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Article(id=1217529315802137513, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250110007, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1736438400000, receivedDateStr=2025-01-10, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768211209488, onlineDateStr=2026-01-12, pubDate=1752508800000, pubDateStr=2025-07-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768211209488, onlineIssueDateStr=2026-01-12, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768211209488, creator=13701087609, updateTime=1768211209488, updator=13701087609, issue=Issue{id=1217529305693864468, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='13', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768211207077, creator=13701087609, updateTime=1768212057891, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217532874337730593, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217532874337730594, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=214, endPage=219, ext={EN=ArticleExt(id=1217529316192207816, articleId=1217529315802137513, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Rapid detection of viable Pediococcus pentosaceus in rose jam by propidium monoazide-quantitative real-time polymerase chain reaction method, columnId=1151895321388347923, journalTitle=Journal of Food Safety & Quality, columnName=Food Analysis and Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a rapid detection method of viable Pediococcus pentosaceus in rose jam based on the combination of propidium monoazide (PMA) and quantitative real-time polymerase chain reaction (qPCR). Methods Primers specific for Pediococcus pentosaceus were designed according to the species-specific murI gene. The optimal PMA treatment conditions were determined by optimizing the PMA concentration, dark incubation time, and exposure time. Different ratios of Pediococcus pentosaceus dead/live bacterial mixtures were validated by the PMA-qPCR method to assess the sensitivity and limit of detection of the technique. Results The designed primers exhibited high specificity, and the optimal PMA treatment conditions were determined as 80 µg/mL PMA, 15 min of dark incubation, and 25 min of exposure. Under this condition, a standard curve with correlation coefficient (r2) of 0.9983 was constructed, and the lowest limit of detection was determined to be 3.98×103 CFU/g. Conclusion The PMA-qPCR method established in this study enables the rapid and accurate quantification of viable Pediococcus pentosaceus in rose jam with high sensitivity, providing technical support for the quantitative detection of microorganisms in complex food matrices.

, correspAuthors=Yun-Guo LIU, 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=Yan-Zhen ZHANG, Wei-Jie YUAN, Chao MA, Li-Dan MA, Yun-Guo LIU), CN=ArticleExt(id=1217529319111442564, articleId=1217529315802137513, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=叠氮溴化丙锭结合实时定量聚合酶链式反应法快速检测玫瑰酱中戊糖片球菌活菌, 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 探究基于叠氮溴化丙锭(propidium monoazide, PMA)与实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)技术联用, 建立一种快速检测玫瑰酱中戊糖片球菌活菌的方法。方法 根据特异性基因murI设计戊糖片球菌特异性引物, 通过优化PMA浓度、暗孵育时间和曝光时间, 确定最佳PMA处理条件, 并通过PMA-qPCR方法验证不同比例的戊糖片球菌死菌/活菌混合菌液, 评估该方法的灵敏度和检出限。结果 设计的引物具有良好的特异性, PMA的最佳处理条件为质量浓度80 µg/mL, 暗孵育时间为15 min, 曝光时间为25 min, 此条件下, 构建的标准曲线相关系数(r2)为0.9983, 最低检出限为3.98×103 CFU/g。结论 本研究建立的PMA-qPCR方法可有效实现玫瑰酱中戊糖片球菌活菌数的快速检测, 灵敏度高, 为复杂食品基质中微生物的定量检测提供了技术支持。

, correspAuthors=刘云国, authorNote=null, correspAuthorsNote=
*刘云国(1977—), 男, 博士, 教授, 主要研究方向为食品微生物。E-mail:
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张艳珍(1996—), 女, 博士研究生, 主要研究方向为食品微生物。E-mail:

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张艳珍(1996—), 女, 博士研究生, 主要研究方向为食品微生物。E-mail:

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Journal of Food Safety & Quality, 2021, 12(12): 4867-4875., articleTitle=Quantitative detection of Lactobacillus plantarum by propidium monoazide-qPCR, refAbstract=null)], funds=[Fund(id=1217901255649841790, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, awardId=YDZX2023081, language=CN, fundingSource=中央引导地方科技发展资金项目(YDZX2023081), fundOrder=null, country=null), Fund(id=1217901255779865224, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, awardId=2024HK014, language=CN, fundingSource=海关总署科研项目(2024HK014), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1217901247496114340, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, xref=1, ext=[AuthorCompanyExt(id=1217901247504502949, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, companyId=1217901247496114340, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 College of Life Sciences, Linyi University, Linyi 276005, China), AuthorCompanyExt(id=1217901247512891559, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, companyId=1217901247496114340, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 临沂大学生命科学学院, 临沂 276005)]), AuthorCompany(id=1217901247621943470, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, xref=2, ext=[AuthorCompanyExt(id=1217901247651303602, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, companyId=1217901247621943470, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 College of Engineering, University of Suwon, Suwon 18323, Korea), AuthorCompanyExt(id=1217901247663886516, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, companyId=1217901247621943470, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 水原大学工程学院, 水原 18323)]), AuthorCompany(id=1217901247785521340, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, xref=3, ext=[AuthorCompanyExt(id=1217901247798104254, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, companyId=1217901247785521340, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3 Gongbei Customs Technical Center, Zhuhai 519075, China), AuthorCompanyExt(id=1217901247810687169, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, companyId=1217901247785521340, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3 拱北海关技术中心, 珠海 519075)])], figs=[ArticleFig(id=1217901252386673090, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=EN, label=Fig.1, caption=Primer specificity verification for Pediococcus pentosaceus, figureFileSmall=7DuM1Ui3dw+OdiYqXrYa9Q==, figureFileBig=GIi1jCm+0h5yJIr0h+uzmQ==, tableContent=null), ArticleFig(id=1217901252512502221, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=CN, label=图1, caption=戊糖片球菌引物特异性验证

注: 红色曲线为戊糖片球菌, 其他曲线为参考菌株。

, figureFileSmall=7DuM1Ui3dw+OdiYqXrYa9Q==, figureFileBig=GIi1jCm+0h5yJIr0h+uzmQ==, tableContent=null), ArticleFig(id=1217901253867262429, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=EN, label=Fig.2, caption=Effects of different mass concentrations of PMA treatment on the qPCR amplification of heat-killed and live Pediococcus pentosaceus, figureFileSmall=/uInSZJOPKyASViIlGFCDQ==, figureFileBig=zFuhnM1lu5rhHGdotnBcRQ==, tableContent=null), ArticleFig(id=1217901253988897256, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=CN, label=图2, caption=不同质量浓度PMA处理对戊糖片球菌死菌活菌qPCR扩增的影响

注: 不同字母表示同种物质组间具有显著性差异(P<0.05), 图3~5同。

, figureFileSmall=/uInSZJOPKyASViIlGFCDQ==, figureFileBig=zFuhnM1lu5rhHGdotnBcRQ==, tableContent=null), ArticleFig(id=1217901254106337783, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=EN, label=Fig.3, caption=Effects of different dark incubation time treatments on the qPCR amplification of heat-killed and live Pediococcus pentosaceus, figureFileSmall=/GRz5yRAa5DICDGFKuPkww==, figureFileBig=ThyEr/QuQhBFxC7BUSsq0Q==, tableContent=null), ArticleFig(id=1217901254253138432, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=CN, label=图3, caption=不同暗孵育时间处理对戊糖片球菌死菌活菌qPCR扩增的影响, figureFileSmall=/GRz5yRAa5DICDGFKuPkww==, figureFileBig=ThyEr/QuQhBFxC7BUSsq0Q==, tableContent=null), ArticleFig(id=1217901254408327687, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=EN, label=Fig.4, caption=Effects of different exposure incubation time treatments on the qPCR amplification of heat-killed and live Pediococcus pentosaceus, figureFileSmall=pE8kGLqZ2cV7zGtQpHS+8Q==, figureFileBig=3mNA6XVR7sSNdwQyk91LVw==, tableContent=null), ArticleFig(id=1217901254542545429, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=CN, label=图4, caption=不同曝光时间处理对戊糖片球菌死菌/活菌qPCR扩增的影响, figureFileSmall=pE8kGLqZ2cV7zGtQpHS+8Q==, figureFileBig=3mNA6XVR7sSNdwQyk91LVw==, tableContent=null), ArticleFig(id=1217901254672568867, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=EN, label=Fig.5, caption=Effects of PMA treatment on the samples with different ratios of heat-killed and live bacteria, figureFileSmall=fAiLH5qznNu7OWgE6A2XkQ==, figureFileBig=DPDq6ucRW1/pWuV8VmSGWg==, tableContent=null), ArticleFig(id=1217901254802592305, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=CN, label=图5, caption=PMA处理对不同死/活菌比例样品的影响, figureFileSmall=fAiLH5qznNu7OWgE6A2XkQ==, figureFileBig=DPDq6ucRW1/pWuV8VmSGWg==, tableContent=null), ArticleFig(id=1217901254995530306, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=EN, label=Fig.6, caption=qPCR amplification map of Pediococcus pentosaceus in rose jam samples, figureFileSmall=G9sL4zZmCPWNCzw23oFNYg==, figureFileBig=l0BQtzTcnm15m1pgV1LfPw==, tableContent=null), ArticleFig(id=1217901255163302480, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=CN, label=图6, caption=玫瑰酱样品中戊糖片球菌的qPCR扩增图谱, figureFileSmall=G9sL4zZmCPWNCzw23oFNYg==, figureFileBig=l0BQtzTcnm15m1pgV1LfPw==, tableContent=null), ArticleFig(id=1217901255310103136, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=EN, label=Table 1, caption=

Primers and probes for Pediococcus pentosaceus

, figureFileSmall=null, figureFileBig=null, tableContent=
引物与探针 序列(5’-3’)
上游引物 ATTGCTACACTAGGCACA
下游引物 GAACGGTCTCCACTCC
TaqMan探针 HEX-ACGGCTGCTTGGATACCTTCT-TAMRA
), ArticleFig(id=1217901255427543659, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529315802137513, language=CN, label=表1, caption=

戊糖片球菌引物与探针

, figureFileSmall=null, figureFileBig=null, tableContent=
引物与探针 序列(5’-3’)
上游引物 ATTGCTACACTAGGCACA
下游引物 GAACGGTCTCCACTCC
TaqMan探针 HEX-ACGGCTGCTTGGATACCTTCT-TAMRA
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叠氮溴化丙锭结合实时定量聚合酶链式反应法快速检测玫瑰酱中戊糖片球菌活菌
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张艳珍 1, 2 , 袁伟杰 1 , 马超 1 , 麻丽丹 3 , 刘云国 1, *
食品安全质量检测学报 | 食品分析与检测 2025,16(13): 214-219
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食品安全质量检测学报 | 食品分析与检测 2025, 16(13): 214-219
叠氮溴化丙锭结合实时定量聚合酶链式反应法快速检测玫瑰酱中戊糖片球菌活菌
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张艳珍1, 2 , 袁伟杰1, 马超1, 麻丽丹3, 刘云国1, *
作者信息
  • 1 临沂大学生命科学学院, 临沂 276005
  • 2 水原大学工程学院, 水原 18323
  • 3 拱北海关技术中心, 珠海 519075

    • 张艳珍(1996—), 女, 博士研究生, 主要研究方向为食品微生物。E-mail:

通讯作者:

*刘云国(1977—), 男, 博士, 教授, 主要研究方向为食品微生物。E-mail:
Rapid detection of viable Pediococcus pentosaceus in rose jam by propidium monoazide-quantitative real-time polymerase chain reaction method
Yan-Zhen ZHANG1, 2 , Wei-Jie YUAN1, Chao MA1, Li-Dan MA3, Yun-Guo LIU1, *
Affiliations
  • 1 College of Life Sciences, Linyi University, Linyi 276005, China
  • 2 College of Engineering, University of Suwon, Suwon 18323, Korea
  • 3 Gongbei Customs Technical Center, Zhuhai 519075, China
出版时间: 2025-07-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250110007
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摘要
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目的 探究基于叠氮溴化丙锭(propidium monoazide, PMA)与实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)技术联用, 建立一种快速检测玫瑰酱中戊糖片球菌活菌的方法。方法 根据特异性基因murI设计戊糖片球菌特异性引物, 通过优化PMA浓度、暗孵育时间和曝光时间, 确定最佳PMA处理条件, 并通过PMA-qPCR方法验证不同比例的戊糖片球菌死菌/活菌混合菌液, 评估该方法的灵敏度和检出限。结果 设计的引物具有良好的特异性, PMA的最佳处理条件为质量浓度80 µg/mL, 暗孵育时间为15 min, 曝光时间为25 min, 此条件下, 构建的标准曲线相关系数(r2)为0.9983, 最低检出限为3.98×103 CFU/g。结论 本研究建立的PMA-qPCR方法可有效实现玫瑰酱中戊糖片球菌活菌数的快速检测, 灵敏度高, 为复杂食品基质中微生物的定量检测提供了技术支持。

玫瑰酱  /  戊糖片球菌  /  叠氮溴化丙锭  /  实时定量聚合酶链式反应  /  活菌

Objective To establish a rapid detection method of viable Pediococcus pentosaceus in rose jam based on the combination of propidium monoazide (PMA) and quantitative real-time polymerase chain reaction (qPCR). Methods Primers specific for Pediococcus pentosaceus were designed according to the species-specific murI gene. The optimal PMA treatment conditions were determined by optimizing the PMA concentration, dark incubation time, and exposure time. Different ratios of Pediococcus pentosaceus dead/live bacterial mixtures were validated by the PMA-qPCR method to assess the sensitivity and limit of detection of the technique. Results The designed primers exhibited high specificity, and the optimal PMA treatment conditions were determined as 80 µg/mL PMA, 15 min of dark incubation, and 25 min of exposure. Under this condition, a standard curve with correlation coefficient (r2) of 0.9983 was constructed, and the lowest limit of detection was determined to be 3.98×103 CFU/g. Conclusion The PMA-qPCR method established in this study enables the rapid and accurate quantification of viable Pediococcus pentosaceus in rose jam with high sensitivity, providing technical support for the quantitative detection of microorganisms in complex food matrices.

rose jam  /  Pediococcus pentosaceus  /  propidium monoazide  /  quantitative real-time polymerase chain reaction  /  viable
张艳珍, 袁伟杰, 马超, 麻丽丹, 刘云国. 叠氮溴化丙锭结合实时定量聚合酶链式反应法快速检测玫瑰酱中戊糖片球菌活菌. 食品安全质量检测学报, 2025 , 16 (13) : 214 -219 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250110007
Yan-Zhen ZHANG, Wei-Jie YUAN, Chao MA, Li-Dan MA, Yun-Guo LIU. Rapid detection of viable Pediococcus pentosaceus in rose jam by propidium monoazide-quantitative real-time polymerase chain reaction method[J]. Journal of Food Safety & Quality, 2025 , 16 (13) : 214 -219 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250110007
正文
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0 引言
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戊糖片球菌(Pediococcus pentosaceus)是一种革兰氏阳性、厌氧或微需氧的乳酸菌, 近年来因其益生功能而备受关注[1-2], 广泛存在于植物及多种发酵食品中, 如发酵乳制品[3]、水果[4]、蔬菜[5]、肉制品[6]和谷物[7], 在延长保质期、提升食品风味以及赋予食品营养价值方面具有重要作用[8-10]。此外, 研究表明, 戊糖片球菌具有显著的抗菌活性[11]、抗氧化活性[12], 并可通过调节免疫系统促进肠道健康等[13-14], 这些特性不仅增强了其在食品工业中改善产品安全性和质量的应用潜力, 还为功能性食品的研发提供了重要的理论依据。
玫瑰酱是我国传统发酵食品, 主要由玫瑰花瓣和糖等辅料发酵制作, 具有独特的花香和丰富的口感[15], 富含酚类和黄酮类化合物, 具有抗氧化作用并能增强免疫力, 深受消费者青睐[16]。微生物在玫瑰酱的发酵过程中起核心作用, 不仅延长了产品的贮藏期, 还促进风味物质的产生并赋予其保健功能[17]。实验室前期从发酵玫瑰酱中分离鉴定了一株具备益生菌性能的戊糖片球菌菌株MP13, 并用于玫瑰酱发酵工艺研究。结果表明, 戊糖片球菌菌株MP13能提升玫瑰酱的感官特性, 显著提高总黄酮、总酚和花青素含量, 并增强抗氧化性能, 赋予玫瑰酱功能性潜力, 为开发功能性玫瑰酱提供了技术支持[18]。
在发酵食品生产及贮藏过程中, 活菌数量是衡量其品质和安全性的关键指标。传统微生物计数方法主要依赖于菌落平板计数法, 存在耗时长、难以有效检测处于存活但不可培养(viable but non-culturable, VBNC)状态的微生物等局限性。此外, 该方法在复杂的微生物群落中准确定量单一菌种也面临挑战[19]。近年来, 实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)因其特异性强、灵敏度高、检测周期短等优势, 广泛应用于定量检测发酵食品中微生物数量[20-21]。然而, 该技术在区分活菌和死菌方面具有局限性, 可能引起检测结果偏差[22]。叠氮溴化丙锭(propidium monoazide, PMA)作为一种光敏性DNA结合染料, 能选择性地与死菌DNA结合并抑制其扩增, 但对活菌DNA却无影响, 显著提高检测准确性, 在活菌定量检测中具有广阔应用前景[23-24]。
本研究结合PMA染料与qPCR技术, 建立了一种用于快速检测发酵玫瑰酱中戊糖片球菌活菌的方法, 基于美国国家生物技术信息中心(National Center of Biotechnology Information, NCBI)数据库设计戊糖片球菌的特异性引物, 优化PMA处理条件(包括PMA浓度、暗孵育时间和曝光时间), 并进一步构建玫瑰酱样品的标准曲线, 确定戊糖片球菌的最低检出限, 以期为发酵玫瑰酱的质量控制提供技术支持。
1 材料与方法
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1.1 材料与试剂
玫瑰酱: 平阴玫瑰产业园; 戊糖片球菌(Pediococcus pentosaceus MP13): 实验室前期从发酵玫瑰酱中分离保藏; 植物乳杆菌(Lactobacillus plantarum) ATCC 14917、金黄色葡萄球菌(Staphylococcus aureus) ATCC 25923、大肠埃希氏菌(Escherichia coli) ATCC 25922对照菌株, 均由实验室保存。
MRS液体培养基(北京陆桥技术股份有限公司); 2×Taq PCR MasterMix Ⅱ、D 2000和细菌基因组DNA提取试剂盒、引物、探针[生工生物工程(上海)有限公司]; PMA(美国Biotium公司); qPCR反应体系试剂(中国BBI生命科学有限公司)。
1.2 仪器与设备
BSA224S电子分析天平(精度0.1 mg, 北京赛多利斯仪器有限公司); LC-LX-H165A型离心机(上海力辰仪器科技有限公司); THZ-15恒温培养摇床(上海一恒科学仪器有限公司); ABI StepOnePlus实时荧光定量PCR仪(美国ABI公司)。
1.3 实验方法
1.3.1 戊糖片球菌的活菌/死菌悬液制备
戊糖片球菌MP13接种于MRS液体培养基, 于37 ℃振荡培养至对数期(180 r/min), 取1 mL菌液进行梯度稀释, 平板计数得到浓度约为108 CFU/mL的活菌悬液。将10 mL该悬液置于沸水中水浴40 min以实现灭活处理, 随后12000 r/min离心2 min, 弃上清并用10 mL无菌蒸馏水重悬, 即为戊糖片球菌死菌悬液。
1.3.2 DNA提取
取1 mL纯培养菌液, 12000 r/min离心2 min收集菌体, 按照细菌DNA提取试剂盒说明书, 分别进行戊糖片球菌和参考菌株的DNA提取, 提取后于-20 ℃保存备用。
1.3.3 qPCR引物设计及特异性验证
基于NCBI数据库选取murI基因序列作为戊糖片球菌的特异性基因, 通过Beacon Designer和Primer Premier 5.0软件进行特异性引物和探针设计, 并由生工生物工程(上海)有限公司合成, 详细信息见表1。以提取的戊糖片球菌基因组DNA以及对照菌株DNA作为模板, 通过qPCR实验评估引物特异性, 反应体系为20 μL: 2×TaqMan Fast qPCR Master Mix (High Rox) 10 μL, 上、下游引物各1 μL, 探针1 μL, DNA模板1 μL, ddH2O 6 μL。反应条件为: 94 ℃ 3 min, 94 ℃ 5 s, 56 ℃ 45 s, 72 ℃ 30 s, 40个循环。
1.3.4 PMA处理条件优化
(1) PMA质量浓度
分别取活菌/死菌悬液各500 μL置于1.5 mL离心管中, 加入一定量100 μg/mL PMA溶液, 并用无菌水补充至最终PMA质量浓度分别为0、20、40、60、80、100 μg/mL。混匀后20~25 ℃暗孵15 min, 随后置于365 nm紫外灯下曝光20 min。处理后12000 r/min离心2 min, 按照1.3.2步骤提取基因组DNA, 用戊糖片球菌特异性引物进行qPCR检测, 反应体系同1.3.3, 以循环阈值(cycle threshold, Ct)来评估不同浓度PMA处理对戊糖片球菌死菌/活菌qPCR扩增的影响。
(2) PMA暗孵育时间
添加最佳质量浓度PMA溶液分别于500 μL活菌/死菌悬液中, 混匀后设置暗孵育时间分别为0、5、10、15、20、25 min, 随后365 nm紫外灯下曝光20 min, 之后步骤同PMA质量浓度优化。
(3) PMA曝光时间
添加最佳质量浓度PMA溶液分别于500 μL活菌/死菌悬液中, 混匀后于最佳暗孵育时间下孵育。随后将离心管置于365 nm紫外灯下分别曝光0、5、10、15、25、35、45 min, 之后步骤同PMA浓度优化。
1.3.5 不同比例死/活菌混合菌悬液的验证
按照活菌比例分别为0%、5%、10%、25%、50%、100%配制混合菌悬液, 分别取500 μL混合菌液于1.5 mL离心管中, 根据PMA优化条件处理后按照1.3.2步骤提取基因组DNA并进行qPCR检测, 反应体系同1.3.3, 以未加PMA处理的样品作为对照组。
1.3.6 标准曲线的建立
取5 g玫瑰酱样品加入50 mL离心管中, 12000 r/min离心2 min后弃上清。加入无菌水混匀后10倍梯度稀释成浓度分别为107、106、105、104、103、102和10 CFU/mL的菌液。按1.3.4优化的PMA处理条件处理后, 按照1.3.2步骤提取DNA, 并进行qPCR检测测定对应Ct值, 反应体系同1.3.3, 通过菌落浓度对数值与Ct值作线性回归分析。
1.4 数据处理
所有实验均重复3次, 使用StepOneTM v2.3对qPCR数据进行初步分析, Excel 2016和GraphPad Prism 10.4.1进行数据处理分析与制图, 标准曲线的线性关系通过线性回归分析评估, Ct值间差异的统计学分析采用方差分析法。
2 结果与分析
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2.1 戊糖片球菌引物探针特异性验证
基于戊糖片球菌murI基因设计特异性引物和探针, 并以戊糖片球菌DNA和其他对照菌株DNA为模板, 通过qPCR方法对引物特异性进行验证。qPCR检测结果如图1所示, 与其他对照菌株相比, 只在戊糖片球菌样品中检测到明显的荧光信号, 这一结果证实了根据murI基因设计的戊糖片球菌特异性引物和探针的特异性较好, 可用于后续实验检测。
2.2 戊糖片球菌PMA处理条件优化
2.2.1 PMA质量浓度的优化
分别添加不同质量浓度的PMA溶液对两类菌悬液进行处理, 并监测其Ct值的变化, 以探究不同PMA质量浓度对戊糖片球菌活菌/死菌悬液在qPCR扩增中的影响。如图2所示, 不同质量浓度PMA处理的Ct值整体上呈现显著差异, 这说明PMA溶液可以有效抑制死菌DNA扩增。具体而言, PMA质量浓度低于80 μg/mL时, 随PMA质量浓度增加, 死菌Ct值逐渐增大, 可见低浓度PMA不能完全抑制死菌DNA的扩增, 而当PMA质量浓度达到或超过80 μg/mL时, 各PMA浓度间Ct值无显著差异, 说明80 μg/mL的PMA溶液已足够与死菌DNA充分结合, 从而有效抑制其扩增。同时, 不同浓度PMA处理对活菌qPCR扩增均无显著抑制作用, 故戊糖片球菌的最佳PMA处理质量浓度为80 μg/mL。
2.2.2 暗孵育时间的优化
适当增加暗孵育时间可以提升PMA结合死菌细胞DNA的效率, 从而更有效地抑制其qPCR扩增信号。如图3所示, 在暗孵育时间0~15 min范围时, 死菌Ct值随之显著上升, 而暗孵育时间为15、20或25 min时, Ct值间不再表现出显著差异, 这说明暗孵育15 min已足够使PMA充分渗透并完全结合死菌DNA, 实现对其扩增的有效抑制。此外, 不同暗孵育时间处理的活菌Ct值间基本无显著差异, 说明暗孵育处理对活菌DNA扩增无影响, 故戊糖片球菌的最佳暗孵育时间为15 min。
2.2.3 曝光时间的优化
光照条件下, PMA会释放叠氮基团与死菌DNA发生共价交联, 形成稳定的碳氮键, 进而抑制其DNA扩增。如图4所示, PMA曝光时间少于25 min时, 死菌Ct值随曝光时间延长而显著上升, 这说明曝光时间不足以使PMA充分光解, 无法完全抑制死菌DNA扩增, 而当曝光时间超过25 min时, 死菌Ct值间不再表现出显著差异, 意味着进一步的延长曝光时间不会增强交联反应效果, 曝光25 min已足以使PMA充分光解。此外, 不同曝光时间处理的活菌Ct值之间无显著差异, 说明曝光时间处理同样对活菌DNA扩增无影响, 故戊糖片球菌的最佳PMA曝光时间为25 min。
2.3 PMA对戊糖片球菌不同比例混合菌样的影响
如图5所示, 在无PMA处理的对照组中, Ct值不受活菌比例的变化影响, 而在PMA处理的实验组中, Ct值随活菌比例上升而显著下降, 且显著高于无PMA处理的Ct值, 当活菌比例达到100%时, 两者间的Ct值差异不再显著。这一结果表明, PMA处理能够有效抑制死菌DNA扩增, 从而避免出现假阳性结果, 同时该处理对活菌DNA扩增无影响, 防止了假阴性结果产生。
2.4 PMA处理的玫瑰酱样品中戊糖片球菌的最低检出限
通过构建玫瑰酱样品的PMA-qPCR方法标准曲线, 确定了该方法检测玫瑰酱中戊糖片球菌的最低检出限。对戊糖片球菌浓度范围为107至102 CFU/mL的玫瑰酱样品实施PMA-qPCR检测后, 如图6所示, 各菌浓度下的扩增曲线呈现出明显的分离趋势: 高浓度菌样的扩增曲线较早地跨越了阈值线, 而低浓度菌样则需要更多循环才能达到阈值线。细菌浓度的降低伴随着qPCR检测Ct值的逐渐升高, 说明了qPCR技术在检测不同菌浓度时的高灵敏度。标准曲线进一步揭示了Ct值与细菌浓度对数值间的良好线性关系, 其回归方程为Y=-3.4926X+47.68, r2=0.9983, 确定的最低检出限为3.98×103 CFU/g。
3 讨论与结论
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戊糖片球菌因其在发酵食品中的功能性和益生特性而备受关注, 并被报道为多种发酵基质的优势菌种[25-26], 但过量的戊糖片球菌可能会导致不良的发酵效果, 影响产品质量, 据研究报道, 啤酒酿造过程中, 戊糖片球菌作为一种潜在的污染微生物, 可能会引起酒体混浊或出现酸败, 并产生过量的双乙酰[27]。因此, 建立有效的戊糖片球菌活菌检测方法对于玫瑰酱质量控制具有重要意义。
相较于传统的平板菌落计数方法相比, PMA-qPCR技术具有灵敏度高、检测周期短的显著优势, 同时又弥补了传统PCR和qPCR在活菌检测中的局限性[28], 已被广泛应用于发酵微生物的定量检测[29-32]。然而, 目前尚未见其在戊糖片球菌活菌检测中的应用研究。特异性引物设计是PCR技术的关键步骤之一, 能够确保仅针对目标基因进行扩增, 本研究基于NCBI数据库选取murI基因作为戊糖片球菌的目标基因, 设计了特异性引物和探针, 该基因是一种不依赖辅因子的酶, 对细菌肽聚糖的生物合成途径至关重要[33], 实验结果表明PMA-qPCR能够特异性检测戊糖片球菌, 同时对于常见的乳酸菌和食源性致病菌均无非特异性扩增, 证实了设计的戊糖片球菌特异性引物和探针具有良好的特异性和排他性。
但PMA-qPCR技术依赖于PMA与死菌DNA结合的效率, 而这种效率受菌株特性显著影响, 尤其是革兰氏阴性菌与革兰氏阳性菌之间细胞膜结构的差异, 研究表明, 与革兰氏阳性菌相比, 膜受损的革兰氏阴性菌更容易被PMA所渗透[19]。此外, PMA的浓度、暗孵育时间以及曝光处理时间也是影响PMA处理效果的关键因素, 本研究通过优化PMA处理条件, 确定适宜的PMA浓度和处理时间, 即有效去除死菌DNA的干扰, 又确保了活菌信号的完整性, 随着PMA浓度增加以及处理时间延长, 死菌DNA的抑制效果逐渐增强, 但在达到平衡点后进一步提高这些参数对抑制效果的改善不明显, 这与其他研究的优化结果相符[21,31,34]。此外, 在不同死菌/活菌比例混合菌液中, 该方法能够有效抑制死菌DNA的扩增, 特别是在死菌比例较高时, 有效减少了假阳性结果的发生。本研究通过标准曲线的线性回归分析, 验证了PMA-qPCR方法在检测玫瑰酱中戊糖片球菌的稳定性和可靠性, 菌浓度对数值与Ct值之间具有良好的线性关系(r2=0.9983), 最低检出限为3.98×103 CFU/g。然而, 与发酵乳制品基质的相关研究结果相比[31,34], 该检出限略高, 这可能与食品基质的特性差异有关。玫瑰酱作为高糖食品, 其较高的黏稠度可能限制PMA分子与死菌DNA结合的效率; 此外, 其基质中富含的酚类化合物等复杂成分可能干扰qPCR扩增反应, 从而影响检测灵敏度。
本研究建立了玫瑰酱中戊糖片球菌的PMA- qPCR检测方法, 并优化了PMA处理条件(80 µg/mL PMA、暗孵育15 min、曝光处理25 min), 在此条件下, 该方法能够快速、准确地检测玫瑰酱中戊糖片球菌的活菌数, 最低检出限为3.98×103 CFU/g, 这为功能性玫瑰酱的质量控制提供了有效的检测手段, 也为PMA-qPCR技术在其他高糖食品基质中的应用奠定了基础, 未来研究可进一步评估该方法在复杂食品基质中的适用性, 并结合自动化和高通量检测技术推动其在食品工业中的大规模应用。
基金
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  • 中央引导地方科技发展资金项目(YDZX2023081)
  • 海关总署科研项目(2024HK014)
文献
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2025年第16卷第13期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250110007
  • 接收时间：2025-01-10
  • 首发时间：2026-01-12
  • 出版时间：2025-07-15
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  • 收稿日期：2025-01-10
基金
中央引导地方科技发展资金项目(YDZX2023081)
海关总署科研项目(2024HK014)
作者信息
    1 临沂大学生命科学学院, 临沂 276005
    2 水原大学工程学院, 水原 18323
    3 拱北海关技术中心, 珠海 519075

通讯作者:

*刘云国(1977—), 男, 博士, 教授, 主要研究方向为食品微生物。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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