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Article(id=1216517521096753282, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241118003, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1731859200000, receivedDateStr=2024-11-18, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1767969978832, onlineDateStr=2026-01-09, pubDate=1755187200000, pubDateStr=2025-08-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1767969978832, onlineIssueDateStr=2026-01-09, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1767969978832, creator=13701087609, updateTime=1767969978832, updator=13701087609, issue=Issue{id=1216517514570417012, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='15', pageStart='1', pageEnd='322', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1767969977276, creator=13701087609, updateTime=1768211590858, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217530915467743720, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217530915467743721, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=301, endPage=307, ext={EN=ArticleExt(id=1216517524108263693, articleId=1216517521096753282, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Visual rapid detection of Aspergillus niger in Zea mays L. by loop-mediated isothermal amplification, columnId=1151895321388347923, journalTitle=Journal of Food Safety & Quality, columnName=Food Analysis and Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a rapid visual detection method for Aspergillus niger in Zea mays L. by loop-mediated isothermal amplification (LAMP) technology. Methods Based on LAMP technology, specific primers targeting the key ochratoxin A (OTA) biosynthesis gene OTAhal were designed. Through screening of the primers and optimization of conditions such as primer sequence, primer ratio, reaction temperature and visual dye, the optimal detection conditions were determined. Using the total DNA of Aspergillus niger and artificially contaminated corn samples as templates, the sensitivity of the detection method and the feasibility of its practical application were validated. Results The optimal primer ratio was 8:4:1, the optimal reaction temperature was 64 °C, and the method could complete the amplification of target DNA within 30 minutes. The color of the positive sample changed from violet to blue-purple. This method could detect DNA concentrations as low as 6.87×10⁻3 ng/μL. When applied to actual Zea mays L. sample testing, it achieved a detection limit as low as 10¹ spores/mL. Conclusion This method is simple to operate, highly sensitive, and does not require sophisticated instruments. It can be applied to the detection of OTA in actual Zea mays L. samples, indicating broad application prospects.

, correspAuthors=Jia-Wen LEI, 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=Shao-Zhen CHA, Shuang-Yan YANG, Shu-Lin LU, Jia-Wen LEI), CN=ArticleExt(id=1216517532303934153, articleId=1216517521096753282, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=环介导等温扩增法可视化快速检测玉米中的黑曲霉, columnId=1151895321958773274, journalTitle=食品安全质量检测学报, columnName=食品分析与检测, runingTitle=null, highlight=null, articleAbstract=目的 建立环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)快速可视化检测玉米中黑曲霉(Aspergillus niger)的方法。方法 基于LAMP技术, 针对赭曲霉毒素A (ochratoxin A, OTA)产毒关键基因OTAhal设计特异性引物, 通过对引物序列、引物比例、反应温度、目视染料等条件的优化, 确定最佳检测条件; 以黑曲霉总DNA和人工污染玉米样品为模板, 验证方法的检测灵敏度和实际应用的可行性。结果 最佳引物比例为8:4:1, 最佳反应温度为64 ℃, 方法可在30 min内完成目标DNA扩增, 阳性样本的颜色由紫罗兰色转变为蓝紫色。该方法可检测低至6.87×10-3 ng/μL的DNA质量浓度, 应用于玉米实际样品测定时, 可检测低至101个/mL的孢子浓度。结论 该方法操作简单、灵敏度高、无需大型仪器, 能应用于玉米实际样品中OTA的检测, 具有广阔的应用前景。, correspAuthors=雷佳文, authorNote=null, correspAuthorsNote=
*雷佳文(1987—), 男, 博士, 讲师, 主要研究方向为食品安全快速检测技术。E-mail:
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茶绍桢(2001—), 女, 硕士研究生, 主要研究方向为食品安全快速检测技术。E-mail:

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茶绍桢(2001—), 女, 硕士研究生, 主要研究方向为食品安全快速检测技术。E-mail:

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keyword=环介导等温扩增), Keyword(id=1217127895210836950, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, orderNo=3, keyword=赭曲霉毒素), Keyword(id=1217127895282140119, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, orderNo=4, keyword=可视化), Keyword(id=1217127895445717985, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, orderNo=5, keyword=快速检测)], refs=[Reference(id=1217127899904262270, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, doi=null, pmid=null, pmcid=null, year=2008, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[1], rfOrder=0, authorNames=杨家玲, journalName=我国主要食品中赭曲霉毒素a调查与风险评估, refType=null, unstructuredReference=杨家玲. 我国主要食品中赭曲霉毒素a调查与风险评估[D]. 咸阳: 西北农林科技大学, 2008., articleTitle=null, refAbstract=null), Reference(id=1217127900009119876, 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Journal of Dairy Science and Technology, 2022, 45(1): 20-25., articleTitle=High-throughput detection of ochratoxin a-producing fungi in dairy products using gene probe scanning technology, refAbstract=null)], funds=[Fund(id=1217127899648409715, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, awardId=CZQ22014, language=CN, fundingSource=中南民族大学中央高校基本科研业务费项目(CZQ22014), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1217127891809256341, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, xref=null, ext=[AuthorCompanyExt(id=1217127891813450646, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, companyId=1217127891809256341, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=College of Life Sciences, South-Central Minzu University, Wuhan 430074, China), AuthorCompanyExt(id=1217127891821839255, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, companyId=1217127891809256341, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=中南民族大学生命科学学院, 武汉 430074)])], figs=[ArticleFig(id=1217127895668016105, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.1, caption=Gene bands of OTAhal (800 bp), figureFileSmall=fTtzLKXdTeLWZtHcfr5acQ==, figureFileBig=ZmR773WJ1uiSovvDOe3MeA==, tableContent=null), ArticleFig(id=1217127895785456623, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图1, caption=OTAhal基因条带(800 bp)

注: M. DNA Marke; 1. OTAhal基因条带。

, figureFileSmall=fTtzLKXdTeLWZtHcfr5acQ==, figureFileBig=ZmR773WJ1uiSovvDOe3MeA==, tableContent=null), ArticleFig(id=1217127895902897141, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.2, caption=LAMP response curves of 7 sets of primers, figureFileSmall=Ww8U1hT0ZlTo9nETNDzWmQ==, figureFileBig=8tspy1A58KzFEQ899cGUjg==, tableContent=null), ArticleFig(id=1217127896028726269, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图2, caption=7套引物LAMP反应曲线

注: a. 阴性对照组; b. 阳性对照组; 其中-代表阴性, +代表阳性。

, figureFileSmall=Ww8U1hT0ZlTo9nETNDzWmQ==, figureFileBig=8tspy1A58KzFEQ899cGUjg==, tableContent=null), ArticleFig(id=1217127896162942979, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.3, caption=Optimization of LAMP reaction system, figureFileSmall=JPaKxYIi9X9WtsxjIauYxQ==, figureFileBig=9G86CugDdQJaHO8ZCKfZcw==, tableContent=null), ArticleFig(id=1217127896255217673, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图3, caption=LAMP反应体系优化

注: a. 5种引物比例阴性对照, b. 5种引物比例阳性对照, c. 5种温度阴性反应曲线, d. 5种温度阳性反应曲线。

, figureFileSmall=JPaKxYIi9X9WtsxjIauYxQ==, figureFileBig=9G86CugDdQJaHO8ZCKfZcw==, tableContent=null), ArticleFig(id=1217127896343298061, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.4, caption=Limit of detection of OTAhal gene copy number, figureFileSmall=f0N/6mexyjlZnWUAP4HTtQ==, figureFileBig=P6eW/3r/T1684isTtdWEEw==, tableContent=null), ArticleFig(id=1217127896439767061, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图4, caption=OTAhal基因拷贝数检出限

注: a不同拷贝数LAMP反应曲线, b不同拷贝数Ct值线性方程。

, figureFileSmall=f0N/6mexyjlZnWUAP4HTtQ==, figureFileBig=P6eW/3r/T1684isTtdWEEw==, tableContent=null), ArticleFig(id=1217127897719029785, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.5, caption=Limit of detection of total DNA, figureFileSmall=Bi9fJj5uQG4Qycd0tmR2kw==, figureFileBig=CI8TvXn3jxCb+n+Kt3G1rw==, tableContent=null), ArticleFig(id=1217127897815498785, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图5, caption=总DNA检出限, figureFileSmall=Bi9fJj5uQG4Qycd0tmR2kw==, figureFileBig=CI8TvXn3jxCb+n+Kt3G1rw==, tableContent=null), ArticleFig(id=1217127897903579174, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.6, caption=Screening of LAMP visual dyes, figureFileSmall=S81F54hLNsNqI6wzzdYpmA==, figureFileBig=QSIdB6y0p3owCQzrCuaJFw==, tableContent=null), ArticleFig(id=1217127898029408304, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图6, caption=LAMP可视化染料筛选, figureFileSmall=S81F54hLNsNqI6wzzdYpmA==, figureFileBig=QSIdB6y0p3owCQzrCuaJFw==, tableContent=null), ArticleFig(id=1217127898130071605, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.7, caption=Sensitivity of the visual LAMP reaction, figureFileSmall=wLjIsz9Wy2KNo+Q1UBRrjg==, figureFileBig=FZjdfoSyW/ZPhi7PDX459Q==, tableContent=null), ArticleFig(id=1217127898213957688, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图7, caption=目视LAMP反应的灵敏度, figureFileSmall=wLjIsz9Wy2KNo+Q1UBRrjg==, figureFileBig=FZjdfoSyW/ZPhi7PDX459Q==, tableContent=null), ArticleFig(id=1217127898302038076, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.8, caption=Specificity of visual LAMP reaction, figureFileSmall=Ja5MQFr8T5Dox8PlOX/B+g==, figureFileBig=XUFB41muurgtG1/UqGeaDw==, tableContent=null), ArticleFig(id=1217127898423672897, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图8, caption=目视LAMP反应的特异性, figureFileSmall=Ja5MQFr8T5Dox8PlOX/B+g==, figureFileBig=XUFB41muurgtG1/UqGeaDw==, tableContent=null), ArticleFig(id=1217127898541113414, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Fig.9, caption=Gradient spore contaminated Zea mays L. samples with visual LAMP color variation, figureFileSmall=Py+On4uxEBK3Qa7aRyrZbg==, figureFileBig=h6hnK9UszQVi8P+20c0XAQ==, tableContent=null), ArticleFig(id=1217127898675331148, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=图9, caption=梯度孢子污染的玉米样品目视LAMP颜色变化, figureFileSmall=Py+On4uxEBK3Qa7aRyrZbg==, figureFileBig=h6hnK9UszQVi8P+20c0XAQ==, tableContent=null), ArticleFig(id=1217127898830520402, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Table 1, caption=

qPCR reaction system

, figureFileSmall=null, figureFileBig=null, tableContent=
组分 体积/μL
2×Q3 SYBR qPCR 预混液 10.0
hal-qF 0.4
hal-qR 0.4
ddH2O 8.8
DNA 1
), ArticleFig(id=1217127898943766615, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=表1, caption=

qPCR反应体系

, figureFileSmall=null, figureFileBig=null, tableContent=
组分 体积/μL
2×Q3 SYBR qPCR 预混液 10.0
hal-qF 0.4
hal-qR 0.4
ddH2O 8.8
DNA 1
), ArticleFig(id=1217127899073790043, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Table 2, caption=

Primer name and sequence of Aspergillus.niger CBS513.88 LAMP

, figureFileSmall=null, figureFileBig=null, tableContent=
引物 名称 序列(5'-3')
PM1 F3 TCCGAGCACCACGAGAAG
B3 CCGTACCTCCGCATTGTC
FIP TATCCGGGGTGACTGTCCCGAAACGTCCGTATCTGTTCGC
BIP GGCAATGGATACCGCCGCAGAGCCTTTATCGACCCGTACT
LF CTGGCAGTGGCATTCCCAA
LB CTGCCAAATGCACGCCAGAT
), ArticleFig(id=1217127899174453342, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=表2, caption=

Aspergillus.niger CBS513.88 LAMP引物名称及序列

, figureFileSmall=null, figureFileBig=null, tableContent=
引物 名称 序列(5'-3')
PM1 F3 TCCGAGCACCACGAGAAG
B3 CCGTACCTCCGCATTGTC
FIP TATCCGGGGTGACTGTCCCGAAACGTCCGTATCTGTTCGC
BIP GGCAATGGATACCGCCGCAGAGCCTTTATCGACCCGTACT
LF CTGGCAGTGGCATTCCCAA
LB CTGCCAAATGCACGCCAGAT
), ArticleFig(id=1217127899317059684, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=EN, label=Table 3, caption=

Comparative results of visual LAMP and qPCR methods for actual sample detection

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 孢子浓度/(个/mL)
100 101 102 103 104 105
qPCR - - + + + +
目视LAMP - + + + + +
), ArticleFig(id=1217127899447083112, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517521096753282, language=CN, label=表3, caption=

实际样品检测目视LAMP与qPCR方法对比结果

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 孢子浓度/(个/mL)
100 101 102 103 104 105
qPCR - - + + + +
目视LAMP - + + + + +
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环介导等温扩增法可视化快速检测玉米中的黑曲霉
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茶绍桢 , 杨双艳 , 陆淑林 , 雷佳文 *
食品安全质量检测学报 | 食品分析与检测 2025,16(15): 301-307
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食品安全质量检测学报 | 食品分析与检测 2025, 16(15): 301-307
环介导等温扩增法可视化快速检测玉米中的黑曲霉
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茶绍桢 , 杨双艳, 陆淑林, 雷佳文*
作者信息
  • 中南民族大学生命科学学院, 武汉 430074

    • 茶绍桢(2001—), 女, 硕士研究生, 主要研究方向为食品安全快速检测技术。E-mail:

通讯作者:

*雷佳文(1987—), 男, 博士, 讲师, 主要研究方向为食品安全快速检测技术。E-mail:
Visual rapid detection of Aspergillus niger in Zea mays L. by loop-mediated isothermal amplification
Shao-Zhen CHA , Shuang-Yan YANG, Shu-Lin LU, Jia-Wen LEI*
Affiliations
  • College of Life Sciences, South-Central Minzu University, Wuhan 430074, China
出版时间: 2025-08-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241118003
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摘要
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目的 建立环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)快速可视化检测玉米中黑曲霉(Aspergillus niger)的方法。方法 基于LAMP技术, 针对赭曲霉毒素A (ochratoxin A, OTA)产毒关键基因OTAhal设计特异性引物, 通过对引物序列、引物比例、反应温度、目视染料等条件的优化, 确定最佳检测条件; 以黑曲霉总DNA和人工污染玉米样品为模板, 验证方法的检测灵敏度和实际应用的可行性。结果 最佳引物比例为8:4:1, 最佳反应温度为64 ℃, 方法可在30 min内完成目标DNA扩增, 阳性样本的颜色由紫罗兰色转变为蓝紫色。该方法可检测低至6.87×10-3 ng/μL的DNA质量浓度, 应用于玉米实际样品测定时, 可检测低至101个/mL的孢子浓度。结论 该方法操作简单、灵敏度高、无需大型仪器, 能应用于玉米实际样品中OTA的检测, 具有广阔的应用前景。
黑曲霉  /  环介导等温扩增  /  赭曲霉毒素  /  可视化  /  快速检测

Objective To establish a rapid visual detection method for Aspergillus niger in Zea mays L. by loop-mediated isothermal amplification (LAMP) technology. Methods Based on LAMP technology, specific primers targeting the key ochratoxin A (OTA) biosynthesis gene OTAhal were designed. Through screening of the primers and optimization of conditions such as primer sequence, primer ratio, reaction temperature and visual dye, the optimal detection conditions were determined. Using the total DNA of Aspergillus niger and artificially contaminated corn samples as templates, the sensitivity of the detection method and the feasibility of its practical application were validated. Results The optimal primer ratio was 8:4:1, the optimal reaction temperature was 64 °C, and the method could complete the amplification of target DNA within 30 minutes. The color of the positive sample changed from violet to blue-purple. This method could detect DNA concentrations as low as 6.87×10⁻3 ng/μL. When applied to actual Zea mays L. sample testing, it achieved a detection limit as low as 10¹ spores/mL. Conclusion This method is simple to operate, highly sensitive, and does not require sophisticated instruments. It can be applied to the detection of OTA in actual Zea mays L. samples, indicating broad application prospects.

Aspergillus niger  /  loop-mediated isothermal amplification  /  ochratoxin  /  visualization  /  rapid detection
茶绍桢, 杨双艳, 陆淑林, 雷佳文. 环介导等温扩增法可视化快速检测玉米中的黑曲霉. 食品安全质量检测学报, 2025 , 16 (15) : 301 -307 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241118003
Shao-Zhen CHA, Shuang-Yan YANG, Shu-Lin LU, Jia-Wen LEI. Visual rapid detection of Aspergillus niger in Zea mays L. by loop-mediated isothermal amplification[J]. Journal of Food Safety & Quality, 2025 , 16 (15) : 301 -307 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241118003
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0 引言
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赭曲霉毒素A (ochratoxin A, OTA)广泛存在于小麦、玉米、大麦、豆类、坚果、葡萄、香料和咖啡等食品中, 人类通过进食被OTA污染的农作物而受到危害[1-2], 国际食品法典委员会(Codex Alimentarius Commission, CAC)指出, 谷物是每日摄入的OTA的最重要来源[3-5]。OTA对多种动物具有遗传毒性、致畸性、肝毒性、免疫毒性和致癌性[6-8]。世界卫生组织食品添加剂联合专家委员会(Joint FAO/WHO Expert Committee on Food Additives, JECFA)在2001年将该毒素列为人类可能致癌物(2B)[9-10]。根据以往的研究, 黑曲霉(Aspergillus niger)是中国地区玉米、小麦和水稻等谷物中分离到的最常见的真菌之一, 并被认为是中国谷物中最重要的OTA产毒真菌[11]。
传统产毒真菌的检测方法主要依赖平板计数和生化培养法[12-13], 虽然这些方法在食品安全监管中发挥了重要作用, 但存在检测周期长、操作烦琐、成本高等缺点, 难以满足现代食品安全快速检测的需求。近年来, 分子生物学技术的快速发展为产毒真菌的检测提供了新的思路。聚合酶链式反应(polymerase chain reaction, PCR)、实时荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction, qPCR)等技术通过扩增目标基因, 实现了对产毒真菌的高灵敏度、高特异性检测[14-17]。其中, 环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术因其快速、简便、灵敏度高等特点, 在食品安全快速检测领域备受关注[18-19]。LAMP技术利用6种特异性引物, 在恒温条件下(60~65 ℃)可实现DNA的指数级扩增, 通过肉眼观察即可获得检测结果[20-24]。相较于传统的PCR技术, LAMP技术无需复杂的仪器设备, 操作简便, 更适合现场检测[25-30]。与直接检测OTA相比, 通过检测产毒菌, 可以提前预警食品污染风险, 及时采取措施防止OTA的产生和积累, 从而有效降低食品中OTA的含量, 保障食品安全。此外, 检测产毒菌还可以用于原料筛选、生产过程监控等环节, 为食品安全管理提供科学依据。
目前已报道的产毒霉菌检测方法, 多针对真菌通用基因进行检测[31], 虽能实现菌种鉴定, 却无法精准区分产毒菌株与非产毒菌株。部分方法依赖浊度判读或荧光染料进行终点检测[31-32], 虽实现可视化但需借助荧光读数仪辅助判读, 难以满足现场快速筛查需求。本研究基于LAMP技术, 针对OTA产毒关键基因OTAhal设计特异性引物, 用于检测玉米中的黑曲霉, 可以在OTA污染的早期进行防控和预警, 对保障粮食安全具有重要意义。
1 材料与方法
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1.1 材料与试剂
黑曲霉CBS513.88(广东省微生物菌种保藏中心); 禾谷镰孢(Fusarium graminearum)、绿色木霉(Trichoderma viride)、黄绿青霉(Penicillium citreo-virde)、棒曲霉(Aspergillus clavatus)(中国工业微生物菌种保藏管理中心)。
2×Tolo Super LAMP预混液、50×LAMP荧光染料、2×Q3 SYBR qPCR预混液(上海吐露港生物科技有限公司); 苯酚红、中性红(分析纯, 上海麦克林生化科技有限公司); 钙黄绿素(分析纯, 国药集团化学试剂有限公司); 铬黑T (eriochrome black T, EBT)(分析纯, 天津希恩思生化科技有限公司); 羟基萘酚蓝(hydroxynaphtholblue, HNB)(纯度80%, 北京索莱宝科技有限公司); 琼脂糖凝胶DNA回收试剂盒[B511139, 生工生物工程(上海)股份有限公司]; 马铃薯葡萄糖琼脂培养基(potato dextrose agar, PDA)(青岛海博生物技术有限公司)。
1.2 仪器与设备
T100 PCR仪、CFX Opus 96荧光定量PCR仪[伯乐生命医学产品(上海)有限公司]; DB1恒温金属浴(群安实验仪器有限公司); H1850R高速冷冻离心机(湖南湘仪实验室仪器开发有限公司); HX-21G快速组织细胞破碎仪(天津欧诺仪器股份有限公司); THZ-98A恒温培养箱(上海恒一精密仪器有限公司); Nano Drop one超微量紫外分光光度计(美国赛默飞世尔科技公司)。
1.3 实验方法
1.3.1 引物的设计与合成
几种产OTA的真菌其OTA产量均被证明与各自卤化酶基因OTAhal的表达量有相关性, OTAhal被证实存在多种产OTA的真菌中[33], 是产OTA菌株的关键基因。CBS513.88 OTAhal基因的GenBank登录号为MG701892.1, 选取800 bp大小的序列, 在LAMP引物在线设计平台(http://primerexplorer.jp/lampv5e/index.html)进行引物设计, 每套LAMP引物包括两个外引物(F3, B3)、两个内引物(FIP, BIP)以及两个环引物(LF, LB)。一共设计7套LAMP引物(PM1~PM7), 一组PCR引物(hal-F: GAGTACCTCGCGGG CAGACA, hal-R: TTGAAGAATGTCGCCAGCTGG), 一组qPCR引物(hal-qF: TGTCACATGCTCTGTAGGATTGG, hal-qR: CCCTACCTGATCCGAGGTCAA), 委托生工生物工程(上海)股份有限公司进行引物合成。
1.3.2 真菌的培养和基因组DNA提取
将活化的冻干菌粉接种于活化培养, 在菌丝生长旺盛时刮取新鲜菌丝或者在产孢时洗脱孢子用以提取DNA; 称取约100 g菌丝体加入600 μL溴化十六烷基三甲铵(cetyltrimethylammonium bromide, CTAB)裂解液和6 μL β-巯基乙醇, 用研磨仪研磨后充分振荡1 h, 然后水浴1 h, 加入600 μL苯酚:氯仿:异戊醇(25:24:1, V:V:V)剧烈振荡混匀, 4 ℃下12000 r/min离心10 min, 取上清液转移至新的1.5 mL离心管中, 重复此步骤5次; 向上清液中加入等体积预冷的异丙醇, 置于4 ℃冰箱中过夜, 然后在4 ℃下12000 r/min离心10 min, 弃去上清; 用70%乙醇吹洗沉淀2次, 待残留乙醇挥发后加入30 μL纯水, 得到基因组总DNA, 再通过琼脂糖凝胶DNA回收试剂盒对OTAhal基因片段进行回收, 超微量紫外分光光度计测定DNA浓度后于-20 ℃保存。
1.3.3 LAMP引物筛选、引物比例和反应温度优化
常规LAMP和目视检测体系反应总体积均为25 μL, 按照TOLOBIO反应体系: 2×Tolo Super LAMP 预混液 12.5 μL, 10×LAMP Primers 2.5 μL, 50×LAMP 荧光染料 0.5 μL, Target DNA 1 μL, ddH2O补足至25 μL。为了防止气溶胶污染, 在加样完成后另外添加25 μL矿物油对反应体系进行油封, 封盖后于荧光定量PCR仪上设定30 s为1个循环采集荧光信号, 反应结束后90 ℃终止反应。引物浓度比例(FIP/BIP: LF/LB: F3/B3)按8:1:1、8:4:1、8:6:1、8:8:1进行优化, 在61、62、63、64、65 ℃条件下进行温度优化。
1.3.4 LAMP灵敏度实验
确定最佳引物和最适温度后, 将提取的总DNA(质量浓度为6.87×102 ng/μL)用超纯水(ddH2O)连续10倍梯度稀释至6.87×10-7 ng/μL, 将切胶回收的OTAhal基因片段(浓度为1.37×1010 copies/μL)同样梯度稀释至1.37×104 copies/μL, 分别通过实时荧光LAMP测定灵敏度。
1.3.5 目视LAMP染料筛选
分别向LAMP反应体系中加入钙黄绿素、苯酚红、EBT、中性红、HNB作为目视指示剂, 通过LAMP进行可视化检测分析。以30 min为反应终止时间, 通过反应终点与起点LAMP体系颜色的变化, 筛选最适合的LAMP目视染料。
1.3.6 目视LAMP灵敏度实验
将1.3.5中筛选到的目视染料加入到LAMP体系中, 对梯度稀释的总DNA(原液为6.87×102 ng/μL, ddH2O 10倍梯度稀释至6.87×10-4 ng/μL)进行可视化LAMP检测, 测定目视LAMP灵敏度。
1.3.7 目视LAMP特异性实验
选取玉米和小麦中常见的4种真菌: 禾谷镰孢、绿色木霉、黄绿青霉、棒曲霉, 培养后提取DNA为模板, 同时设置CBS513.88总DNA为阳性对照, ddH2O为阴性对照, 按目视LAMP方法进行扩增反应, 对方法的特异性进行验证。
1.3.8 实际样品检测及方法学对比
精确称取0.2 g空白玉米粉末, 将其转移至无核酸酶的2.0 mL离心管中。将获得的孢子溶液10倍梯度稀释(由105个/mL稀释至100个/mL), 接种200 μL至玉米样品中, 在28 ℃条件下孵育2 h后, 用CTAB法提取总DNA作为检测模板, 同时提取未被污染的玉米DNA作为阴性对照, 用本研究中建立的目视LAMP方法及常规qPCR方法进行检测和对比, qPCR反应体系见表1。
1.4 数据处理
每组实验均设置3个重复, 目视LAMP图片由Canon EOS R6拍摄, 为保证图片效果一致, 所有拍摄参数均固定为: 焦距35 mm, 光圈f/4, 曝光时间1/250 s, 感光度100, 数据分析及绘图由Origin 2024软件进行。
2 结果与分析
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2.1 OTAhal基因片段PCR扩增结果
由图1可知PCR产物达到预期效果, 扩增段为800 bp大小, 未出现非特异性扩增, 说明PCR引物设计合理, 可以通过切胶回收OTAhal基因片段用于下一步实验。
2.2 LAMP引物筛选
根据实时荧光LAMP结果(图2), 引物PM1在4个循环内即发生扩增, 且阴性对照在120个循环内无扩增, 对比其他几套引物, PM1不仅在扩增起始阶段更为迅速, 且没有发生非特异性扩增, 综上, 选取PM1作为后续实验的最佳引物, 引物相关序列见表2。
2.3 LAMP反应体系优化
由图3A~b可以看出, 引物浓度比例(FIP/BIP: LF/LB: F3/B3)为8:4:1时, 荧光信号出现时间最早, 且阴性在120个循环内无扩增, 故选取最佳引物比例为8:4:1。由图3C~d可知, 反应温度为64 ℃时, 相对荧光值峰值明显高于其他温度, 且阴性对照无非特异性扩增, 故选择64 ℃为最佳反应温度。
2.4 LAMP灵敏度实验
将OTAhal基因拷贝数进行梯度稀释后, 如图4A所示, 检出限为1.37×104 copies/μL, 以拷贝数和Ct值做标准曲线, 如图4b所示, r2≥0.98, 拷贝数和Ct值存在良好线性关系。将总DNA梯度稀释后进行LAMP反应, 如图5所示, 最低检出限为6.87×10-4 ng/μL。
2.5 目视LAMP染料筛选
如图6所示, 5种染料在反应30 min后均发生了较为明显的颜色变化。钙黄绿素反应前为无色, 反应后为黄绿色荧光; 苯酚红反应前为深粉色, 反应后为浅粉色; EBT反应前为紫罗兰色, 反应后为蓝紫色; 中性红反应前为黄色, 反应后为橙红色; HNB反应前为浅蓝色, 反应后为天蓝色。其中钙黄绿素荧光需要在黑暗环境下, 由紫外灯照射产生, 不适用于实验室条件外的实际检测。其余4种染料中, 终点颜色变化最为明显, 且最容易被肉眼区分的为EBT, 因此在后续所有LAMP反应中, 选择EBT为最佳目视染料。
2.6 目视LAMP灵敏度
如图7所示, 目视LAMP总DNA检出限为6.87×10-3 ng/μL, 在之后的稀释度下没有明显颜色变化, 与实时荧光LAMP结果基本相符, 但目视LAMP灵敏度要略低于实时荧光LAMP (6.87×10-4 ng/μL)。
2.7 目视LAMP特异性
实验结果表明(图8), 只有黑曲霉CBS513.88发生了明显颜色变化(由紫罗兰色变为蓝紫色), 其他4株菌株均与阴性颜色保持一致(紫罗兰色), 表明本研究建立的黑曲霉可视化LAMP检测方法具有良好的特异性, 不与其他4种真菌发生交叉反应。
2.8 实际样品检测及方法学对比
由图9可以看出, 孢子浓度越高, LAMP反应体系由紫罗兰色转变为天蓝色的时间越快, 以30 min为反应终止时间, 当孢子浓度为100个/mL时, 反应体系颜色与阴性保持一致(紫罗兰色), 本方法能检测到的最低孢子浓度为101个/mL。表3为qPCR与本研究建立的目视LAMP方法对比, 可以看出, qPCR最低能检出102个/mL的孢子浓度, 目视LAMP的实际样品检测灵敏度比普通qPCR法要高出一个数量级。
3 结论
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黑曲霉是中国地区谷物中重要的OTA产毒菌之一, 本研究通过在OTA产毒关键基因OTAhal区域设计特异性引物, 结合LAMP技术, 实现了黑曲霉的可视化快速检测。相比传统方法, 本研究无需昂贵设备, 实验周期短, 通过颜色变化即可直接判断结果, 显著提高了检测效率和便捷性。本研究不仅能提前预警OTA污染, 还有助于及时防控, 减少经济损失, 对于保障粮食安全、控制食品中OTA含量具有重要意义。
基金
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  • 中南民族大学中央高校基本科研业务费项目(CZQ22014)
文献
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241118003
  • 接收时间：2024-11-18
  • 首发时间：2026-01-09
  • 出版时间：2025-08-15
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  • 收稿日期：2024-11-18
基金
中南民族大学中央高校基本科研业务费项目(CZQ22014)
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    中南民族大学生命科学学院, 武汉 430074

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*雷佳文(1987—), 男, 博士, 讲师, 主要研究方向为食品安全快速检测技术。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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