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Article(id=1153433744267535089, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433737141412332, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250110003, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1736438400000, receivedDateStr=2025-01-10, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752929634395, onlineDateStr=2025-07-19, pubDate=1745510400000, pubDateStr=2025-04-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752929634395, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752929634395, creator=13701087609, updateTime=1752929634395, updator=13701087609, issue=Issue{id=1153433737141412332, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='8', pageStart='1', pageEnd='316', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752929632696, creator=13701087609, updateTime=1757293087150, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1171735391666225233, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433737141412332, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1171735391666225234, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433737141412332, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=122, endPage=130, ext={EN=ArticleExt(id=1153433744816988945, articleId=1153433744267535089, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Establishment of visual detection method for Salmonella based on recombinase polymerase amplification-CRISPR/Cas12a, columnId=1151895322692776479, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Analysis and Monitoring of Toxic and Harmful Substances in Food, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a visual detection method of Salmonella based on recombinant enzyme polymerase amplification-CRISPR/Cas12a. Methods In this study, recombinase polymerase amplification (PRA) was combined with clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) system. RPA primers and CRISPR-derived RNA (crRNA) were designed and synthesized based on the target invA gene of Salmonella. Fluorescence and test strip methods were employed to read the results. The optimized RPA-CRISPR/Cas12 detection system were evaluated for specificity and sensitivity, and was applied to the detection of food samples. Results Nano gold test strip base on nucleic acid were developed. The established RPA-CRISPR/Cas12 detection method could specifically detecting Salmonella with a sensitivity of 80 CFU/mL within 1.5 h. Used this method to detect 21 kinds of food samples, the results from fluorescence and test strip read were consistent, with a Salmonella detection rate of 4.8%, which was completely consistent with the results of the national standard method. Conclusion The Salmonella RPA-CRISPR/Cas12a visual detection method established in this study has high specificity and sensitivity and is valuable for on-site rapid detection of Salmonella.

, correspAuthors=Jing CHEN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Jian-Fei HUANG, Jing CHEN, Qian ZHANG, Cheng-Rong XIAO, Yan-Hui WU, Xin-Tian LAI), CN=ArticleExt(id=1153433778639856628, articleId=1153433744267535089, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法的建立, columnId=1151895322898297380, journalTitle=食品安全质量检测学报, columnName=本期专题：食品中有毒有害物质分析与监测, runingTitle=null, highlight=null, articleAbstract=

目的 建立基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法。方法 本研究将重组酶聚合酶扩增(recombinase polymerase amplification, RPA)技术与成簇规律间隔短回文重复序列及其相关蛋白12a (clustered regularly interspaced short palindromic repeats/associated protein 12a, CRISPR/Cas12a)系统相结合, 以沙门氏菌invA作为目标基因, 设计并合成RPA引物和CRISPR引导RNA (CRISPR-derived RNA, crRNA), 通过荧光法和试纸条两种方法读取结果。对优化的RPA-CRISPR/Cas12检测体系进行特异性和灵敏度实验, 并应用于食品样本检测。结果 本研究成功研制纳米金核酸试纸条, 建立的RPA-CRISPR/Cas12a体系可在1.5 h内特异性完成沙门氏菌的检测, 灵敏度为80 CFU/mL。利用此方法对21个食品样本进行检测, 荧光法、试纸条法与国家标准法检测结果完全一致, 沙门氏菌检出率为4.8%。结论 本研究建立的沙门氏菌RPA-CRISPR/Cas12a可视化检测方法具有高特异性和灵敏度, 在沙门氏菌的现场快速检测方面具有应用价值。

, correspAuthors=陈晶, authorNote=null, correspAuthorsNote=
* 陈晶(1976—), 女, 高级工程师, 主要研究方向为食品微生物检测。E-mail:
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黄建飞(1988—), 男, 中级工程师, 主要研究方向为食品微生物检测。E-mail:

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黄建飞(1988—), 男, 中级工程师, 主要研究方向为食品微生物检测。E-mail:

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黄建飞(1988—), 男, 中级工程师, 主要研究方向为食品微生物检测。E-mail:

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Letters in Applied Microbiology, 2022, 75(4): 899-907., articleTitle=Sensitive and high-accuracy detection of Salmonella based on CRISPR/Cas12a combined with recombinase polymerase amplification, refAbstract=null), Reference(id=1171733915661279481, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, doi=null, pmid=null, pmcid=null, year=2024, volume=45, issue=5, pageStart=412, pageEnd=425, url=null, language=null, rfNumber=[29], rfOrder=32, authorNames=贠紫光, 魏勇, 张建, journalName=食品工业科技, refType=null, unstructuredReference=贠紫光, 魏勇, 张建, 等. 基于核酸扩增-侧流层析试纸的食源性致病菌快速检测研究进展[J]. 食品工业科技, 2024, 45(5): 412-425., articleTitle=基于核酸扩增-侧流层析试纸的食源性致病菌快速检测研究进展, refAbstract=null), Reference(id=1171733915749359867, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, doi=null, pmid=null, pmcid=null, year=2024, volume=45, issue=5, pageStart=412, pageEnd=425, url=null, language=null, rfNumber=[29], rfOrder=33, authorNames=YUN ZG, WEI Y, ZHANG J, journalName=Science and Technology of Food Industry, refType=null, unstructuredReference=YUN ZG, WEI Y, ZHANG J, et al. 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Research progress of immunochromatographic test strip detection technology[J]. Laboratory Medicine and Clinic, 2019, 16(22): 3382-3386., articleTitle=Research progress of immunochromatographic test strip detection technology, refAbstract=null), Reference(id=1171733915942297857, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, doi=null, pmid=null, pmcid=null, year=2021, volume=171, issue=3, pageStart=106859, pageEnd=null, url=null, language=null, rfNumber=[31], rfOrder=36, authorNames=FU Q, YUAN L, CAO F, journalName=Microchemical Journal, refType=null, unstructuredReference=FU Q, YUAN L, CAO F, et al. Lateral flow strip biosensor based streptavidin-coated gold nanoparticles with recombinase polymerase amplication for the quantitative point-of-care testing of Salmonella[J]. Microchemical Journal, 2021, 171(3): 106859., articleTitle=Lateral flow strip biosensor based streptavidin-coated gold nanoparticles with recombinase polymerase amplication for the quantitative point-of-care testing of Salmonella, refAbstract=null)], funds=[Fund(id=1171733912603631801, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, awardId=KCXFZ20211020165404007, language=CN, fundingSource=深圳市科创计划资助项目(KCXFZ20211020165404007), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1171733907176202320, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, xref=null, ext=[AuthorCompanyExt(id=1171733907188785234, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, companyId=1171733907176202320, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Shenzhen Academy of Metrology & Quality Inspection, Shenzhen 518131, China), AuthorCompanyExt(id=1171733907201368148, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, companyId=1171733907176202320, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=深圳市计量质量检测研究院, 深圳 518131)])], figs=[ArticleFig(id=1171733910590365847, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Fig.1, caption=Results of PRA amplification, figureFileSmall=knN0GUkljWsAQYtRq6a6LA==, figureFileBig=MZX9fnqikcpKlMSqsAAjEw==, tableContent=null), ArticleFig(id=1171733910707806361, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=图1, caption=RPA扩增结果

注: M. DNA Marker; 1. RPA1产物; 2. RPA2产物; 3. RPA3产物; 4. RPA4产物; 5. RPA5产物; 6. RPA6产物; N. 阴性对照。

, figureFileSmall=knN0GUkljWsAQYtRq6a6LA==, figureFileBig=MZX9fnqikcpKlMSqsAAjEw==, tableContent=null), ArticleFig(id=1171733910795886747, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Fig.2, caption=Screening of RPA primers with crRNA, figureFileSmall=b5JlGghDNQNb45HSgKhy3g==, figureFileBig=ooij7F+E48KjvqGRrdzbHg==, tableContent=null), ArticleFig(id=1171733910883967133, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=图2, caption=RPA引物与crRNA搭配的筛选, figureFileSmall=b5JlGghDNQNb45HSgKhy3g==, figureFileBig=ooij7F+E48KjvqGRrdzbHg==, tableContent=null), ArticleFig(id=1171733911030767775, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Fig.3, caption=Optimization of CRISPR-Cas12a fluorescence detection system, figureFileSmall=NLEuf8DiV/NWQMAYLoxg4A==, figureFileBig=XlajrefKUy6t7x9pF26XaQ==, tableContent=null), ArticleFig(id=1171733911106265248, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=图3, caption=CRISPR-Cas12a荧光检测体系优化

注: A. Cas12a和crRNA的浓度比例对荧光强度的影响; B. Cas12a和crRNA的浓度对荧光强度的影响; 1. Cas12a:crRNA=1:0.5; 2. Cas12a:crRNA=1:1; 3. Cas12a:crRNA=1:2; 4. Cas12a:crRNA=1:3。

, figureFileSmall=NLEuf8DiV/NWQMAYLoxg4A==, figureFileBig=XlajrefKUy6t7x9pF26XaQ==, tableContent=null), ArticleFig(id=1171733911211122850, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Fig.4, caption=Optimization of lateral flow strips reporter concentration, figureFileSmall=b4QTQKizQti7NMJG474XuQ==, figureFileBig=LbbXPI4hoV3lGaGaQDi80g==, tableContent=null), ArticleFig(id=1171733911295008932, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=图4, caption=试纸条报告分子浓度优化

注: N. 阴性对照, 下同; P. 阳性组。

, figureFileSmall=b4QTQKizQti7NMJG474XuQ==, figureFileBig=LbbXPI4hoV3lGaGaQDi80g==, tableContent=null), ArticleFig(id=1171733911567638693, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Fig.5, caption=Specificity of RPA/CRISPR-Cas12a detection method, figureFileSmall=JP3J224tnIHFzpzaDbhQqA==, figureFileBig=Zf34Dh/9cGLajZwRZ2xMCw==, tableContent=null), ArticleFig(id=1171733911747993766, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=图5, caption=RPA/CRISPR-Cas12a检测方法的特异性

注: A. 荧光检测法; B. 试纸条检测法, 下同; 1. 大肠埃希氏菌O157; 2. 唐菖蒲伯克霍尔德氏菌; 3. 鼠伤寒沙门氏菌; 4. 福氏志贺氏菌; 5. 大肠埃希氏菌; 6. 普通变形杆菌; 7. 枯草芽胞杆菌; 8. 蜡样芽胞杆菌; 9. 金黄色葡萄球菌; 10. 肠炎沙门氏菌; 11. 单核细胞增生李斯特氏菌; 12. 副溶血性弧菌; 13. 弗氏柠檬酸杆菌; 14. 肺炎克雷伯氏菌; 15. 粘质沙雷氏菌。

, figureFileSmall=JP3J224tnIHFzpzaDbhQqA==, figureFileBig=Zf34Dh/9cGLajZwRZ2xMCw==, tableContent=null), ArticleFig(id=1171733911827685544, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Fig.6, caption=Sensitivity of RPA/CRISPR-Cas12a detection method, figureFileSmall=1L4RQMLC69nw0l2QyREJ5A==, figureFileBig=JeBWpURt8W34zk+b+e9G7Q==, tableContent=null), ArticleFig(id=1171733911907377322, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=图6, caption=RPA/CRISPR-Cas12a检测方法的灵敏度

注: 1. 8.0×106 CFU/mL; 2. 8.0×105 CFU/mL; 3. 8.0×104 CFU/mL; 4. 8.0×103 CFU/mL; 5. 8.0×102CFU/mL; 6. 80 CFU/mL;7. 8 CFU/mL。

, figureFileSmall=1L4RQMLC69nw0l2QyREJ5A==, figureFileBig=JeBWpURt8W34zk+b+e9G7Q==, tableContent=null), ArticleFig(id=1171733912012234924, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Fig.7, caption=Detection results of samples, figureFileSmall=VZ9X+jFJj2B2M+l6ODnGSw==, figureFileBig=lClDc4UP/qWlNbZZG3IYTg==, tableContent=null), ArticleFig(id=1171733912100315310, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=图7, caption=样品检测结果

注: 1~21. 样品编号。

, figureFileSmall=VZ9X+jFJj2B2M+l6ODnGSw==, figureFileBig=lClDc4UP/qWlNbZZG3IYTg==, tableContent=null), ArticleFig(id=1171733912196784304, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Table 1, caption=

Strains used in the study

, figureFileSmall=null, figureFileBig=null, tableContent=
序号 菌株名称 菌株编号 序号 菌株名称 菌株编号
1 肠炎沙门氏菌 CMCC(B)50335 9 蜡样芽胞杆菌
 ATCC2
2 鼠伤寒沙门氏菌 ATCC14028 10 枯草芽胞杆菌
 ATCC9372
3 大肠埃希氏菌O157 ATCC43895 11 弗氏柠檬酸杆菌
 ATCC43864
4 单核细胞增生李斯特氏菌 CICC21662 12 肺炎克雷伯氏菌 ATCC4352
5 福氏志贺氏菌 CICC21534 13 普通变形杆菌
 ATCC6380
6 副溶血性弧菌 CICC21617
 14 大肠埃希氏菌
 ATCC25922
7 金黄色葡萄球菌
 ATCC6538
 15 粘质沙雷氏菌
 ATCC14041
8 唐菖蒲伯克霍尔德氏菌 CICC10574
), ArticleFig(id=1171733912289058994, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=表1, caption=

实验用菌株

, figureFileSmall=null, figureFileBig=null, tableContent=
序号 菌株名称 菌株编号 序号 菌株名称 菌株编号
1 肠炎沙门氏菌 CMCC(B)50335 9 蜡样芽胞杆菌
 ATCC2
2 鼠伤寒沙门氏菌 ATCC14028 10 枯草芽胞杆菌
 ATCC9372
3 大肠埃希氏菌O157 ATCC43895 11 弗氏柠檬酸杆菌
 ATCC43864
4 单核细胞增生李斯特氏菌 CICC21662 12 肺炎克雷伯氏菌 ATCC4352
5 福氏志贺氏菌 CICC21534 13 普通变形杆菌
 ATCC6380
6 副溶血性弧菌 CICC21617
 14 大肠埃希氏菌
 ATCC25922
7 金黄色葡萄球菌
 ATCC6538
 15 粘质沙雷氏菌
 ATCC14041
8 唐菖蒲伯克霍尔德氏菌 CICC10574
), ArticleFig(id=1171733912372945076, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=EN, label=Table 2, caption=

DNA sequence used in the study

, figureFileSmall=null, figureFileBig=null, tableContent=
名称 序列(5'-3')
RPA1-F AGTGCTCGTTTACGACCTGAATTACTGATTCTGGT
RPA1-R TTAACAGTACCGCAGGAAACGTTGAAAAACTGAG
RPA2-F TCTGGGTAACGCATGAAGAGGGGGAGAAAC
RPA2-R GATATACGTTGTACCGTGGCATGTCTGAGCACTTC
RPA3-F GCATTGTGGGCGCCAAGAGAAAAAGATGTCATTA
RPA3-R CGGGTCAAGGCTGAGGAAGGTACTGCCAGAG
RPA4-F GTCATTCCATTACCTACCTATCTGGTTGATTTCC
RPA4-R GCATCGGCTTCAATCAAGATAAGACGACTGGT
RPA5-F ATGCCGGTGAAATTATCGCCACGTTCGGGCAATTC
RPA5-R CGCGCAGCATCCGCATCAATAATACCGGCCTTCA
crRNA1 UAAUUUCUACUAAGUGUAGAUUCGUUAUUACCAAAGGUUCA
RPA6-F GCTCGCATTGTGGGCGCCAAGAGAAAAAGATG
RPA6-R TCCGGGTCAAGGCTGAGGAAGGTACTGCCAGAG
crRNA2 UAAUUUCUACUAAGUGUAGAUCCAUAAAUUCGCCAAUGGCG
FB-ssDNA AGTACCGATAGATACAGAC
FQ-ssDNA FAM-AGTACCGATAGATACAGAC-BHQ
AP SH-TTTTTTTTGTCTGTAT
TCP CTATCGGTACTATACA-Biotin
CCP Biotin-ATACAGAC
), ArticleFig(id=1171733912465219766, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433744267535089, language=CN, label=表2, caption=

实验用核酸序列

, figureFileSmall=null, figureFileBig=null, tableContent=
名称 序列(5'-3')
RPA1-F AGTGCTCGTTTACGACCTGAATTACTGATTCTGGT
RPA1-R TTAACAGTACCGCAGGAAACGTTGAAAAACTGAG
RPA2-F TCTGGGTAACGCATGAAGAGGGGGAGAAAC
RPA2-R GATATACGTTGTACCGTGGCATGTCTGAGCACTTC
RPA3-F GCATTGTGGGCGCCAAGAGAAAAAGATGTCATTA
RPA3-R CGGGTCAAGGCTGAGGAAGGTACTGCCAGAG
RPA4-F GTCATTCCATTACCTACCTATCTGGTTGATTTCC
RPA4-R GCATCGGCTTCAATCAAGATAAGACGACTGGT
RPA5-F ATGCCGGTGAAATTATCGCCACGTTCGGGCAATTC
RPA5-R CGCGCAGCATCCGCATCAATAATACCGGCCTTCA
crRNA1 UAAUUUCUACUAAGUGUAGAUUCGUUAUUACCAAAGGUUCA
RPA6-F GCTCGCATTGTGGGCGCCAAGAGAAAAAGATG
RPA6-R TCCGGGTCAAGGCTGAGGAAGGTACTGCCAGAG
crRNA2 UAAUUUCUACUAAGUGUAGAUCCAUAAAUUCGCCAAUGGCG
FB-ssDNA AGTACCGATAGATACAGAC
FQ-ssDNA FAM-AGTACCGATAGATACAGAC-BHQ
AP SH-TTTTTTTTGTCTGTAT
TCP CTATCGGTACTATACA-Biotin
CCP Biotin-ATACAGAC
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基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法的建立
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黄建飞 , 陈晶 * , 张倩 , 肖承荣 , 吴燕蕙 , 赖心田
食品安全质量检测学报 | 本期专题：食品中有毒有害物质分析与监测 2025,16(8): 122-130
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食品安全质量检测学报 | 本期专题：食品中有毒有害物质分析与监测 2025, 16(8): 122-130
基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法的建立
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黄建飞 , 陈晶* , 张倩, 肖承荣, 吴燕蕙, 赖心田
作者信息
  • 深圳市计量质量检测研究院, 深圳 518131

    • 黄建飞(1988—), 男, 中级工程师, 主要研究方向为食品微生物检测。E-mail:

通讯作者:

* 陈晶(1976—), 女, 高级工程师, 主要研究方向为食品微生物检测。E-mail:
Establishment of visual detection method for Salmonella based on recombinase polymerase amplification-CRISPR/Cas12a
Jian-Fei HUANG , Jing CHEN* , Qian ZHANG, Cheng-Rong XIAO, Yan-Hui WU, Xin-Tian LAI
Affiliations
  • Shenzhen Academy of Metrology & Quality Inspection, Shenzhen 518131, China
出版时间: 2025-04-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250110003
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摘要
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目的 建立基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法。方法 本研究将重组酶聚合酶扩增(recombinase polymerase amplification, RPA)技术与成簇规律间隔短回文重复序列及其相关蛋白12a (clustered regularly interspaced short palindromic repeats/associated protein 12a, CRISPR/Cas12a)系统相结合, 以沙门氏菌invA作为目标基因, 设计并合成RPA引物和CRISPR引导RNA (CRISPR-derived RNA, crRNA), 通过荧光法和试纸条两种方法读取结果。对优化的RPA-CRISPR/Cas12检测体系进行特异性和灵敏度实验, 并应用于食品样本检测。结果 本研究成功研制纳米金核酸试纸条, 建立的RPA-CRISPR/Cas12a体系可在1.5 h内特异性完成沙门氏菌的检测, 灵敏度为80 CFU/mL。利用此方法对21个食品样本进行检测, 荧光法、试纸条法与国家标准法检测结果完全一致, 沙门氏菌检出率为4.8%。结论 本研究建立的沙门氏菌RPA-CRISPR/Cas12a可视化检测方法具有高特异性和灵敏度, 在沙门氏菌的现场快速检测方面具有应用价值。

沙门氏菌  /  成簇规律间隔短回文重复序列及其相关蛋白12a  /  试纸条  /  重组酶聚合酶扩增  /  可视化检测

Objective To establish a visual detection method of Salmonella based on recombinant enzyme polymerase amplification-CRISPR/Cas12a. Methods In this study, recombinase polymerase amplification (PRA) was combined with clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) system. RPA primers and CRISPR-derived RNA (crRNA) were designed and synthesized based on the target invA gene of Salmonella. Fluorescence and test strip methods were employed to read the results. The optimized RPA-CRISPR/Cas12 detection system were evaluated for specificity and sensitivity, and was applied to the detection of food samples. Results Nano gold test strip base on nucleic acid were developed. The established RPA-CRISPR/Cas12 detection method could specifically detecting Salmonella with a sensitivity of 80 CFU/mL within 1.5 h. Used this method to detect 21 kinds of food samples, the results from fluorescence and test strip read were consistent, with a Salmonella detection rate of 4.8%, which was completely consistent with the results of the national standard method. Conclusion The Salmonella RPA-CRISPR/Cas12a visual detection method established in this study has high specificity and sensitivity and is valuable for on-site rapid detection of Salmonella.

Salmonella  /  clustered regularly interspaced short palindromic repeats/associated protein 12a  /  test strip  /  recombinase polymerase amplification  /  visual detection
黄建飞, 陈晶, 张倩, 肖承荣, 吴燕蕙, 赖心田. 基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法的建立. 食品安全质量检测学报, 2025 , 16 (8) : 122 -130 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250110003
Jian-Fei HUANG, Jing CHEN, Qian ZHANG, Cheng-Rong XIAO, Yan-Hui WU, Xin-Tian LAI. Establishment of visual detection method for Salmonella based on recombinase polymerase amplification-CRISPR/Cas12a[J]. Journal of Food Safety & Quality, 2025 , 16 (8) : 122 -130 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250110003
正文
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0 引言
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沙门氏菌是一种革兰氏阴性肠杆菌, 生存能力强, 营养需求低, 广泛存在于自然环境中。世界卫生组织(World Health Organization, WHO)公布的数据显示, 沙门氏菌是全球范围内食源性疾病爆发的主要诱因, 全球每年有超过1亿人感染, 对人类健康构成重大威胁, 常见的临床表现有发热、腹痛、腹泻、发烧、恶心、呕吐等, 严重的会导致休克死亡[1-3]。美国疾病预防和控制中心(Centers for Disease Control and Prevention, CDC)估计沙门氏菌每年造成约135万人感染, 26500人住院, 420人死亡[4]。2023年欧盟报告了77486例人类感染沙门氏菌病例, 发病率约为每10万人中有18例, 沙门氏菌是欧盟第二大食源性致病菌, 仅次于弯曲杆菌[5]。YUE等[6]研究评估中国非伤寒沙门氏菌的发病率为每10万人626.6例。因此, 沙门氏菌是食源性致病菌的重点监控对象, 对其进行准确、快速检测是预防和控制沙门氏菌病的有效手段。
目前, 实验室主要通过传统的生化分离法检测食品中的沙门氏菌, 耗时长, 需要4~7 d, 不适合沙门氏菌的快速检测。随着技术的发展, 开发出各种食源性致病菌快速检测技术, 如免疫学、分子生物学、生物传感器等技术。基于核酸水平的分子生物学方法因具有灵敏度高、特异性强、快速等优点被广泛用于沙门氏菌的检测, 如聚合酶链式反应(polymerase chain reaction, PCR)[7]、多重PCR[8]、荧光定量PCR[9]、数字PCR (digital PCR, dPCR)[10]等。PCR技术需要昂贵的仪器, 不适合缺少设备的基层实验室以及现场检测。基于核酸检测的等温扩增技术如重组酶聚合酶扩增(recombinase polymerase amplification, RPA)[11]和环介导等温扩增(loop-mediated isothermal amplification, LAMP)[12]是在等温条件下工作的方法, 不需要价格昂贵的仪器, 对现场检测具有重要的意义, 但扩增产物需要后续凝胶电泳进行分析, 难以真正实现快速检测。因此, 需要开发更简单、更方便和更少依赖仪器的技术来实现在现场条件下进行沙门氏菌的即时诊断。
成簇规律间隔短回文重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeats/associated protein, CRISPR-Cas)系统是古细菌和细菌长期进化形成的免疫防疫系统, 具有高效特异序列识别及切割活性, 基因编辑功能强大, 为食源性致病菌的快速、特异、精准检测提供了一种新的技术, 根据Cas效应蛋白类型的不同可将CRISPR-Cas分为两类[13]。Cas13和Cas12a 属于第二类, 两者相同之处是它们都具有非特异性切割活性, 不同之处是Cas12a在crRNA的引导下识别靶标DNA, 激发非特异性切割ssDNA的活性, 而Cas13在CRISPR引导RNA (CRISPR-derived RNA, crRNA)的引导下识别靶标RNA, 激发非特异性切割单链RNA (single-stranded RNA, ssRNA)的活性, 这种不加区分的切割能力使CRISPR-Cas成为信号放大和输出的重要工具, 例如以荧光团和淬灭剂标记的ssDNA作为报告。虽然CRISPR-Cas系统可以利用反式裂解活性方法信号, 但其灵敏度不足以直接检测出致病菌, 结合RPA可构建一种高特异性、高灵敏度的方法, 目前基于RPA和CRISPR-Cas技术结合的系统在检测核酸方面具有巨大的潜力。CHEN等[14]将CRISPR-Cas12a与RPA相结合, 开发了一种基于CRISPR的诊断方法, 简称为DETECTR系统。该技术应可应用于人乳头瘤病毒(human papillomavirus, HPV)检测, 能够有效区分HPV16和HPV18人乳头瘤病毒。GOOTENBERG等[15]将CRISPR-Cas13a与RPA相结合, 利用荧光探针作为信号输出, 建立了SHERLOCK系统。近年来, 研究者将RPA与CRISPR/Cas12a结合实现目的基因进行双重特异性识别, 极大地提高检测特异性和灵敏度, 在细菌、病毒、支原体、转基因生物、寄生虫等检测领域具有广阔的前景[16]。
本研究采用RPA与CRISPR-Cas12技术相结合开发出沙门氏菌的快速检测方法, 特异性基因经过RPA扩增后, Cas12a蛋白在crRNA介导下特异性结合扩增产物, 通过荧光和侧流层析试纸条两种方法进行结果判读, 优化建立的RPA/CRISPR-Cas12a检测体系, 为食品中沙门氏菌的快速可视化检测提供新的技术。
1 材料与方法
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1.1 材料与试剂
鸡爪、牛肉、猪肉脯、鸡蛋、鸭肉、方便面、鸡腿、牛奶等检测样品来自深圳市商场。
DNA片段纯化试剂盒、细菌基因组提取试剂盒(型号: 9763)、DNA Marker[宝日医生物技术(北京)有限公司]; 聚氯乙烯(polyvinyl chloride, PVC)背板、结合垫、样品垫、吸收垫、硝酸纤维素膜(德国Millipore公司); RPA基础扩增试剂盒(英国TwistDX公司); EnGen Lab Cas12a(美国NEB公司); 营养肉汤培养基(北京陆桥技术有限公司); 牛血清蛋白、氯金酸、链霉亲和素(美国Sigma公司); 实验用菌株见表1; 实验用核酸序列见表2[生工生物工程(上海)股份有限公司]。
1.2 仪器与设备
BAX Q7全自动病原微生物检测系统(美国DuPont公司); GelDoc XR+凝胶成像系统、Powerpac Universal 电泳仪、C1000 PCR扩增仪(美国Bio-Rad公司); XYZ3060胶体金试纸条三维喷金划膜仪(美国Biodot公司); HGS201可编程切条机(杭州峰航科技有限公司); LA2-6A1生物安全柜(新加坡Esco公司); INE600培养箱(德国Memmert公司)。
1.3 RPA引物和crRNA的设计与筛选
根据RPA引物设计原则, 以沙门氏菌invA基因(GenBank号: AE014613.1)设计特异性扩增引物, 选取RPA扩增结果特异性强且条带明亮引物所在的基因序列区域设计crRNA。根据不同实验方法设计ssDNA, 荧光法5'端标记FAM荧光基团, 3'端标记BHQ1淬灭剂, 试纸条法两端无修饰。
1.4 基因组提取
将表1中2株沙门氏菌和13株非沙门氏菌分别接种到营养肉汤培养基中, 37 ℃培养24 h, 用细菌基因组提取试剂盒提取细菌基因组, 置于-20 ℃保存。对于灵敏度检测实验, 采用水煮法提取菌株基因组, 取1 mL菌液于1.5 mL离心管中, 8000 r/min离心5 min, 吸弃上清; 加入100 µL无菌水, 将细菌重悬于无菌水中, 100 ℃加热10 min, 12000 r/min离心5 min, 取上清保存于-20 ℃备用。
1.5 纳米金核酸试纸条的制备
纳米金粒子(gold nanoparticles, AuNPs)根据LI等[17]建立的方法制备, 利用冷冻法将DNA连接在AuNPs上。取100 µL AuNPs与20 µL AP探针(100 µmol/L)混合, -80 ℃冷冻15 min, 取出后室温解冻, 10000 g离心5 min, 弃上清, 加入45 µL AuNP-DNA复溶液复溶(20 nmol/L Na3PO4+5% BSA+0.25% Tween-20+10%蔗糖), 制备AuNP-AP。
试纸条由样品垫、结合垫、硝酸纤维素膜、吸水垫和PVC背衬5部分组成, 其中样品垫和结合垫根据张倩[18]建立的方法进行处理。将20 µL链霉亲和素(streptavidin, SA)分别与20 µL TCP探针(100 µmol/L)和20 µL CCP探针(100 µmol/L)混合并孵育1 h, 然后用划线器以1 µL/cm的速度分别将SA-TCP和SA-CCP混合物喷洒在硝酸纤维素膜上, 形成测试线TL和质控线CL。37 ℃烘箱中干燥1 h, 将样品垫、结合垫、硝酸纤维素膜和吸水垫按照顺序组装在和PVC背衬上, 使用可编程切条器将其切割成宽度为3 mm的成品备用。
1.6 RPA-CRISPR/Cas12a检测体系建立和优化
RPA扩增体系(50 µL): 将29.5 µL Twist Amp再水化液、11.2 µL无菌水、上下游引物(10 µmol/L)各2.4 µL加入至离心管中, 混合后加入到RPA酶冻干粉中, 充分溶解后加入2 µL样品 DNA, 再加入2.5 µL醋酸镁(280 mmol/L), 混匀后放入恒温反应器中, 39 ℃加热20 min。RPA反应结束后, 采用胶回收试剂盒回收扩增产物, 取5 µL进行电泳。
CRISPR-Cas12荧光检测体系(20 µL): 2 μL NEBufferr 2.1 (10×), 2 μL Cas12a (1 μmol/L), 2 μL crRNA (1 μmol/L), 2.5 μL RPA扩增产物, 0.8 μL荧光报告分子FQ-ssDNA (5 μmol/L), 用无菌水补足至20 μL, 将配制好的体系离心混匀, 转移到全自动病原微生物检测系统中, 反应温度37 ℃, 每隔1 min采集一次荧光, 测量实时荧光曲线。
CRISPR-Cas12a试纸条检测体系(100 μL): 2 μL NEBufferr 2.1 (10×), 2 μL Cas 12a (1 μmol/L), 2 μL crRNA (1 μmol/L), 2.5 μL RPA扩增产物, 2 μL试纸条报告分子FB-ssDNA (5 μmol/L), 用无菌水补足至20 μL, 将配制好的体系离心混匀放入便携式恒温反应器中, 于37 ℃孵育30 min。反应完毕后加入80 μL缓冲液(4×SSC+1% BSA+0.05% Tween 20), 混匀后把试纸条的样品端浸于溶液中, 其中2.5 μL AuNP-AP预先滴到试纸条的结合垫上, 观察控制条带和检测条带的强弱。
CRISPR-Cas12a荧光检测体系优化: 对Cas12a和crRNA的浓度比例进行优化, 保持体系中Cas12a的浓度为100 nmol/L, crRNA的浓度为50、100、150和200 nmol/L, 使其Cas12a:crRNA比例为1:0.5、1:1、1:2和1:3共计4个比例, 其他件不变, 以无菌水为阴性对照, 观察荧光强度变化情况。对Cas12a和crRNA的浓度进行优化, 设置CRISPR体系中Cas12a与crRNA的浓度分别为: ① 50 nmol/L和50 nmol/L; ② 100 nmol/L和100 nmol/L; ③ 150 nmol/L和150 nmol/L; ④ 200 nmol/L和200 nmol/L, 其他条件不变, 以无菌水为阴性对照, 观察荧光强度变化情况。
CRISPR-Cas12a试纸条检测体系优化: 针对试纸条报告分子浓度进行优化, 保持体系中Cas12a和crRNA的浓度为100 nmol/L, 加入5个试纸条报告分子浓度, 使其在100 μL检测体系(20 μL反应体系+80 μL缓冲液)中终浓度分别为50、100、150、200和300 nmol/L, 其他条件保持不变, 以无菌水为阴性对照, 观察不同条件下控制条带和检测条带的强弱。
1.7 RPA-CRISPR/Cas12a检测体系特异性
选取表1中的15株菌株进行RPA-CRISPR/Cas12a检测方法的特异性实验。将提取的基因组作为RPA模板, 扩增结束后后将RPA产物加入到优化的CRISPR/Cas12a检测体系中, 通过荧光和试纸条两种方法判读检测结果。
1.8 RPA-CRISPR/Cas12a检测体系灵敏度
将鼠伤寒沙门氏菌ATCC14028接种到营养肉汤培养基中, 37 ℃培养18 h, 对菌悬液进行10倍梯度稀释(稀释梯度10-1~10-6), 同时对各梯度进行平板计数。吸取1 mL各梯度菌液提取基因组DNA, 取2 μL DNA作为模板进行RPA-CRISPR/Cas12a检测, 确定方法的灵敏度, 通过荧光和试纸条两种方法判读检测结果。
1.9 实际样品检测
在市场随机购买21份市售样品为检测样本, 分别取25 g样品, 加入盛有225 mL BPW增菌液的无菌袋中, 36 ℃培养18 h, 取1 mL增菌液, 采用水煮法提取基因组DNA, 用本研究所建立的方法进行检测; 同时使用GB 4789.4—2024《食品安全国家标准 食品微生物学检验沙门氏菌检验》进行检测, 对2种方法的检测结果进行对比分析, 进一步验证所建立方法的准确性和实际应用效果。
1.10 数据处理
本研究的所有图像均采用SmartDraw 7进行处理, 采用Excel 2019进行表格制作。
2 结果与分析
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2.1 RPA引物与crRNA的筛选
设计的6对引物分别为RPA1-F/R、RPA2-F/R、RPA3-F/R、RPA4-F/R、RPA5-F/R和RPA6-F/R, 对应目标片段长度为196、220、195、200、229和201 bp。结果显示(图1), 6对引物都能扩增出与目的条带大小相符的DNA片段, 其中PRA5和RPA6引物的扩增产物单一且条带明亮, 扩增效率最高; RPA1、RPA3和RPA4引物的扩增产物存在非特异性扩增条带, RPA2扩增产物单一但条带亮度不够。根据RPA5和RPA6引物所在的序列设计crRNA1和crRNA2, 将RPA引物与crRNA搭配: RPA5-crRNA1和RPA6-crRNA2进行检测, 结果显示RPA5-crRNA1起峰时间和荧光曲线均优于RPA6-crRNA2(图2), 因此, 本研究选取RPA5-crRNA1作为后续实验。
2.2 CRISPR-Cas12a检测体系优化
2.2.1 Cas12a和crRNA的浓度比例优化
反应体系中Cas12a和crRNA的浓度比例对CRISPR-Cas12a荧光检测强度的实验结果(图3A)显示, Cas12a:crRNA=1:0.5下荧光强度较低, Cas12a:crRNA=1:1下荧光强度较高, 产生的荧光信号最为明显, 本研究选取Cas12a和crRNA的浓度比为1:1用于后续实验。
2.2.2 Cas12a和crRNA浓度优化
反应体系中Cas12a和crRNA浓度对荧光强度的实验结果(图3B)显示, 荧光信号随着Cas12a与crRNA浓度的增加而增强, 在Cas12a和crRNA分别为100 nmol/L时效果已非常明显, 能满足实验要求, 故以Cas12a和crRNA浓度分别为100 nmol/L和100 nmol/L进行后续实验。
2.2.3 试纸条报告分子浓度进行优化
试纸条检测是基于Cas12a反式切割活性切割体系中的FB-ssDNA, 当有沙门氏菌存在时, 其RPA产物会激活Cas12a的反式切割活性, 导致FB-ssDNA被切断, 从而使AuNP-AP不能通过FB-ssDNA连接到TL上, 即TL上不显红色。当不存在沙门氏菌时, 不能激活Cas12a的反式切割活性, 体系中的FB-ssDNA完整存在, 其3'端可以与AuNP-AP互补配对形成稳定的双链, 其5'端可以与TL上的TCP探针互补配对, 从而引导AuNP-AP探针结合到TL上, TL显红色。如果只出现CL线, 说明体系中的FB-ssDNA被完全切割, 判为存在目标产物; 如果同时出现CL线和TL线且样本TL线比对照组颜色浅, 说明FB-ssDNA部分被切割, 判为存在目标产物; 如果同时出现CL线和TL线且样本TL线跟对照组颜色强度相近, 说明FB-ssDNA未被切割, 判为不存在目标产物。
CRISPR-Cas12a试纸条报告分子浓度优化结果(图4)显示, 随着FB-ssDNA浓度的增加, 阴性对照和阳性组的TL线都逐渐变红, 其中浓度在150 nmol/L以上时阳性组不能完全切割, 浓度为50 nmol/L时试纸条TL线较淡, 容易出现假阳性结果, 最终选择浓度为100 nmol/L作为试纸条报告分子最优浓度。
2.3 RPA-Cas12a检测方法的特异性实验
荧光检测法和测流层析试纸条检测结果(图5)显示, 本研究建立的RPA-CRISPR/Cas12a检测方法能够检测出沙门氏菌, 荧光检测法结果(图5A)显示沙门氏菌出现明显的荧光信号, 其他菌株则无明显荧光信号。侧流层析试纸条检测结果(图5B)显示, 沙门氏菌只出现质控线, 未出现检测线, 其他菌株同时出现质控线和检测线, 且样品检测线跟阴性对照检测线颜色强度接近, 表现为阴性。结果表明本检测体系中的引物与其他病原核酸无交叉反应, 特异性强。
2.4 RPA-Cas12a检测方法的灵敏度实验
菌落计数测定菌株沙门氏菌菌悬液的初始浓度为8×106 CFU/mL, 10倍梯度稀释对应的各稀释度菌悬液浓度理论上分别为8×105、8×104、8×103、8×102、80和8 CFU/mL。用优化的RPA/CRISPR-Cas12a荧光法检测时, 梯度为80 CFU/mL能生可被检测的荧光信号, 8 CFU/mL无法检测到荧光信号(图6A), 表明RPA/CRISPR-Cas12a荧光法的灵敏度为80 CFU/mL。试纸条结果(图6B)显示随着菌量浓度的增加, TL红色逐渐变淡, 与阴性相比, 其相对颜色强度变化越来越明显, 当浓度为80 CFU/mL时与阴性对比TL条带有弱的检测带, 表明CRISPR-Cas12a试纸条的灵敏度为80 CFU/mL, 与RPA/CRISPR-Cas12a荧光法灵敏度一致。
2.5 样品检测
采用本研究中建立的荧光方法和试纸条法对21个样品进行检测, 结果表明荧光方法(图7)、试纸条法跟国家标准法检测结果一致, 检出1份阳性样品(编号7), 检出率为4.8%, 表明本研究中所建立的方法可用于食品的检测。
3 讨论
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沙门氏菌是导致食物微生物中毒的重要要因, 目前国内外标准规定25 g食品中不得含有沙门氏菌。基于核酸的检测方法因特异性强、灵敏度高而被广泛使用, 常用的靶标有invA[19]、fimY[20]、rfbS[21]、sefA[22]、viaB[23]、fliC[24]等, 其中研究最多基因是invA。RPA是一种等温扩增技术, 不需要复杂仪器即可完成扩增, 但会出现非特异性扩增问题, 该技术对温度波动较为敏感, 最佳反应温度在37~42 ℃。CHEN等[25]建立了基于RPA荧光探针的沙门氏菌检测方法, 菌液检测灵敏度为2.2×103 CFU/mL, 灵敏度有进一步提升空间。随着检测技术的不断革新, CRISPR/Cas系统由于具有极高的灵敏度与特异性, 在核酸检测领域逐渐显示出巨大优势, 并被誉为2022年最受瞩目的7项技术之一[26]。将RPA技术跟CRISPR/Cas相结合既可以避免RPA的非特异性扩增, 又可以提高检测的灵敏度, 在现场检测方面具有巨大的的潜力, 基于CRISPR-Cas快速检测技术将会是未来分子诊断领域重要研究方向之一。
本研究成功建立RPA-CRISPR/Cas12a可视化检测方法, 整个过程可在1.5 h内完成。通过筛选RPA引物和crRNA得到最佳的引物组合RPA5-crRNA1, 保证了检测方法的特异性和准确性。CRISPR/Cas12a检测体系中Cas12a和crRNA的浓度决定了同源重组解离酶C (recombination uv repair C, RuvC)活性剪切位点的数量和反式裂解的性能, 体系优化结果为Cas12a和crRNA浓度分别为100 nmol/L, 浓度比例为1:1, 基于荧光法检测的菌液灵敏度为80 CFU/mL。LIU等[27]建立基于FimY基因RPA-CRISPR/Cas12a荧光法检测沙门氏菌, Cas12和crRNA浓度分别为100 nmol/L, 菌液的检出限为102 CFU/mL。MAO等[28]将RPA与CRISPR-LbCas12a相结合建立一种RPA-LbCas12a- TTECDS荧光法检测沙门氏菌, 该方法可在1 h特异性检测出50 CFU/mL的沙门氏菌。HIGGINSON等[23]建立了基于荧光定量PCR的鼠伤寒沙门氏菌检测方法, 该方法特异性强, 灵敏度为7.4×102 CFU/mL。本研究基于荧光法检测特异性强、菌液灵敏度为80 CFU/mL, 较CHEN等[25]建立的RPA荧光方法灵敏度提升2个数量级, 证实了RPA与CRISPR/Cas结合可提高检测灵敏度。
侧流层析试纸条是一种纸基平台的生物传感器, 具有快速简单、特异性强、成本低等特点而被广泛用于食品、医疗、环境等领域。目前, 已有商业化的CRISPR/Cas专用试纸条用于检测领域, 主要是基于抗体技术, 但价格相对昂贵, 不利于控制成本[29-30]。本研究将核酸探针技术与纳米金层析技术相结合, 成功研制一种基于核酸探针的侧流层析试纸条, 并应用于核酸检测中。本研究采用建立的CRISPR/Cas12a试纸条方法特异性强, 该方法与其他致病菌无交叉反应。灵敏度为菌液灵敏度为80 CFU/mL。FU等[31]分别利用地高辛和生物素修饰肠炎沙门氏菌的特异性上下游引物, RPA扩增目标产物后利用试纸条检测, 该方法检出限为91.4 CFU/mL。SN/T5439.1—2022《出口食品中食源性致病菌快速检测方法PCR-试纸条法》第1部分: 沙门氏菌建立PCR-试纸条法快速检测沙门氏菌, 在上游和下游引物中分别标记地高辛和异硫氰酸, 扩增后跟试纸条上进行结合标准中规定的最低检出限为102 CFU/mL。本研究方法的最低检出限低于该标准, 具有更高的灵敏度, 达到标准检测水平, 可用于沙门氏菌的检测。
4 结论
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本研究成功制备基于核酸探针的侧流层析试纸条并建立沙门氏菌RPA-CRISPR/Cas12a可视化检测方法。该方法特异性强、灵敏度高、简单快速, 1.5 h内完成检测, 可利用便携式荧光检测仪或侧流层析试纸条读取结果, 不需要大型设备, 适用于基层检测机构, 有助于向现场检测发展, 具有广阔的应用前景。
基金
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  • 深圳市科创计划资助项目(KCXFZ20211020165404007)
文献
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2025年第16卷第8期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250110003
  • 接收时间：2025-01-10
  • 首发时间：2025-07-19
  • 出版时间：2025-04-25
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  • 收稿日期：2025-01-10
基金
深圳市科创计划资助项目(KCXFZ20211020165404007)
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    深圳市计量质量检测研究院, 深圳 518131

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* 陈晶(1976—), 女, 高级工程师, 主要研究方向为食品微生物检测。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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