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Article(id=1241036252322001352, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241036242561855785, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202410457, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1730131200000, receivedDateStr=2024-10-29, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773815699892, onlineDateStr=2026-03-18, pubDate=1756051200000, pubDateStr=2025-08-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773815699892, onlineIssueDateStr=2026-03-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773815699892, creator=13701087609, updateTime=1773815699892, updator=13701087609, issue=Issue{id=1241036242561855785, tenantId=1146029695717560320, journalId=1227665162245664772, year='2025', volume='52', issue='16', pageStart='2881', pageEnd='3072', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773815697565, creator=13701087609, updateTime=1773840190562, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241138973712634304, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241036242561855785, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241138973712634305, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241036242561855785, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=3013, endPage=3021, ext={EN=ArticleExt(id=1241036254649840127, articleId=1241036252322001352, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Establishment of a detection method for five respiratory viruses based on RPA-CRISPR/Cas12a, columnId=1228016572065837304, journalTitle=Modern Preventive Medicine, columnName=Experimental Technology and Applications, runingTitle=null, highlight=null, articleAbstract=
Objective

To promptly identify common respiratory viruses causing infections and facilitate effective therapeutic interventions in resource-limited settings such as grassroots, clinics, border areas, and border defense, this study established a visual detection method based on recombinase polymerase amplification (RPA) technology and CRISPR/Cas12a (clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a) system.

Methods

Initially, conservative sequences of each virus target were designed, and RPA primers along with single-strand guide RNAs (sgRNAs) were selected. Subsequently, the RPA technology was integrated with the CRISPR/Cas12a detection method for visual detection of influenza A virus, influenza B virus, respiratory syncytial virus, Severe Acute Respiratory Syndrome Coronavirus 2, and human rhinovirus.

Results

The detection method yielded results within 1.5 hours, with a sensitivity of 3.5 copies/μl plasmid, and exhibited no cross-reactivity between each virus target. In terms of detection accuracy, this method demonstrated higher consistency compared to the control Quantitative Reverse Transcription Polymerase Chain Reaction, (qRT-PCR) method.

Conclusion

The visual detection method established in this study possesses good specificity and high sensitivity for the common five respiratory virus infections. It is suitable for on-site detection of common respiratory virus infections, especially in resource-limited environments, and holds promising clinical application prospects.

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目的

为了在基层、诊所、边防和野外等资源匮乏环境中及早明确常见导致呼吸道感染的病毒，从而采取有效的治疗方法和应对措施，本研究建立了一种基于重组酶聚合酶扩增技术（recombinase polymerase amplification，RPA）和CRISPR/Cas12a（clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a）系统的可视化检测方法。

方法

针对每种病毒靶标的保守序列设计和筛选RPA引物及单链向导RNA（single-strand guide RNA，sgRNA），然后将RPA技术与CRISPR/Cas12a检测方法相结合，建立呼吸道病毒即时可视化检测方法，并通过检测甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、新型冠状病毒和人鼻病毒临床样品对该方法进行验证。

结果

所建立的方法可在1.5 h内获得结果，灵敏度达3.5 copies/μl，各病毒靶标之间无交叉反应。该方法在检测正确率方面与对照的定量逆转录-聚合酶链式反应（Quantitative Reverse Transcription Polymerase Chain Reaction，qRT-PCR）法比具有较高的一致性。

结论

本研究建立的可视化检测方法对常见5种呼吸道感染病毒的8种型别具有较好的特异性和高灵敏度，适用于常见呼吸道病毒感染的现场检测，尤其是资源有限的地区，具有良好的临床应用前景。

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杨春晖，E-mail：
, copyrightStatement=本刊刊出的所有文章不代表中华预防医学会和本刊编委会的观点，除非特别声明。, copyrightOwner=中华预防医学会和四川大学华西公共卫生学院, extLink=null, articleAbsUrl=null, sourceXml=tJW/3tNewXkccXpC77pBPw==, magXml=sxMbrxTYSXJeRLG4shYQgw==, pdfUrl=null, pdf=78Cjs46yD6mh3UPr3eXqhw==, pdfFileSize=3035937, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=wkruTNTsKTCU7QZfyzIZKw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=kd+Pvhyj2sAjHUVXQZEUcA==, mapNumber=null, authorCompany=null, fund=null, authors=

王清华（1976—），女，硕士，副主任中医师，研究方向：输血医学

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王清华（1976—），女，硕士，副主任中医师，研究方向：输血医学

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Journal of Medical Virology, 2023, 95(11): e29215., articleTitle=Development of a rapid, sensitive detection method for SARS-CoV-2 and influenza virus based on recombinase polymerase amplification combined with CRISPR-Cas12a assay, refAbstract=null)], funds=[Fund(id=1241057516277264672, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, awardId=2021331, language=CN, fundingSource=成都市卫健委医学科研课题(2021331), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241057510078084032, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, xref=1., ext=[AuthorCompanyExt(id=1241057510086472641, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, companyId=1241057510078084032, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Chengdu Wenjiang District Hospital of Traditional Chinese Medicine, Chengdu, Sichuan 611130, China), AuthorCompanyExt(id=1241057510111638466, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, companyId=1241057510078084032, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.成都市温江区中医医院，四川 成都 611130)]), AuthorCompany(id=1241057510212301768, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, xref=2., ext=[AuthorCompanyExt(id=1241057510220690377, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, companyId=1241057510212301768, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.中国医学科学院输血研究所)])], figs=[ArticleFig(id=1241057514456936646, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Fig.1, caption=Screening results of RPA primers for eight viral targets, figureFileSmall=9RKpSXO2oBoqLqTjDaVYHA==, figureFileBig=9HmvUoQSTZOjJfLQk1GnBw==, tableContent=null), ArticleFig(id=1241057514582765774, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=图1, caption=8种病毒靶标的RPA引物筛选结果

注：NTC: negative test control，下同。

, figureFileSmall=9RKpSXO2oBoqLqTjDaVYHA==, figureFileBig=9HmvUoQSTZOjJfLQk1GnBw==, tableContent=null), ArticleFig(id=1241057514704400595, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Fig.2, caption=Screening results of sgRNA for eight viral targets, figureFileSmall=tl2iTQepa7HCpHg9fRmb7Q==, figureFileBig=dbmVrf5+sRaP5TZ6/rCEkw==, tableContent=null), ArticleFig(id=1241057514805063896, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=图2, caption=8种病毒靶标的sgRNA筛选结果

注：A: 8种靶标的终点蓝光拍照图; B: 8种靶标的终点荧光强度图。

, figureFileSmall=tl2iTQepa7HCpHg9fRmb7Q==, figureFileBig=dbmVrf5+sRaP5TZ6/rCEkw==, tableContent=null), ArticleFig(id=1241057514930893023, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Fig.3, caption=Sensitivity evaluation of the RPA-CRISPR/Cas12a detection method for eight viral targets, figureFileSmall=LeW6FjRAc8E1Vpwc6FDghw==, figureFileBig=r53pjJR31aBm+hM4T0Xzww==, tableContent=null), ArticleFig(id=1241057515039944932, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=图3, caption=8种病毒靶标RPA-CRISPR/Cas12a检测方法的灵敏度评价, figureFileSmall=LeW6FjRAc8E1Vpwc6FDghw==, figureFileBig=r53pjJR31aBm+hM4T0Xzww==, tableContent=null), ArticleFig(id=1241057515144802541, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Fig.4, caption=Specificity evaluation of the RPA-CRISPR/Cas12a detection method for eight viral targets, figureFileSmall=gG+rNKgMacJCeGSG2CuzAw==, figureFileBig=KZTfr/5Wc0sY17fcXgr0qg==, tableContent=null), ArticleFig(id=1241057515249660142, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=图4, caption=8种病毒靶标RPA-CRISPR/Cas12a检测方法的特异度评价, figureFileSmall=gG+rNKgMacJCeGSG2CuzAw==, figureFileBig=KZTfr/5Wc0sY17fcXgr0qg==, tableContent=null), ArticleFig(id=1241057515333546227, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Fig.5, caption=Clinical application of the RPA-CRISPR/Cas12a detection method, figureFileSmall=hB4J96G4cAer9m5GZB3BNQ==, figureFileBig=+DhDdEuEEBS9N8v5mHFJOw==, tableContent=null), ArticleFig(id=1241057515421626617, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=图5, caption=RPA-CRISPR/Cas12a检测方法的临床应用, figureFileSmall=hB4J96G4cAer9m5GZB3BNQ==, figureFileBig=+DhDdEuEEBS9N8v5mHFJOw==, tableContent=null), ArticleFig(id=1241057515572621569, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Table 1, caption=

Primer sequences of RPA

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称序列（5’—3’）
H1N1-HA-1FTAGACACAGTACTAGAAAAGAATGTAACAGTA
H1N1-HA-1RGAATATCTCAAACCTTTCAAATGATGACACTG
H1N1-HA-2FCAAATCCTACATTAATGATAAAGGGAAAGAAGT
H1N1-HA-2RACTAGATTTCCAGTTGCTTCGAATGTTATTTT
H1N1-HA-3FAAAAGCTTCTACAAAAATTTAATATGGCTAGTT
H1N1-HA-3RCTCTACTAGTGTCCAGTAATAGTTCATTCTC
H3N2-HA-1FAATAACAGTTTCTTTAGTAGATTGAATTGGTTG
H3N2-HA-1RTTAATCAAAAGTATGTCTCCCGGTTTTACTATT
H3N2-HA-2FTAACAGTTTCTTTAGTAGATTGAATTGGTTGA
H3N2-HA-2RTGTTAATCAAAAGTATGTCTCCCGGTTTTACTA
H3N2-HA-3FGATATGTTAAGCAAAACACTCTGAAATTGG
H3N2-HA-3RTTCTACTTCTGAGAATTCTTTTTCAATCTGATG
FluB-HA-1FCAATAAATCCAGTAACAGTAGAAGTACCATAC
FluB-HA-1RCAGGTTTTTGTACCATGTAATCAACAACAATTC
FluB-HA-2FTAAATCCAGTAACAGTAGAAGTACCATACATT
FluB-HA-2RGGTTTTTGTACCATGTAATCAACAACAATTC
FluB-HA-3FTCTAATATCCACAAAATGAAGGCAATAATTGTA
FluB-HA-3RGTTTGACTTCATGGAGTATTGAGACTTTTG
FluB-NS1-1FAAACGAAAATTAGAATCAAGAATAAAGACTCAC
FluB-NS1-1RTTTATTGTTCATGTCCCTTAATACTACCTCAAT
FluB-NS1-2FCACAGACTAAAACGAAAATTAGAATCAAGAATA
FluB-NS1-2RATCTTTATTGTTCATGTCCCTTAATACTACCTC
FluB-NS1-3FTACACAGACTAAAACGAAAATTAGAATCAAGAA
FluB-NS1-3RCATCTTTATTGTTCATGTCCCTTAATACTACC
RSV-A-1FTTAACAACAGTTAAAGATCTCACTATGAAAACA
RSV-A-1RTAATATATACTTTCTTTTTCTAGGTAGGCTCCA
RSV-A-2FCTCATAAAAGAACTAGCCAATGTCAATATACTA
RSV-A-2RTGTTACTATATTTTCAAATTCACATAAAGCAATG
RSV-A-3FTACTCATAAAAGAACTAGCCAATGTCAATATAC
RSV-A-3RTTACTATATTTTCAAATTCACATAAAGCAATGA
RSV-B-1FATAGATTTAACTTTGTATTTAGTTCCACAGGAT
RSV-B-1RAAAGATACTAATAGGTTCTGCAAATTTTATGTG
RSV-B-2FGAACTTCATCCTGACATAAGATATATTTACAGA
RSV-B-2RTTACATGCTTACTCCATTCAATTATGATTTTAC
RSV-B-3FCTTGTACAATTTATTTCCAATTGTTGTGATAGA
RSV-B-3RCTTCTGTAAATATATCTTATGTCAGGATGAAGT
COV2-ORF1ab-1FCTCAGAGTAGAAATTTACAAGAATTTAAACCCA
COV2-ORF1ab-1RATCATCAAGTAATAAATCAATAACAGAACACAC
COV2-ORF1ab-2FAAATGGAAATTGATTTCTTAGAATTAGCTATGG
COV2-ORF1ab-2RATAAATCAATAACAGAACACACACACTTAGATG
COV2-ORF1ab-3FAGTTTCTATCATTAATAACACTGTTTACACAAA
COV2-ORF1ab-3RTTTCTAAATAAGTCTACTTGACCATCAACTCTA
RV-Poly-1FTTATTAATTATTACAAGGATGCAGCAAGTTCAT
RV-Poly-1RTTTGATAAATTTGTCCATGTTTTACTCTCTAGG
RV-Poly-2FACACTTTACTGTTATTAATTATTACAAGGATGC
RV-Poly-2RTTTTGTTTACATCACTTGCATCAGTATCTGTTA
RV-Poly-3FTTACTGTTATTAATTATTACAAGGATGCAGCAA
RV-Poly-3RGTTTACATCACTTGCATCAGTATCTGTTAAGTA
), ArticleFig(id=1241057515677479175, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=表1, caption=

RPA引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称序列（5’—3’）
H1N1-HA-1FTAGACACAGTACTAGAAAAGAATGTAACAGTA
H1N1-HA-1RGAATATCTCAAACCTTTCAAATGATGACACTG
H1N1-HA-2FCAAATCCTACATTAATGATAAAGGGAAAGAAGT
H1N1-HA-2RACTAGATTTCCAGTTGCTTCGAATGTTATTTT
H1N1-HA-3FAAAAGCTTCTACAAAAATTTAATATGGCTAGTT
H1N1-HA-3RCTCTACTAGTGTCCAGTAATAGTTCATTCTC
H3N2-HA-1FAATAACAGTTTCTTTAGTAGATTGAATTGGTTG
H3N2-HA-1RTTAATCAAAAGTATGTCTCCCGGTTTTACTATT
H3N2-HA-2FTAACAGTTTCTTTAGTAGATTGAATTGGTTGA
H3N2-HA-2RTGTTAATCAAAAGTATGTCTCCCGGTTTTACTA
H3N2-HA-3FGATATGTTAAGCAAAACACTCTGAAATTGG
H3N2-HA-3RTTCTACTTCTGAGAATTCTTTTTCAATCTGATG
FluB-HA-1FCAATAAATCCAGTAACAGTAGAAGTACCATAC
FluB-HA-1RCAGGTTTTTGTACCATGTAATCAACAACAATTC
FluB-HA-2FTAAATCCAGTAACAGTAGAAGTACCATACATT
FluB-HA-2RGGTTTTTGTACCATGTAATCAACAACAATTC
FluB-HA-3FTCTAATATCCACAAAATGAAGGCAATAATTGTA
FluB-HA-3RGTTTGACTTCATGGAGTATTGAGACTTTTG
FluB-NS1-1FAAACGAAAATTAGAATCAAGAATAAAGACTCAC
FluB-NS1-1RTTTATTGTTCATGTCCCTTAATACTACCTCAAT
FluB-NS1-2FCACAGACTAAAACGAAAATTAGAATCAAGAATA
FluB-NS1-2RATCTTTATTGTTCATGTCCCTTAATACTACCTC
FluB-NS1-3FTACACAGACTAAAACGAAAATTAGAATCAAGAA
FluB-NS1-3RCATCTTTATTGTTCATGTCCCTTAATACTACC
RSV-A-1FTTAACAACAGTTAAAGATCTCACTATGAAAACA
RSV-A-1RTAATATATACTTTCTTTTTCTAGGTAGGCTCCA
RSV-A-2FCTCATAAAAGAACTAGCCAATGTCAATATACTA
RSV-A-2RTGTTACTATATTTTCAAATTCACATAAAGCAATG
RSV-A-3FTACTCATAAAAGAACTAGCCAATGTCAATATAC
RSV-A-3RTTACTATATTTTCAAATTCACATAAAGCAATGA
RSV-B-1FATAGATTTAACTTTGTATTTAGTTCCACAGGAT
RSV-B-1RAAAGATACTAATAGGTTCTGCAAATTTTATGTG
RSV-B-2FGAACTTCATCCTGACATAAGATATATTTACAGA
RSV-B-2RTTACATGCTTACTCCATTCAATTATGATTTTAC
RSV-B-3FCTTGTACAATTTATTTCCAATTGTTGTGATAGA
RSV-B-3RCTTCTGTAAATATATCTTATGTCAGGATGAAGT
COV2-ORF1ab-1FCTCAGAGTAGAAATTTACAAGAATTTAAACCCA
COV2-ORF1ab-1RATCATCAAGTAATAAATCAATAACAGAACACAC
COV2-ORF1ab-2FAAATGGAAATTGATTTCTTAGAATTAGCTATGG
COV2-ORF1ab-2RATAAATCAATAACAGAACACACACACTTAGATG
COV2-ORF1ab-3FAGTTTCTATCATTAATAACACTGTTTACACAAA
COV2-ORF1ab-3RTTTCTAAATAAGTCTACTTGACCATCAACTCTA
RV-Poly-1FTTATTAATTATTACAAGGATGCAGCAAGTTCAT
RV-Poly-1RTTTGATAAATTTGTCCATGTTTTACTCTCTAGG
RV-Poly-2FACACTTTACTGTTATTAATTATTACAAGGATGC
RV-Poly-2RTTTTGTTTACATCACTTGCATCAGTATCTGTTA
RV-Poly-3FTTACTGTTATTAATTATTACAAGGATGCAGCAA
RV-Poly-3RGTTTACATCACTTGCATCAGTATCTGTTAAGTA
), ArticleFig(id=1241057515799113997, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Table 2, caption=

sgRNA Sequences

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称序列（5’—3’）
influenza A virus-H1N1-HA-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUUGGGGUCAUCAAGAUACAGCAAGA
influenza A virus-H1N1-HA-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCCUUUAUCAUUAAUGUAGGAUUUG
influenza A virus-H3N2-HA-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUAUGCCUGAAACCGUACCAACCGUC
influenza A virus-H3N2-HA-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCCGAUCAACCUAUUCAGCUUCCCA
influenza B virus-HA-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUGUCUUCCCCUUCUGAACAAAUGUA
influenza B virus-HA-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUGGGGUUCCACUCUGAUGACAAAAC
influenza B virus-NS1-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUACACAGUAUGGCUCAAACCCUUCA
influenza B virus-NS1-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUUGGAUCCCUCUGCUGGAAUUGAAG
RSV-A-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUACAGGGUGUGGUUACAUCAUAUAC
RSV-A-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCCAUAUGUGCCAAUGUGUCCUUGG
RSV-B-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUACCAUUCCUGCUACAGAUGCAACU
RSV-B-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUUAUGCCCGUUGUAUAACCUUAGAA
SARS-CoV-2-ORF1ab-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUCAUCUACUGAUUGGACUAGCUAAA
SARS-CoV-2- ORF1ab-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUGUCAUAGUCAGUUAGGUGGUUUAC
RhV-Poly-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUCUGAGCCUGUAAAGGAUAUCAUGU
RhV- Poly-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCAUCACUUGCAUCAGUAUCUGUUA
), ArticleFig(id=1241057515874611473, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=表2, caption=

sgRNA序列

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称序列（5’—3’）
influenza A virus-H1N1-HA-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUUGGGGUCAUCAAGAUACAGCAAGA
influenza A virus-H1N1-HA-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCCUUUAUCAUUAAUGUAGGAUUUG
influenza A virus-H3N2-HA-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUAUGCCUGAAACCGUACCAACCGUC
influenza A virus-H3N2-HA-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCCGAUCAACCUAUUCAGCUUCCCA
influenza B virus-HA-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUGUCUUCCCCUUCUGAACAAAUGUA
influenza B virus-HA-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUGGGGUUCCACUCUGAUGACAAAAC
influenza B virus-NS1-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUACACAGUAUGGCUCAAACCCUUCA
influenza B virus-NS1-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUUGGAUCCCUCUGCUGGAAUUGAAG
RSV-A-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUACAGGGUGUGGUUACAUCAUAUAC
RSV-A-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCCAUAUGUGCCAAUGUGUCCUUGG
RSV-B-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUACCAUUCCUGCUACAGAUGCAACU
RSV-B-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUUAUGCCCGUUGUAUAACCUUAGAA
SARS-CoV-2-ORF1ab-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUCAUCUACUGAUUGGACUAGCUAAA
SARS-CoV-2- ORF1ab-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUGUCAUAGUCAGUUAGGUGGUUUAC
RhV-Poly-sgRNA1GGGGUAAUUUCUACUAAGUGUAGAUCUGAGCCUGUAAAGGAUAUCAUGU
RhV- Poly-sgRNA2GGGGUAAUUUCUACUAAGUGUAGAUCAUCACUUGCAUCAGUAUCUGUUA
), ArticleFig(id=1241057515979469078, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=EN, label=Table 3, caption=

Comparison of qRT-PCR and RPA-CRISPR/Cas12a detection results in clinical samples

, figureFileSmall=null, figureFileBig=null, tableContent=
病原体qRT-PCR检测阳性数（CT≤35）RPA-CRISPR/Cas12a检测阳性数一致率a
influenza A virus-H1N1-HA88100
influenza A virus-H3N2-HA88100
influenza B virus-HA88100
influenza B virus-NS188100
Respiratory syncytial virus-A88100
Respiratory syncytial virus-B88100
SARS-CoV-2-ORF1ab88100
Rhinovirus-Poly6233
), ArticleFig(id=1241057516067549466, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241036252322001352, language=CN, label=表3, caption=

临床样本的qRT-PCR与RPA-CRISPR/Cas12a检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
病原体qRT-PCR检测阳性数（CT≤35）RPA-CRISPR/Cas12a检测阳性数一致率a
influenza A virus-H1N1-HA88100
influenza A virus-H3N2-HA88100
influenza B virus-HA88100
influenza B virus-NS188100
Respiratory syncytial virus-A88100
Respiratory syncytial virus-B88100
SARS-CoV-2-ORF1ab88100
Rhinovirus-Poly6233
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基于RPA-CRISPR/Cas12a的5种呼吸道病毒检测方法的建立
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王清华 1 , 张雅路 2 , 谭琪 2 , 石耀强 2 , 王攀 1 , 白明明 1 , 杨春晖 2
现代预防医学 | 实验技术及其应用 2025,52(16): 3013-3021
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现代预防医学 | 实验技术及其应用 2025, 52(16): 3013-3021
基于RPA-CRISPR/Cas12a的5种呼吸道病毒检测方法的建立
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王清华1, 张雅路2, 谭琪2, 石耀强2, 王攀1, 白明明1, 杨春晖2
作者信息
  • 1.成都市温江区中医医院，四川 成都 611130
  • 2.中国医学科学院输血研究所

    • 王清华（1976—），女，硕士，副主任中医师，研究方向：输血医学

通讯作者:

杨春晖，E-mail：
Establishment of a detection method for five respiratory viruses based on RPA-CRISPR/Cas12a
Qing-hua WANG1, Ya-lu ZHANG2, Qi TAN2, Yao-qiang SHI2, Pan WANG1, Ming-ming BAI1, Chun-hui YANG2
Affiliations
  • Chengdu Wenjiang District Hospital of Traditional Chinese Medicine, Chengdu, Sichuan 611130, China
出版时间: 2025-08-25 doi: 10.20043/j.cnki.MPM.202410457
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摘要
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目的

为了在基层、诊所、边防和野外等资源匮乏环境中及早明确常见导致呼吸道感染的病毒，从而采取有效的治疗方法和应对措施，本研究建立了一种基于重组酶聚合酶扩增技术（recombinase polymerase amplification，RPA）和CRISPR/Cas12a（clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a）系统的可视化检测方法。

方法

针对每种病毒靶标的保守序列设计和筛选RPA引物及单链向导RNA（single-strand guide RNA，sgRNA），然后将RPA技术与CRISPR/Cas12a检测方法相结合，建立呼吸道病毒即时可视化检测方法，并通过检测甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、新型冠状病毒和人鼻病毒临床样品对该方法进行验证。

结果

所建立的方法可在1.5 h内获得结果，灵敏度达3.5 copies/μl，各病毒靶标之间无交叉反应。该方法在检测正确率方面与对照的定量逆转录-聚合酶链式反应（Quantitative Reverse Transcription Polymerase Chain Reaction，qRT-PCR）法比具有较高的一致性。

结论

本研究建立的可视化检测方法对常见5种呼吸道感染病毒的8种型别具有较好的特异性和高灵敏度，适用于常见呼吸道病毒感染的现场检测，尤其是资源有限的地区，具有良好的临床应用前景。

呼吸道病毒感染  /  RPA  /  CRISPR/Cas12a  /  可视化检测  /  现场检测
Objective

To promptly identify common respiratory viruses causing infections and facilitate effective therapeutic interventions in resource-limited settings such as grassroots, clinics, border areas, and border defense, this study established a visual detection method based on recombinase polymerase amplification (RPA) technology and CRISPR/Cas12a (clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a) system.

Methods

Initially, conservative sequences of each virus target were designed, and RPA primers along with single-strand guide RNAs (sgRNAs) were selected. Subsequently, the RPA technology was integrated with the CRISPR/Cas12a detection method for visual detection of influenza A virus, influenza B virus, respiratory syncytial virus, Severe Acute Respiratory Syndrome Coronavirus 2, and human rhinovirus.

Results

The detection method yielded results within 1.5 hours, with a sensitivity of 3.5 copies/μl plasmid, and exhibited no cross-reactivity between each virus target. In terms of detection accuracy, this method demonstrated higher consistency compared to the control Quantitative Reverse Transcription Polymerase Chain Reaction, (qRT-PCR) method.

Conclusion

The visual detection method established in this study possesses good specificity and high sensitivity for the common five respiratory virus infections. It is suitable for on-site detection of common respiratory virus infections, especially in resource-limited environments, and holds promising clinical application prospects.

Respiratory virus infection  /  RPA  /  CRISPR/Cas12a  /  Visual detection  /  On-site detection
王清华, 张雅路, 谭琪, 石耀强, 王攀, 白明明, 杨春晖. 基于RPA-CRISPR/Cas12a的5种呼吸道病毒检测方法的建立. 现代预防医学, 2025 , 52 (16) : 3013 -3021 . DOI: 10.20043/j.cnki.MPM.202410457
Qing-hua WANG, Ya-lu ZHANG, Qi TAN, Yao-qiang SHI, Pan WANG, Ming-ming BAI, Chun-hui YANG. Establishment of a detection method for five respiratory viruses based on RPA-CRISPR/Cas12a[J]. Modern Preventive Medicine, 2025 , 52 (16) : 3013 -3021 . DOI: 10.20043/j.cnki.MPM.202410457
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呼吸道病毒感染是全球范围内导致呼吸道疾病的主要原因之一，对公共卫生构成了严重威胁[1]。这些病毒包括流感病毒（Influenza Virus）、呼吸道合胞病毒（Respiratory Syncytial Virus，RSV）、冠状病毒（如SARS-CoV-2）和鼻病毒（Rhinovirus）[2]等。这些病毒的传播迅速且难以控制，尤其是在人口密集的地区。尽早检测和确诊呼吸道病毒感染对于控制疾病传播、指导临床治疗和保护公众健康至关重要[3]。
传统的呼吸道病毒检测方法主要包括病毒培养、免疫荧光检测（immunofluorescence assays，IFA）、酶联免疫吸附试验（enzyme-linked immunosorbent assays，ELISA）和聚合酶链式反应（polymerase chain reaction，PCR）[4]等。这些方法虽能够提供较为准确的检测结果，但也存在一些不足。病毒培养方法耗时较长，操作复杂，且对实验室条件要求较高[5]；免疫荧光和ELISA方法的特异性和灵敏度受限，可能出现假阳性或假阴性结果[6]；PCR方法虽然灵敏度和特异性较高，但需要昂贵的仪器和耗材，且实验过程繁琐[7]。因此，迫切需要开发一种快速、准确、简便且成本效益高的呼吸道病毒检测方法，以满足基层临床和公共卫生需求。
近年来，CRISPR/Cas（Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein）技术的发展为分子诊断领域带来了革命性的变化。CRISPR/Cas系统不仅在基因编辑方面取得了重要进展，还展示了在分子诊断中的巨大潜力[8]。特别是CRISPR/Cas12a（也称Cpf1）系统，通过其独特的核酸切割特性，可以实现高灵敏度和高特异性的核酸检测[9]。此外，重组酶聚合酶扩增（Recombinase Polymerase Amplification，RPA）技术因其操作简便、反应速度快且无需热循环仪的优势，成为一种理想的等温扩增方法[10]。将RPA与CRISPR/Cas12a结合，能够在短时间内实现目标核酸的高效扩增和检测，具有极大的应用潜力。Jennifer Doudna团队开发的DETECTR方法是两步法即先进行RPA扩增，然后将部分RPA产物转移到含有Cas12a、crRNA和报告分子的另一管中进行检测[11]。
本研究的目的在于开发一种基于RPA-CRISPR/Cas12a的检测平台，用于快速、灵敏地检测五种常见呼吸道病毒：甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、新型冠状病毒、人鼻病毒。本研究设计了一系列针对这些病毒的特异性引物和sgRNA（single-strand guide RNA，sgRNA），通过对温度、反应时间、试剂浓度等试验条件的摸索，建立了适用于多种呼吸道病毒的即时检验方法，并评估了该方法的灵敏度、特异性和实际样本中的应用效果。
1　材料与方法
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1.1　阳性质粒与病毒样本来源
本实验所用的5种病毒对应的8种靶标的阳性标准质粒（甲型流感病毒H1N1型HA靶标（influenza A virus-H1N1-HA）、甲型流感病毒H3N2型HA靶标（influenza A virus-H3N2-HA）、乙型流感病毒HA靶标（influenza B virus-HA）、乙型流感病毒NS1靶标（influenza B virus-NS1）、呼吸道合胞病毒A型（Respiratory syncytial virus-A）、呼吸道合胞病毒B型（Respiratory syncytial virus-B）、新型冠状病毒ORF1ab靶标（Severe Acute Respiratory Syndrome Coronavirus 2-ORF1ab，SARS-CoV-2-ORF1ab）、人鼻病毒Poly靶标（rhinovirus-Poly））均购自上海生工有限公司；呼吸道咽拭子病毒样本由成都市温江区中医医院提供。样品获取经过医院的伦理委员会审批，病人知情同意由医院完成。
1.2　主要试剂与仪器
RPA引物、qPCR引物、ssDNA探针、琼脂糖粉均在上海生工有限公司合成和购买；PCR反应试剂、一步法qRT-PCR反应试剂、T7高效体外转录试剂盒、极简RNA纯化回收试剂盒均购自北京全式金有限公司；RT-RPA基础型扩增试剂盒购自杭州众测生物有限公司；缓冲液r2.1、缓冲液3、Cas12a蛋白均购自北京纽英伦生物技术有限公司；qPCR试剂购自近岸蛋白有限公司。荧光定量PCR仪（型号为CFX96）购买自上海伯乐生命医学产品有限公司；凝胶成像仪（型号为Jena）北京耶拿分析仪器有限公司；分光光度计（型号为NanoDrop）和PCR仪（型号为VeritiPro）均购买自上海赛默飞世尔科技有限公司；蓝光仪（型号为TGreen Transilluminator）购买自北京全式金有限公司。PRA引物序列和sgRNA序列见表1和表2。
1.3　方法
1.3.1　RPA引物设计和筛选
在NCBI（http://www.ncbi.nlm.nih.gov）中下载不同病原的基因序列，并分段比对查找保守基因片段。将筛选出来的5种病毒8个靶标的保守序列合成阳性标准质粒。同时通过NCBI对这8条保守序列分别设计RPA引物，选取评分较高的前3对引物合成。
以8种阳性标准质粒为模板，按照RPA核酸扩增试剂盒说明书，分别对每种靶标的三对RPA引物进行RPA反应。将A buffer 25 μl，上下游引物（10 μM）各2 μl，B buffer 2.5 μl，阳性标准质粒5 μl，无酶水补齐到50 μl，混合均匀后加入装有干粉的检测单元管中，上下颠倒充分混匀5~6次，低速离心10s后，37 ℃反应30 min，同时设置阴性对照组。扩增结束后取5 μl扩增产物进行1.5%琼脂糖凝胶电泳检测扩增效果。
1.3.2　sgRNA的设计与制备
在CHOPCHOP（http://chopchop.cbu.uib.no/）上设计sgRNA的序列，然后转换为一对互补的带有骨架序列的DNA链，交于上海生工有限公司合成。对DNA链在95 ℃加热5 min，梯度降温到室温以合成DNA双链。对退火后的DNA双链进行体外转录得到RNA产物，最后将RNA产物通过RNA纯化回收试剂盒纯化以得到sgRNA，对每种靶标设计合成了2种sgRNA。
1.3.3　sgRNA筛选和CRISPR/Cas12a反应的建立
CRISPR/Cas12a反应体系包括200 nM Cas12a，36 ng sgRNA，1 000 nM ssDNA探针，10×NEBuffer r2.1 3 μl，10 μl RPA扩增产物，无酶水补齐至30 μl。将该体系充分混匀后放到PCR仪或金属浴中37 ℃加热60 min，然后在蓝光灯下通过肉眼观察或拍照记录终点荧光强度。也可通过荧光采集仪器如实时荧光定量PCR仪，按照37 ℃加热，每1 min记录一次荧光，记录60次的方法，来记录实时荧光强度的变化。以阳性标准质粒为模板，按照上述方法建立CRISPR/Cas12a反应体系，对每种靶标的sgRNA进行筛选，同时设置空白对照组，根据终点荧光强度高低选择最佳的sgRNA。
1.3.4　RPA-CRISPR/Cas12a检测方法的灵敏度检测
将每种靶标的阳性标准质粒进行10倍梯度稀释，取5个浓度（3.5×100～3.5×104 copies/μl）作为模板，空白对照组以无酶水作为模板，按照上述方法建立10 μl RPA反应体系加在反应管管底。同时按照上述方法建立30 μl CRISPR/Cas12a反应体系加于反应管管盖上，先在37 ℃反应30 min使得靶序列充分扩增，然后将反应管离心20 s，使管盖上的CRISPR/Cas12a反应体系与RPA产物混合，于37 ℃反应60 min，使Cas12a酶识别和切割靶序列，并切割体系中的ssDNA以产生荧光。在蓝光灯下通过肉眼观察或拍照记录终点荧光强度，或在实时荧光定量PCR仪中实时记录荧光强度的变化。
1.3.5　RPA-CRISPR/Cas12a检测方法的特异度检测
以每种靶标的阳性标准质粒为模板，将其对应的RPA-CRISPR/Cas12a检测方法作为阳性对照组，其他7种RPA-CRISPR/Cas12a检测方法作为阴性对照组，同时设置水为扩增模板作为空白依照组，以进行8种RPA-CRISPR/Cas12a检测方法的特异度评价。
1.3.6　临床样本的RPA-CRISPR/Cas12a检测与qRT-PCR方法对比
将8份呼吸道咽拭子病毒样本经核酸提取后作为模板，按照上述建立的8种RPA-CRISPR/Cas12a检测方法进行临床样本的检测，通过实时荧光定量PCR仪实时记录荧光强度变化，并通过蓝光灯记录终点荧光强度。同时通过qRT-PCR的方法对临床样本进行检测，比较两种方法应用于临床样本的一致性。
2　结果
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2.1　RPA引物的筛选
以8种靶标的阳性标准质粒作为模板在37 ℃反应30 min，反应结束后，取5 μl RPA扩增产物进行1.5%琼脂糖凝胶电泳，根据能否扩增出与目的条带大小相符的DNA片段以及条带的亮度与单一性，对每种病毒靶标选择合适的RPA产物，以用于后续的反应。结果如图1，对每个病毒靶标分别进行了3对RPA引物筛选，最终对甲型流感病毒H1N1型HA靶标、甲型流感病毒H3N2型HA靶标、乙型流感病毒HA靶标、乙型流感病毒NS1靶标、呼吸道合胞病毒A型、呼吸道合胞病毒B型、新型冠状病毒ORF1ab靶标、人鼻病毒Poly靶标分别选择引物3，引物3，引物2，引物3，引物3，引物1，引物1，引物3为后续RPA反应的引物。
2.2　sgRNA的筛选
本研究对每种病毒靶标分别设计和合成了2种sgRNA，以8种亚型或靶标的阳性标准质粒作为模板，通过CRISPR/Cas12a标准反应体系，根据荧光强度高低选择合适的sgRNA以用于后续反应。图2为8种病毒靶标的sgRNA筛选结果，根据肉眼观察蓝光灯下反应管的荧光强度和荧光定量PCR仪记录到的荧光强度，选择荧光强度高的一组sgRNA。最终对甲型流感病毒H1N1型HA靶标、甲型流感病毒H3N2型HA靶标、乙型流感病毒HA靶标、乙型流感病毒NS1靶标、呼吸道合胞病毒A型、呼吸道合胞病毒B型、新型冠状病毒ORF1ab靶标、人鼻病毒Poly靶标分别选择sgRNA1，sgRNA1，sgRNA1，sgRNA1，sgRNA2，sgRNA1，sgRNA1，sgRNA1用于后续反应。
2.3　RPA-CRISPR/Cas12a检测方法的灵敏度评价
将8种靶标的阳性标准质粒分别进行10倍梯度稀释，取5个浓度梯度（3.5×100～3.5×104 copies/μl）作为模板，空白对照组以无酶水作为模板，按照上述方法建立一管化RPA-CRISPR/Cas12a反应体系对RPA-CRISPR/Cas12a检测方法进行灵敏度评价，如图3所示，每种病毒靶标的RPA-CRISPR/Cas12a检测方法灵敏度都能达到3.5×100copies/μl质粒。
2.4　RPA-CRISPR/Cas12a检测方法的特异度评价
以8种亚型或靶标的阳性标准质粒作为模板，按照上述建立的方法，分别对8种RPA-CRISPR/Cas12a检测方法进行特异度评价，结果如图4所示，每种病毒靶标的RPA-CRISPR/Cas12a检测方法特异性良好，与其他7种病毒型别RPA-CRISPR/Cas12a检测方法无交叉反应。
2.5　临床样本的RPA-CRISPR/Cas12a检测与qRT-PCR方法对比
采用本研究建立的5种呼吸道病毒的8种亚型或靶标的RPA-CRISPR/Cas12a检测方法对8份临床样本进行检测，同时与qRT-PCR的方法进行比较。8份样本的RPA-CRISPR/Cas12a方法检测结果如图5。表3为RPA-CRISPR/Cas12a检测方法与qRT-PCR法的检测结果对比，可以看出本研究针对5种呼吸道病毒的8种亚型或靶标建立的RPA-CRISPR/Cas12a检测方法的检测结果与qRT-PCR法相比，除人鼻病毒Poly靶标一致率为33%外，其余亚型或靶标的一致率较高，能用于实际临床样本的检测。
3　讨论
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随着全球公共卫生事件的频发，快速、准确的病毒检测技术成为防控疫情、保障公共健康的关键。传统检测方法如病毒培养、免疫学检测及PCR技术等，在灵敏度、特异性、检测速度及成本效益等方面各有优劣[12]。近年来，CRISPR/Cas系统与等温扩增技术（如RPA）的联合应用为病毒检测领域带来了革命性的变化。本研究建立的基于RPA-CRISPR/Cas12a可视化方法对5种常见呼吸道感染病毒甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、新型冠状病毒和人鼻病毒有高灵敏度和特异性，该检测方法1.5 h内可得结果，灵敏度达3.5 copies/μl，病毒靶标间无交叉反应，与qRT-PCR法检测正确率一致。适用于现场检测，尤其在资源有限环境下，临床应用前景良好。
CRISPR/Cas系统与等温扩增技术的联合应用能够实现对病毒核酸的高灵敏度和特异性检测。具体而言，CRISPR/Cas系统负责精准定位和切割目标核酸序列，而等温扩增技术则负责在体外快速扩增这些序列。这种组合不仅提高了检测的灵敏度和特异性，还大大缩短了检测时间，降低了对复杂设备的依赖，使得检测更加便捷和经济。近年来，基于RPA结合CRISPR-Cas12a的检测方法在呼吸道病毒检测中取得了显著成果。例如，亚辉龙研发团队开发的检测方法能够在1h内成功检测出SARS-CoV-2、甲型流感和乙型流感3种呼吸道病毒，检测限低至102 copies/μl[13]。本研究相比既往的基于RPA结合CRISPR-Cas12a的检测方法新颖性在于，将扩增和检测放在同一管中，开发了单管反应，灵敏度达3.5 copies/μl，从而提升便捷性，提高灵敏度，减少污染风险。
尽管CRISPR/Cas系统与等温扩增技术具有诸多优势，但仍也存在一些不足，如本方法目前暂时仅能实现定性检测，下一步打算进一步优化反应体系和结果判读方式，以实现半定量或定量解读，达到更全面准确的诊断结果。另外，本实验还需设置质控品，以排除某些假阳性和假阴性结果。未来希望本研究建立的One-Pot-RPA-CRISPR/Cas12a方法能在其他更多的细菌菌株和病毒突变体上检测和进一步验证。随着技术的不断发展，需要建立相应的标准化体系和监管机制来确保检测结果的准确性和可靠性。这包括制定统一的技术标准、建立质量控制体系以及加强监管力度等方面。未来，随着CRISPR/Cas系统和等温扩增技术的不断发展和完善，本研究可以期待更创新性的应用出现。例如，可以探索将其他类型的CRISPR/Cas系统（如Cas13a、CasX等）与等温扩增技术相结合，以实现对不同类型病毒或病原体的检测；还可以开发更加高效、稳定的扩增酶和sgRNA设计工具，以提高检测的灵敏度和特异性。为了满足现场检测和基层医疗机构的需求，未来应继续推动检测平台的便携化和自动化发展。通过集成更多的功能模块和优化操作流程，可以实现检测设备的小型化、轻量化以及一键式操作等功能；同时，还可以利用物联网、大数据等先进技术实现检测结果的远程传输和智能分析等功能。
通过本研究，我们期望提供一种便捷、高效的检测工具，能够显著提升呼吸道病毒感染的早期诊断能力，为疾病防控和临床管理提供有力支持。未来，我们还将进一步扩展该平台的检测范围，开发针对更多病原体的快速检测方法，以应对可能出现的新型呼吸道病毒威胁。
基金
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  • 成都市卫健委医学科研课题(2021331)
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2025年第52卷第16期
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doi: 10.20043/j.cnki.MPM.202410457
  • 接收时间：2024-10-29
  • 首发时间：2026-03-18
  • 出版时间：2025-08-25
补充材料
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  • 收稿日期：2024-10-29
基金
成都市卫健委医学科研课题(2021331)
作者信息
    1.成都市温江区中医医院，四川 成都 611130
    2.中国医学科学院输血研究所

通讯作者:

杨春晖，E-mail：
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
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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