应结束时对所有反应孔进行拍照，对应检测孔可见明显绿色荧光，且不同病毒crRNA与ssRNA之间无交叉反应，特异性良好，见图2B。使用核酸检测试纸法替代荧光法重复上述检测，结果与荧光检测法一致，各病毒crRNA可检测到对应的病毒核酸，且不与其他三种病毒核酸发生交叉反应，特异性良好，见图2C。
2.3　CRISPR/Cas13a检测法灵敏度检测
将四种病毒核酸按照10倍梯度稀释，稀释后的RNA样本，使用RT-RAA法扩增后分别进行CRISPR/Cas13a荧光法和CRISPR/Cas13a核酸检测试纸法检测，再使用RT-qPCR方法检测。RAA-CRISPR/Cas13a荧光法检测阳性的判定标准为检测样品的荧光值达到或超过阴性对照最大荧光值两倍[19]。ZIKV样本的检测结果显示，反应进行15 min后，CRISPR/Cas13a荧光法最低可检测到101 copies/μl的目标核酸，CRISPR/Cas13a核酸检测试纸法的最低检测限为102 copies/μl，见图3B；RT-qPCR法最低可检出浓度为102 copies/μl的目标核酸，见图3C；对于ZIKV的CRISPR/Cas13a荧光检测法灵敏度高于RT-qPCR，CRISPR/Cas13a试纸条检测法则与RT-qPCR相当。DENV样本的检测结果显示，检测10 min时CRISPR/Cas13a荧光法、试纸法以及RT-qPCR法最低可检出浓度均为102 copies/μl，见图3D～F，其灵敏度与RT-qPCR一致。JEV检测结果如图3G～I所示，检测进行10 min时，CRISPR/Cas13a荧光法即可检测101 copies/μl浓度核酸，与CRISPR/Cas13a核酸检测试纸法及RT-qPCR法灵敏度一致。CHIKV样本的检测结果显示，反应至15 min时CRISPR/Cas13a荧光法和试纸法的最低检测下限皆为102 copies/μl，见图3J、K；RT-qPCR法可检出浓度为101 copies/μl的目标核酸，见图3L，表明对于CHIKV病毒的CRISPR/Cas13a检测的灵敏度比RT-qPCR低一个数量级，有待后续改进。
2.4　免提取核酸检测方法的建立
本研究优化了基于TCEP/EDTA的加热裂解法，可通过直接裂解媒介唾液等样本中病毒颗粒，释放病毒RNA后通过CRISPR方法检测。本研究以病毒悬液模拟液体样本进行倍比稀释，获得7个10倍梯度浓度的病毒样本，取相同体积的病毒进行免提取方法处理和试剂盒提取病毒RNA，随后使用RT-RAA进行检测。结果如图4A、D、G所示，免提取方法能成功检测到病毒核酸，但灵敏度比常规核酸提取方法低一个数量级。将免提取方法结合RT-RAA-CRISPR/Cas13a荧光法检测，结果显示，ZIKV、DENV和JEV最低可分别检测到105、104和106倍稀释的病毒，而试剂盒提取病毒RNA进行RT-qPCR检测，三种病毒最低可分别检测到107、106和107倍稀释的病毒。整体来说本研究建立的免提取核酸检测方法能检测到较高浓度的病毒颗粒，但灵敏度较低，还需后续继续改进。
3　讨论
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本研究成功构建了一种基于RT-RAA与CRISPR/Cas13a技术联用的虫媒病毒快速检测方法，可在40 min内完成ZIKV、DENV、JEV和CHIKV四种虫媒病毒的高效检测。该方法通过将T7启动子序列引入RT-RAA扩增引物，利用T7 RNA聚合酶将扩增产物转录为RNA。crRNA引导Cas13a蛋白特异性识别目标RNA并激活其切割活性，同时降解荧光标记的报告RNA释放荧光信号，从而实现目标基因检测[20]。实验数据显示，该方法对ZIKV、DENV和CHIKV的最低检测限均达到102 copies/μl，而对JEV的检测灵敏度更高，可达101 copies/μl，高于LAMP-CRISPR方法的检测灵敏度[21]，与现有的使用横向色谱（690 copies/μl）的RT-RPA联合CRISPR-Cas13a的AIV检测方法相比[22]，此方法表现出更好的灵敏度。此外，通过优化crRNA和引物设计，充分发挥CRISPR/Cas13a系统的特异性识别功能，成功实现四种病毒的特异性检测，避免交叉反应的发生。该结果验证了检测体系在实验室检测中的可靠性，但实际应用场景如唾液、血液样本等通常存在多重干扰因素，复杂样本的检测可能面临样本中非目标成分影响荧光信号以及目标物浓度差异过大时高丰度组分可能掩盖低丰度信号的挑战，仍需进一步完善复杂样本的特异性检测。最后该方法采用试纸条形式，无需复杂设备，结果可肉眼判读，进一步提高其适用性。
RT-RAA作为新型核酸扩增技术，相比传统PCR无需高温循环，可在较低温下30 min内完成高效扩增[23]。CRISPR/Cas13a系统则凭独特的RNA识别机制，能实现精准的RNA检测[24]。实验结果显示，除CHIKV外，其他三种病毒的RT-RAA-CRISPR/Cas13a检测灵敏度与RT-qPCR方法相当。CRISPR/Cas13a检测和RT-RAA反应可在恒温条件下进行，仅需使用一台便携式加热仪器即可完成检测流程，适合野外现场开展工作。此外，本研究在Myhrvold C[25]的HUDSON核酸免提法基础上进一步优化反应条件，通过加入TCEP/EDTA并设置温度梯度加热样本进行RNA酶灭活后，成功实现对ZIKV、DENV和JEV三种病毒的免核酸提取和RT-RAA扩增。该方法无需复杂的RNA提取步骤，缩短检测时间并简化过程，提高检测效率并降低了成本。但与经RNA试剂盒提取后的核酸RT-qPCR检测结果相比，其灵敏度低1～2个数量级，还需继续优化才能应用于实际检测。尽管本研究的方法在灵敏度、特异性、检测速度等方面均表优异，但实际应用中仍面临挑战。一方面，尽管免提取方法简化了检测过程，但其稳定性与实用性仍需在现场环境下通过真实样本的检测进一步验证。另一方面，在利用CRISPR-Cas13a系统检测RT-RAA扩增产物的过程中，需进行开盖操作，存在气溶胶污染的风险。
综上所述，本研究提出的RT-RAA-CRISPR/Cas13a荧光及试纸条检测方法，在四种虫媒病毒的高效、特异性检测方面表现出了良好的性能。该方法不仅具有与RT-qPCR相当的灵敏度，还在检测速度、操作简便性及免提取RNA样本的应用等方面展现出独特优势。未来的研究可以通过进一步优化免提取技术、提高检测设备的便携性等措施，推动这一方法在现场快速检测中的应用。
基金
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  • 国家重点研发计划(2023YFF0724601)
文献
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参考文献 引证文献
排序方式：
[1]
Power GM, Vaughan AM, Qiao LX, et al. Socioeconomic risk markers of arthropod-borne virus (arbovirus) infections: a systematic literature review and meta-analysis[J]. BMJ Glob Health, 2022, 7(4): e007735.
[2]
Sun JF, Wu D, Zhou HQ, et al. The epidemiological characteristics and genetic diversity of dengue virus during the third largest historical outbreak of dengue in Guangdong, China, in 2014[J]. Journal of Infection, 2016, 72(1): 80-90.
[3]
Zhang J, Shu Y, Shan XY, et al. Co-circulation of three dengue virus serotypes led to a severe dengue outbreak in Xishuangbanna, a border area of China, Myanmar, and Laos, in 2019[J]. International Journal of Infectious Diseases, 2021, 107(6): 15-17.
[4]
胡学锋，李勃，韩辉，等.黄热病和寨卡病毒病输入性病例流行病学特征比较[J].中华卫生杀虫药械，2021，27（3）：254-257.
Hu XF, Li B, Han H, et al. Comparison of epidemiological features of imported cases between yellow fever and Zika virus disease[J]. Chinese Journal of Hygienic Insecticides & Equipments, 2021, 27(3): 254-257.(In Chinese)
[5]
刘克娜，马杨岭，王钦，等.2010-2019年云南省流行性乙型脑炎流行特征分析[J].中国预防医学杂志，2021，22（1）：66-69.
Liu KN, Ma YL, Wang Q, et al. Epidemiological characteristics of Epidemic encephalitis B in Yunnan in 2010-2019[J]. China Preventive Medicine, 2021, 22(1): 66-69.(In Chinese)
[6]
蒋凤，梁禄祥，芮莉萍，等.2007-2019年贵州省流行性乙型脑炎流行病学特征[J].中国疫苗和免疫，2020，26（3）：280-282.
Jiang F, Liang LX, Rui LP, et al. Epidemiological characteristics of Japanese encephalitis in Guizhou province, 2007-2019[J]. Chinese Journal of Vaccines and Immunization, 2020, 26(3): 280-282.(In Chinese)
[7]
金安松， 钟莲梅.2016-2022年云南省流行性乙型脑炎分子流行病学、实验室及影像学检查[J].中国国境卫生检疫杂志，2024，47（1）：79-83.
Jin AS, Zhong LM. Molecular epidemiology, laboratory and imaging of Japanese encephalitis in Yunnan Province from 2016 to 2022[J]. Chinese Journal of Frontier Health and Quarantine, 2024, 47(1): 79-83.(In Chinese)
[8]
刘丽珺，漆琪，刘宇，等.2015-2018年四川省542例流行性乙型脑炎病例流行病学特征分析[J].现代预防医学，2020，47（8）：1345-1348.
Liu LJ, Qi Q, Liu Y, et al. Characteristics of 542 Japanese encephalitis cases in Sichuan province from 2015 to 2018[J]. Modern Preventive Medicine, 2020, 47(8): 1345-1348.(In Chinese)
[9]
袁达康， 黄勇， 陈平华.我国首起基孔肯雅热社区暴发疫情的流行病学调查分析[C]//中华医学会医学病毒学分会.新发和再发传染病防治热点研讨会论文集.珠海，2011，东莞：东莞市疾病预防控制中心，2011：100-103.
Yuan DK, Huang Y, Chen PH. Epidemiological investigation and analysis of the first community outbreak of chikungunya fever in our country[C]//Medical Virology Branch of Chinese Medical Association. Proceedings of the Symposium on the Prevention and Treatment of Emerging and Re-emerging Infectious Diseases. Zhuhai, 2011, Dongguan: Dongguan Center for Disease Control and Prevention, 2011:100-103.(In Chinese)
[10]
曹国平，钟建跃，郑灿杰，等.中国第二起基孔肯雅热暴发疫情调查结果分析[C]//浙江省医学会热带病与寄生虫病分会.2018年浙江省医学会热带病与寄生虫病学术年会论文汇编.绍兴，2018，衢州：衢州市疾病预防控制中心，2018.
Cao GP, Zhong JY, Zheng CJ, et al. Analysis of the results of the investigation of the second chikungunya outbreak in China[C]//Tropical Diseases and Parasitic Diseases Branch of Zhejiang Medical Association.Proceedings of the 2018 Annual Meeting of Tropical Diseases and Parasitic Diseases of Zhejiang Medical Association. Shaoxing, 2018, Quzhou:Quzhou Center for Disease Control and Prevention, 2018.(In Chinese)
[11]
陈禹希， 余方友.即时检测与虫媒传染病的实验诊断：应用与挑战[J].中华检验医学杂志，2023，46（2）：113-118.
Chen YX, Yu FY. Point-of-care testing technology and experimental diagnosis of insect-borne diseases: applications and challenges[J]. Chinese Journal of Laboratory Medicine, 2023, 46(2): 113-118.(In Chinese)
[12]
Kevadiya BD, Machhi J, Herskovitz J, et al. Diagnostics for SARS-CoV-2 infections[J]. Nature Materials, 2021, 20(5): 593-605.
[13]
Udugama B, Kadhiresan P, Kozlowski HN, et al. Diagnosing COVID-19: the disease and tools for detection[J]. ACS Nano, 2020,14(4): 3822-3835.
[14]
Boonbanjong P, Treerattrakoon K, Waiwinya W, et al.Isothermal amplification technology for disease diagnosis[J].Biosensors-Basel, 2022, 12(9): 677.
[15]
Li LX, Shen GW, Wu M, et al. CRISPR-Cas-mediated diagnostics[J]. Trends in Biotechnology, 2022, 40(11): 1326-1345.
[16]
Kostyusheva A, Brezgin S, Babin Y, et al. CRISPR-Cas systems for diagnosing infectious diseases[J]. Methods, 2022, 203(7): 431-446.
[17]
Fu SH, Song S, Liu H, et al. ZIKA virus isolated from mosquitoes: a field and laboratory investigation in China, 2016[J]. Science China.Life Sciences, 2017, 60(12): 1364-1371.
[18]
丁康慧，黄俊，范娇，等.基于CRISPR/Cas13a系统的发热伴血小板减少综合征病毒快速检测方法的建立[J].寄生虫与医学昆虫学报，2024，31（3）：153-162.
Ding KH, Huang J, Fan J, et al. A rapid CRISPR/Cas13a system-based method for detecting severe fever with thrombocytopenia syndrome virus[J].Acta Parasitology et Medica Entomologica Sinica, 2024, 31(3): 153-162.(In Chinese)
[19]
蒋静，林颖峥，蔡一村，等.基于RAA-CRISPR/Cas13a的口蹄疫病毒检测方法的建立[J].中国动物传染病学报，2023，32（3）：1-9.
Jiang J, Lin YZ, Cai YC, et al. Establishment of a RAA-CRISPR/Cas13a-based detection method for foot-and-mouth disease virus[J]. Chinese Journal of Animal Infectious Diseases, 2023, 32(3): 1-9.(In Chinese)
[20]
Zhao LX, Qiu MY, Li XJ, et al. CRISPR-Cas13a system: A novel tool for molecular diagnostics[J]. Frontiers in Microbiology, 2022, 13:1060947.
[21]
Qiu XT, Liu XP, Ma X, et al. One-Pot isothermal LAMP-CRISPR-Based assay for klebsiella pneumoniae detection[J]. Microbiol Spectr,2022, 10(4): e0154522.
[22]
Wu Y, Zhan J, Shan Z, et al. CRISPR-Cas13a-based detection method for avian influenza virus[J]. Frontiers in Microbiology, 2023, 14: 1288951.
[23]
Mao X, Xu M, Luo S, et al.Advancements in the synergy of isothermal amplification and CRISPR-cas technologies for pathogen detection[J]. Front Bioeng Biotechnol, 2023, 11: 1273988.
[24]
Zhang H, Wang YN, Chen CG, et al.A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick[J]. International Journal of Biological Macromolecules, 2024, 275(Pt 2): 133720.
[25]
Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science, 2018, 360(6387):444-448.
2025年第52卷第17期
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doi: 10.20043/j.cnki.MPM.202503292
  • 接收时间：2025-03-17
  • 首发时间：2026-03-18
  • 出版时间：2025-09-10
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  • 收稿日期：2025-03-17
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国家重点研发计划(2023YFF0724601)
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    1.贵州医科大学公共卫生与健康学院，环境污染与疾病监控教育部重点实验室，贵州 贵阳 561113
    2.病原微生物生物安全全国重点实验室，北京 100071
    3.贵州医科大学基础医学院，贵州 贵阳 561113

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2种不同金属材料的力学参数
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Number of
genus		 种数
Number of
species		 占总种数比例
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total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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