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Article(id=1320882330571493660, tenantId=1146029695717560320, journalId=1260987677001138203, issueId=1320882022160126288, articleNumber=null, orderNo=null, doi=10.13386/j.issn1002-0306.2024120184, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1734278400000, receivedDateStr=2024-12-16, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1792852488414, onlineDateStr=2026-10-24, pubDate=1767196800000, pubDateStr=2026-01-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792852488414, onlineIssueDateStr=2026-10-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792852488414, creator=13701087609, updateTime=1792852488414, updator=13701087609, issue=Issue{id=1320882022160126288, tenantId=1146029695717560320, journalId=1260987677001138203, year='2026', volume='47', issue='1', pageStart='1', pageEnd='498', issueExtLink='null', onlineDate='null', pubDate='1767196800000', pubDateStr='2026-01-01', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792852414884, creator='13701087609', updateTime=1792852414884, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=311, endPage=317, ext={EN=ArticleExt(id=1320882330785403165, articleId=1320882330571493660, tenantId=1146029695717560320, journalId=1260987677001138203, language=EN, title=Establishment and Application of an SDS-PMA-mRT-qPCR Assay for the Detection of Three Food-borne Pathogenic Bacteria in Dairy Products, columnId=1261343843585241413, journalTitle=Science and Technology of Food Industry, columnName=Analysis and Determination, runingTitle=null, highlight=null, articleAbstract=

This study employed a multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) method incorporating SDS (sodium dodecyl sulfate) and PMA (propidium monoazide) pretreatment to enable rapid and accurate detection of three food-borne pathogens Salmonella spp. (SA), Listeria monocytogenes (LM), and Shiga toxin-producing Escherichia coli (STE) in dairy products. Three sets of specific primers and TaqMan probes were designed targeting conserved regions of the invA gene (SA), hly gene (LM), and stx gene (STE), facilitating the development of an optimized mRT-qPCR assay. Furthermore, PMA pretreatment efficacy was evaluated for eliminating false-positive results caused by dead bacterial cells, and the evaluation of the specificity, detection limit, stability, and detection effect in the simulated dairy samples was conducted in this study. The results revealed that pretreatment with PMA combined with SDS prevented the interference of dead bacterial cells, and the optimal effective PMA concentration was 20 μmol/L. The assay showed high specificity and sensitivity and good stability in detecting the three target strains amplified in the presence of 18 non-target strains, with the lowest limits of detection for the three target strains being up to 102 CFU/mL and the variation coefficients of inter-batch and intra-batch being less than 1%. The results obtained for the detection of causal organisms in bacterially contaminated dairy products using the SDS-PMA-mRT-qPCR assay were comparable to those obtained using the national standard method, with a test cycle of 7 h. These findings indicate that the SDS-PMA-mRT-qPCR assay can be used for the rapid and accurate detection of Salmonella spp., L. monocytogenes, and Shiga toxin-producing E. coli in dairy products and represents an appropriate technique for ensuring the safety of dairy products.

, authors=Peilin LIU, Chenpeng SHI, Yanwei WANG, Pengfei AI*, authorsList=Peilin LIU, Chenpeng SHI, Yanwei WANG, Pengfei AI, authorCompany=null, correspAuthors=Pengfei AI, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright © 2026 Science and Technology of Food Industry. All rights reserved., 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, fund=null), CN=ArticleExt(id=1320882332073054504, articleId=1320882330571493660, tenantId=1146029695717560320, journalId=1260987677001138203, language=CN, title=3种食源性致病菌SDS-PMA-mRT-qPCR检测方法建立及其在乳品中的应用, columnId=1261343846139572559, journalTitle=食品工业科技, columnName=分析检测, runingTitle=null, highlight=null, articleAbstract=

本研究利用十二烷基硫酸钠（sodium dodecyl sulfate，SDS）联合叠氮溴化丙锭（propidium monoazide，PMA）预处理样品后，采用多重实时荧光定量PCR（multiple real-time quantitative polymerase chain reaction，mRT-qPCR）方法快速、准确检测乳制品中沙门氏菌、单增李斯特菌和产志贺毒素大肠埃希氏菌3种食源性致病菌。通过对沙门氏菌中的invA基因、单增李斯特菌中的hly基因和产志贺毒素大肠埃希氏菌中的stx基因的保守序列分别设计特异性引物和探针，建立mRT-qPCR反应体系，探讨PMA预处理消除由死菌造成的检测的假阳性结果，并对检测的特异性、检出限、稳定性以及模拟样品中的检测效果进行了研究。结果表明，PMA结合SDS的预处理能有效排除死菌对mRT-qPCR检测结果的干扰，最佳PMA浓度为20 μmol/L；建立的mRT-qPCR方法特异性强，在18株非目标菌的干扰下仅对3种目标菌进行特异性扩增；灵敏度高，3种目标菌的检出限均为102 CFU/mL；稳定性好，不同批次内和批次间的重复性检测的变异系数均小于1%；对不同污染乳品的检测结果与国家标准中的培养法一致，检测周期约7 h。本研究建立的SDS-PMA-mRT-qPCR方法可以实现对沙门氏菌、单增李斯特菌和产志贺毒素大肠埃希氏菌的快速检测，为乳品安全提供技术支持。

, authors=刘霈霖, 匙辰鹏, 王雁伟, 艾鹏飞*, authorsList=刘霈霖, 匙辰鹏, 王雁伟, 艾鹏飞, authorCompany=null, correspAuthors=艾鹏飞, authorNote=

刘霈霖（1998−），女，硕士研究生，研究方向：微生物分子生物学，E-mail：

, correspAuthorsNote=
艾鹏飞（1974−），男，博士，教授，研究方向：生物化学与分子生物学，E-mail：。
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刘霈霖（1998−），女，硕士研究生，研究方向：微生物分子生物学，E-mail：

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刘霈霖（1998−），女，硕士研究生，研究方向：微生物分子生物学，E-mail：

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Frontiers in Microbiology, 2019, 10: 661., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1320882332307935530, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, xref=null, ext=[AuthorCompanyExt(id=1320882332316324139, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, companyId=1320882332307935530, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=College of Food and Bioscience, Hebei University of Science and Technology, Shijiazhuang 050018, China), AuthorCompanyExt(id=1320882332324712748, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, companyId=1320882332307935530, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=河北科技大学食品与生物学院，河北石家庄 050018)])], figs=[ArticleFig(id=1320882335738876235, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Fig.1, caption=Electrophoresis profiles of PCR amplifications for objective strains, figureFileSmall=0gPWP+C7clDnyPQN2dlPsA==, figureFileBig=oZJAYaBE+GS593DLm3Orhw==, tableContent=null), ArticleFig(id=1320882335797596492, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=图1, caption=目标菌株PCR扩增结果

注：M为50 bp DNA Ladder，A1~A3依次为目标菌株SA、LM、STE，A’为ddH2O。

, figureFileSmall=0gPWP+C7clDnyPQN2dlPsA==, figureFileBig=oZJAYaBE+GS593DLm3Orhw==, tableContent=null), ArticleFig(id=1320882335977951565, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Fig.2, caption=Optimization of usages for primers and probes, figureFileSmall=VBX6uiUBnq65WEdx1d7M0g==, figureFileBig=Zq+6vjEXHNRsHd/3QdHHHg==, tableContent=null), ArticleFig(id=1320882336040866126, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=图2, caption=引物和探针用量优化结果

注：A、B、C依次为SA、LM和STE，④、⑤、⑥依次为引物和探针组合0.4 μL和0.4 μL、0.6 μL和0.4 μL、0.8 μL和0.4 μL。D为三种目标菌在同一优化后的反应体系。

, figureFileSmall=VBX6uiUBnq65WEdx1d7M0g==, figureFileBig=Zq+6vjEXHNRsHd/3QdHHHg==, tableContent=null), ArticleFig(id=1320882336116363599, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Fig.3, caption=Optimization of concentration for PMA, figureFileSmall=UDN3PGLtYarv1Hymi7vpCg==, figureFileBig=+bqNDp5HbixZsgndmtGrgA==, tableContent=null), ArticleFig(id=1320882336179278160, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=图3, caption=PMA浓度优化

注：A、B、C依次为SA、LM和STE。

, figureFileSmall=UDN3PGLtYarv1Hymi7vpCg==, figureFileBig=+bqNDp5HbixZsgndmtGrgA==, tableContent=null), ArticleFig(id=1320882336254775633, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Fig.4, caption=Specificity of triplex RT-qPCR, figureFileSmall=FMQTsVoAUnvzdkkwQsmkhQ==, figureFileBig=kWakWaXo/dnxk8kdiy5gFw==, tableContent=null), ArticleFig(id=1320882336313495890, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=图4, caption=三重RT-qPCR特异性检测, figureFileSmall=FMQTsVoAUnvzdkkwQsmkhQ==, figureFileBig=kWakWaXo/dnxk8kdiy5gFw==, tableContent=null), ArticleFig(id=1320882336388993363, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Fig.5, caption=Sensitivity of triplex RT-qPCR, figureFileSmall=Z2J3mYpiG6dQkztyQQmdEg==, figureFileBig=hLSTJfZv9Mn/Tef9xG1qeQ==, tableContent=null), ArticleFig(id=1320882336451907924, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=图5, caption=三重RT-qPCR灵敏度评价

注：A为mRT-qPCR灵敏度测试结果，图中①~⑥依次代表菌液浓度107~102 CFU/mL；B为mRT-qPCR扩增标准曲线图。

, figureFileSmall=Z2J3mYpiG6dQkztyQQmdEg==, figureFileBig=hLSTJfZv9Mn/Tef9xG1qeQ==, tableContent=null), ArticleFig(id=1320882336535794005, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Table 1, caption=

Primers and probes for RT-qPCR

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株引物/探针碱基序列（5’-3’）Tm值（℃）产物长度（bp）
SAinvA-FTGATGCGAGTGGTAAATTATTCC60.2110
invA-RCTCCCCCTCTTCATGCGTTA62.4
invA-PROX-CCAACAATCCATCAGCAAGGTAGCAGTCA-BHQⅡ70.1
LMhly-FCATGGCACCACCAGCATC62.265
hly-RCATCCGCGTGTTTCTTTTCG62.8
hly-PFAM-CCGCCTGCAAGTCCTAAGACGCCA-BHQⅠ72.8
STEstx-FCATCGCTCTTGCCACAGA61.384
stx-RTTTCGCCATTCGTTGACTACTTC61.0
stx-PCy5-CGCATAGCGGAACCTCACTGACGC-BHQⅢ71.7
), ArticleFig(id=1320882336598708566, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=表1, caption=

RT-qPCR引物和探针序列

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株引物/探针碱基序列（5’-3’）Tm值（℃）产物长度（bp）
SAinvA-FTGATGCGAGTGGTAAATTATTCC60.2110
invA-RCTCCCCCTCTTCATGCGTTA62.4
invA-PROX-CCAACAATCCATCAGCAAGGTAGCAGTCA-BHQⅡ70.1
LMhly-FCATGGCACCACCAGCATC62.265
hly-RCATCCGCGTGTTTCTTTTCG62.8
hly-PFAM-CCGCCTGCAAGTCCTAAGACGCCA-BHQⅠ72.8
STEstx-FCATCGCTCTTGCCACAGA61.384
stx-RTTTCGCCATTCGTTGACTACTTC61.0
stx-PCy5-CGCATAGCGGAACCTCACTGACGC-BHQⅢ71.7
), ArticleFig(id=1320882336674206039, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Table 2, caption=

Reproducibility test results of SDS-PMA-mRT-qPCR

, figureFileSmall=null, figureFileBig=null, tableContent=
菌液浓度
（CFU/mL）		目标菌批次内变异系数（%）批次间变异系数（%）
第1批次第2批次第3批次
104SA0.410.680.530.25
LM0.620.470.720.20
STE0.510.460.370.16
103SA0.710.600.810.15
LM0.270.410.650.43
STE0.540.350.670.31
102SA0.280.450.570.34
LM0.680.340.520.33
STE0.400.510.460.12
), ArticleFig(id=1320882336766480728, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=表2, caption=

SDS-PMA-mRT-qPCR重复性测试结果

, figureFileSmall=null, figureFileBig=null, tableContent=
菌液浓度
（CFU/mL）		目标菌批次内变异系数（%）批次间变异系数（%）
第1批次第2批次第3批次
104SA0.410.680.530.25
LM0.620.470.720.20
STE0.510.460.370.16
103SA0.710.600.810.15
LM0.270.410.650.43
STE0.540.350.670.31
102SA0.280.450.570.34
LM0.680.340.520.33
STE0.400.510.460.12
), ArticleFig(id=1320882336841978201, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=EN, label=Table 3, caption=

Test results of various dairy samples by SDS-PMA-mRT-qPCR and culture method respectively

, figureFileSmall=null, figureFileBig=null, tableContent=
添加目标菌样品数三重RT-qPCR（Ct值）培养法（CFU/mL）
SALMSTESALMSTEC
04000−−−
SA426.24±0.45−−1.1×102−−
LM4−26.55±0.37−−8.9×101−
STE4−−25.74±0.52−−9.6×101
SA+LM429.45±0.1929.54±0.23−9.8×1011.0×102−
SA+STE429.34±0.26−29.17±0.319.1×101−8.7×101
LM+STE4−29.73±0.4529.38±0.28−9.1×1018.3×101
SA+LM+STE429.52±0.2729.68±0.3629.32±0.428.5×1018.9×1019.1×101
), ArticleFig(id=1320882336913281370, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882330571493660, language=CN, label=表3, caption=

不同乳品样品信息及检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
添加目标菌样品数三重RT-qPCR（Ct值）培养法（CFU/mL）
SALMSTESALMSTEC
04000−−−
SA426.24±0.45−−1.1×102−−
LM4−26.55±0.37−−8.9×101−
STE4−−25.74±0.52−−9.6×101
SA+LM429.45±0.1929.54±0.23−9.8×1011.0×102−
SA+STE429.34±0.26−29.17±0.319.1×101−8.7×101
LM+STE4−29.73±0.4529.38±0.28−9.1×1018.3×101
SA+LM+STE429.52±0.2729.68±0.3629.32±0.428.5×1018.9×1019.1×101
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3种食源性致病菌SDS-PMA-mRT-qPCR检测方法建立及其在乳品中的应用
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刘霈霖 , 匙辰鹏 , 王雁伟 , 艾鹏飞 *
食品工业科技 | 分析检测 2026,47(1): 311-317
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食品工业科技 |分析检测 2026 , 47 (1) : 311 -317
3种食源性致病菌SDS-PMA-mRT-qPCR检测方法建立及其在乳品中的应用
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刘霈霖（1998−），女，硕士研究生，研究方向：微生物分子生物学，E-mail：

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刘霈霖 , 匙辰鹏, 王雁伟, 艾鹏飞*
作者信息
  • 河北科技大学食品与生物学院，河北石家庄 050018
通讯作者:
艾鹏飞（1974−），男，博士，教授，研究方向：生物化学与分子生物学，E-mail：。
作者简介:

刘霈霖（1998−），女，硕士研究生，研究方向：微生物分子生物学，E-mail：

Establishment and Application of an SDS-PMA-mRT-qPCR Assay for the Detection of Three Food-borne Pathogenic Bacteria in Dairy Products
Peilin LIU , Chenpeng SHI, Yanwei WANG, Pengfei AI*
Affiliations
  • College of Food and Bioscience, Hebei University of Science and Technology, Shijiazhuang 050018, China
出版时间: 2026-01-01 doi: 10.13386/j.issn1002-0306.2024120184
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摘要
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本研究利用十二烷基硫酸钠（sodium dodecyl sulfate，SDS）联合叠氮溴化丙锭（propidium monoazide，PMA）预处理样品后，采用多重实时荧光定量PCR（multiple real-time quantitative polymerase chain reaction，mRT-qPCR）方法快速、准确检测乳制品中沙门氏菌、单增李斯特菌和产志贺毒素大肠埃希氏菌3种食源性致病菌。通过对沙门氏菌中的invA基因、单增李斯特菌中的hly基因和产志贺毒素大肠埃希氏菌中的stx基因的保守序列分别设计特异性引物和探针，建立mRT-qPCR反应体系，探讨PMA预处理消除由死菌造成的检测的假阳性结果，并对检测的特异性、检出限、稳定性以及模拟样品中的检测效果进行了研究。结果表明，PMA结合SDS的预处理能有效排除死菌对mRT-qPCR检测结果的干扰，最佳PMA浓度为20 μmol/L；建立的mRT-qPCR方法特异性强，在18株非目标菌的干扰下仅对3种目标菌进行特异性扩增；灵敏度高，3种目标菌的检出限均为102 CFU/mL；稳定性好，不同批次内和批次间的重复性检测的变异系数均小于1%；对不同污染乳品的检测结果与国家标准中的培养法一致，检测周期约7 h。本研究建立的SDS-PMA-mRT-qPCR方法可以实现对沙门氏菌、单增李斯特菌和产志贺毒素大肠埃希氏菌的快速检测，为乳品安全提供技术支持。

多重实时荧光定量PCR（mRT-qPCR）  /  叠氮溴化丙锭  /  沙门氏菌  /  单增李斯特菌  /  产志贺毒素大肠埃希氏菌

This study employed a multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) method incorporating SDS (sodium dodecyl sulfate) and PMA (propidium monoazide) pretreatment to enable rapid and accurate detection of three food-borne pathogens Salmonella spp. (SA), Listeria monocytogenes (LM), and Shiga toxin-producing Escherichia coli (STE) in dairy products. Three sets of specific primers and TaqMan probes were designed targeting conserved regions of the invA gene (SA), hly gene (LM), and stx gene (STE), facilitating the development of an optimized mRT-qPCR assay. Furthermore, PMA pretreatment efficacy was evaluated for eliminating false-positive results caused by dead bacterial cells, and the evaluation of the specificity, detection limit, stability, and detection effect in the simulated dairy samples was conducted in this study. The results revealed that pretreatment with PMA combined with SDS prevented the interference of dead bacterial cells, and the optimal effective PMA concentration was 20 μmol/L. The assay showed high specificity and sensitivity and good stability in detecting the three target strains amplified in the presence of 18 non-target strains, with the lowest limits of detection for the three target strains being up to 102 CFU/mL and the variation coefficients of inter-batch and intra-batch being less than 1%. The results obtained for the detection of causal organisms in bacterially contaminated dairy products using the SDS-PMA-mRT-qPCR assay were comparable to those obtained using the national standard method, with a test cycle of 7 h. These findings indicate that the SDS-PMA-mRT-qPCR assay can be used for the rapid and accurate detection of Salmonella spp., L. monocytogenes, and Shiga toxin-producing E. coli in dairy products and represents an appropriate technique for ensuring the safety of dairy products.

multiple real-time quantitative polymerase chain reaction (mRT-qPCR)  /  propidium monoazide  /  Salmonella  /  Listeria monocytogenes  /  Shiga toxin-producing Escherichia coli
刘霈霖, 匙辰鹏, 王雁伟, 艾鹏飞. 3种食源性致病菌SDS-PMA-mRT-qPCR检测方法建立及其在乳品中的应用. 食品工业科技, 2026 , 47 (1) : 311 -317 . DOI: 10.13386/j.issn1002-0306.2024120184
Peilin LIU, Chenpeng SHI, Yanwei WANG, Pengfei AI. Establishment and Application of an SDS-PMA-mRT-qPCR Assay for the Detection of Three Food-borne Pathogenic Bacteria in Dairy Products[J]. Science and Technology of Food Industry, 2026 , 47 (1) : 311 -317 . DOI: 10.13386/j.issn1002-0306.2024120184
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随着社会经济的发展和人们生活水平的提高，市场对乳品的需求量日益增加，乳品安全越来越受到重视。乳品中含有丰富的营养物质，易受病原菌污染而腐败变质[1−2]。近年来，因误食病原菌污染的乳品引起食物中毒或诱发疾病的事件时有发生，且呈现持续上升趋势[3−4]。乳品中食源性病原菌主要包括单增李斯特菌[5]、蜡样芽孢杆菌[6]、沙门氏菌[7]、产志贺毒素大肠埃希氏菌[8]、金黄色葡萄球菌[9]等，其中沙门氏菌、单增李斯特菌和产志贺毒素大肠埃希氏菌更为常见[10]。沙门氏菌（Salmonella spp.，SA）作为人畜共患的肠道致病菌，引起的食物中毒事件常年位居我国食源性致病菌榜首[11]。单增李斯特菌（Listeria monocytogenes，LM）致病性强，临床症状主要为败血症、脑膜炎和流产等，是世界卫生组织（WHO）食品安全工作计划中重点检测的四大食源性致病菌之一[12]。产志贺毒素大肠埃希氏菌（Shiga toxin-producing Escherichia coli，STE）可引起包括出血性腹泻、溶血性尿毒症、肾衰竭在内的严重疾病甚至死亡，曾在世界范围内引发多起食物中毒事件[13]。因此，亟需建立起这三种病原菌快速高效的检测方法，以便可以及时监测乳品的污染情况，有效预防和控制由食源性致病菌引起的疾病发生。
对于食源性病原菌的检测，常用的是国家标准中的微生物培养法，但该方法存在操作步骤多、过程繁琐、检测周期长（5~7 d）且不易定量等弊端[14−15]，难以应用于保鲜期短的乳品中致病菌的实时检测。后续发展起来的检测方法，如腺苷三磷酸发光技术[16]、免疫学技术[17]、生物传感器技术[18]、流式细胞技术[19]等，在灵敏度、稳定性、检测效率等方面尚有欠缺。近年来，基于TaqMan探针的多重实时荧光定量PCR（multiple real-time quantitative polymerase chain reaction，mRT-qPCR）检测技术具有特异性强、灵敏度高、稳定性好、快速高效等优点而备受青睐[20−21]。
叠氮溴化丙锭（propidium monoazide，PMA）是一种DNA结合剂，能透过受损细胞或死菌细胞的细胞膜后与DNA结合。十二烷基硫酸钠（sodium dodecyl sulfate，SDS）是一种辅助PMA透过受损细胞膜的增强剂[22−23]。在进行mRT-qPCR检测过程中，采用PMA结合SDS对菌悬液的预处理能够消除死菌细胞带来的假阳性检测结果，从而对活菌的检测结果更加准确可靠，因而在实际检测中得到广泛应用[24−25]。本研究针对沙门氏菌、单增李斯特菌和产志贺毒素大肠埃希氏菌的特异性基因序列设计引物和探针，结合PMA和SDS对菌液的预处理，建立一种可同时对这3种致病菌活细胞高效快速检测的SDS-PMA-mRT-qPCR方法。
1　材料与方法
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1.1　材料与仪器
单增李斯特菌（54001）、蜡样芽孢杆菌（63001）、鼠伤寒沙门氏菌（50115）、表皮葡萄球菌（26069）、多黏芽孢杆菌（63512）　中国医学菌种保藏中心（CMCC）；沙门氏菌（21484）、产志贺毒素埃希氏菌（10670）、福氏志贺氏菌（21534）、大肠埃希氏菌（10372）、金黄色葡萄球菌（21600）、肠炎沙门氏菌（10982）、副溶血性弧菌（21617）、枯草芽孢杆菌（10002）、巴氏醋杆菌（23561）、大肠杆菌O157:H7（21530）、伊氏李斯特氏菌（21633）　中国工业微生物菌种保藏中心（CICC）；苏云金芽孢杆菌（51024）、弗氏柠檬酸杆菌（43864）、阪崎肠杆菌（51024）、英诺克李斯特菌（33090）、空肠弯曲杆菌（29428）　美国标准菌种收藏所（ATCC）；pGM19-T质粒载体、大肠杆菌JM109　本实验室；牛肉膏、蛋白胨、营养肉汤、琼脂等细菌培养基组分　天根生化科技（北京）有限公司；细菌质粒提取试剂盒、DNA纯化回收试剂盒、5×Super RT-qPCR PreMix试剂盒　北京索莱宝科技有限公司；十二烷基硫酸钠（SDS）、叠氮溴化丙锭（PMA）等其它试剂　北京赛百盛生物工程有限公司。
VeritiPro PCR仪　Thermo Fisher公司；Gel Doc XR凝胶成像系统　Bio Rad公司；7900实时荧光定量PCR仪　ABI公司；超微量紫外可见分光光度计 Mettler Toledo公司；5417R高速冷冻离心机 Eppendorf公司；LUV-260AD紫外透射切胶仪　上海路阳仪器有限公司；DYY-6D电泳仪、DYCP-31BN电泳槽　北京六一生物科技有限公司。
1.2　实验方法
1.2.1　菌悬液制备和DNA提取
本实验中所使用的21种菌株在-80 ℃超低温冰箱中长期冻存。活化培养时从超低温冰箱中取出菌株，用牙签蘸取菌液在LB平板培养基上划线培养，37 ℃培养箱中培养12 h后备用。菌悬液制备时挑取单菌落到LB液体培养基中在160 r/min摇床上培养16 h后，将菌液浓度调整至107 CFU/mL。使用0.01 mol/L无菌磷酸盐缓冲液（PBS，pH7.4）制备10倍梯度稀释菌悬液。为了获得部分细胞死亡或受损的菌悬液，将沙门氏菌在60 ℃下孵育6 min，单增李斯特菌和产志贺毒素大肠埃希氏菌在52 ℃热激10 min[26]，热处理后的菌悬液冰浴5 min后，涂布于LB平板培养（37 ℃）过夜后计数。采用Liang等[27]的沸水浴法提取细菌基因组DNA，用双蒸水（ddH2O）溶解DNA至50 ng/μL，-20 ℃冰箱中保存备用。
1.2.2　引物和探针的设计与合成
根据GenBank收录的SA invA基因（NC_0031997.2）、LM hly基因（NC_003210.1）和STE stx基因（NC_017060.1）的保守序列，采用Premier 5.0软件设计引物，经BLAST和DNAMAN软件分析引物的可行性[28]；采用设计的引物（具体序列见表1）进行目标菌PCR试验，扩增产物经2%琼脂糖电泳，回收纯化目的条带，构建重组pGM19-T质粒载体，转入JM109感受态细胞中，37 ℃平板培养过夜后，挑选阳性克隆测序，依据测序结果设计TaqMan探针[24]；基因测序、引物和探针合成委托生工生物工程（上海）有限公司完成。
1.2.3　mRT-qPCR反应体系优化
在DNA模板相同的情况下，引物浓度和探针浓度是影响整个反应体系的关键因素[29]。参照试剂盒说明书分别对3种目标菌的引物和探针的添加量进行9种组合RT-qPCR试验（引物和探针组合为①0.4、0.3 μL，②0.6、0.3 μL，③0.8、0.3 μL，④0.4、0.4 μL，⑤0.6、0.4 μL，⑥0.8、0.4 μL，⑦0.4、0.5 μL，⑧0.6、0.5 μL，⑨0.8、0.5 μL），即总反应体系20 μL中，5×Super RT-qPCR PreMix 4 μL，各模板DNA 0.5 μL，浓度为10 µmol/L的引物及探针采用上述的不同用量组合，ddH2O补足至20 μL。分别用以上引物和探针不同用量组合对3种目标菌进行mRT-qPCR试验，扩增曲线为标准的S型的相应引物和探针用量组合确认为良好体系。mRT-qPCR扩增条件：95 ℃预变性8 min；94 ℃变性3 s，退火/延伸温度60 ℃时间30 s，40个循环。
1.2.4　菌悬液预处理
取经过热处理后部分细胞死亡或损伤的菌悬液在室温下8000 r/min离心8 min，弃上清，每个样品加入300 μmol/L的SDS溶液约500 μL后，在水浴锅中37 ℃孵育30 min[30]。将SDS处理后的菌悬液添加不同体积的PMA溶液至终浓度分别为0、5、10、20、30和50 μmol/L，室温（25 ℃）下处理20 min（期间颠倒混匀2~3次）后，将样品放置在距离500 W卤素灯20 cm处的冰浴中处理20 min（期间颠倒混匀4~5次）。将处理后的菌悬液于8000 r/min离心8 min，弃上清后加入0.01 mol/L PBS缓冲液，重复2次以洗去多余的PMA，将菌体重悬于100 μL无菌水备用。
1.2.5　特异性验证试验
以3株目标菌（沙门氏菌、单增李斯特菌、产志贺毒素大肠埃希氏菌）和18株非目标菌（蜡样芽孢杆菌、鼠伤寒沙门氏菌、表皮葡萄球菌、多黏芽孢杆菌、福氏志贺氏菌、大肠埃希氏菌、金黄色葡萄球菌、肠炎沙门氏菌、副溶血性弧菌、枯草芽孢杆菌、巴氏醋杆菌、大肠杆菌O157:H7、伊氏李斯特氏菌、苏云金芽孢杆菌、弗氏柠檬酸杆菌、阪崎肠杆菌、英诺克李斯特菌、空肠弯曲杆菌）的DNA混合液为模板，参照1.2.3方法进行mRT-qPCR试验，以验证建立的mRT-qPCR检测方法的特异性。试验结果以只出现3个目标菌株的S型扩增曲线且Ct值小于35判定为特异性良好。
1.2.6　检出限的确定
采用细菌平板计数法，分别对培养过夜的3种目标菌进行10倍的梯度稀释后计数，取各浓度梯度的菌液2 mL，按照1.2.4预处理后按照1.2.1方法提取DNA，溶解于50 μL ddH2O中。分别以各相同稀释梯度的DNA为模板进行mRT-qPCR扩增，以菌液浓度的对数为横坐标和Ct值为纵坐标制作标准曲线，确定检出限。各反应管中模板DNA浓度=菌液梯度浓度（CFU/mL）×取样量（2 mL）/DNA溶解体积（50 μL）×模板DNA添加量（0.5 μL）/反应总体积（20 μL）。
1.2.7　重复性试验
分别对各目标菌的3个浓度梯度（104、103、102 CFU/mL）进行3个批次的SDS-PMA-mRT-qPCR试验，每个浓度梯度重复3次，每次3个平行，根据批次内和批次间Ct值变异系数评估建立的SDS-PMA-mRT-qPCR检测方法的稳定性。
1.2.8　模拟样品检测
目前，我国乳品中病原菌的检测主要采用国家标准中的“培养法”[28]。为了验证建立的SDS-PMA-mRT-qPCR检测方法的有效性，从市场上购买新鲜的牛奶、酸奶、奶粉和奶酪各8份（共32份），其中28份100 mL（g）乳品样品中添加104 CFU/mL不同目标菌液2 mL，其余4份为对照（不添加任何菌液）。样品均质后取2 mL进行细菌基因组DNA提取和SDS-PMA-mRT-qPCR检测。同时按照《GB 4789 食品安全国家标准 食品微生物学检验》中的培养法分别对样品中的SA、LM和STE进行检测，比对2种方法的检测结果。
1.3　数据处理
本文中每份样品或每种菌液重复三次平行实验，数据处理及T检验使用Excel 2019和SPSS v20.0软件进行归纳与整理。
2　结果与分析
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2.1　靶标序列检测结果
分别以3株目标菌基因组DNA为模板，采用上述表1中针对靶基因设计的引物进行PCR扩增，扩增产物经2%琼脂糖电泳结果如图1所示，扩增出与预期大小一致的3个DNA片段。将这些目的片段回收后构建重组质粒，转入大肠杆菌获得阳性克隆。对阳性克隆的重组质粒进行测序和序列分析，发现扩增出的3个DNA片段的序列与GenBank中的完全一致，说明本试验设计的引物是准确可行的。分别依据这3个DNA片段的核苷酸序列设计的TaqMan探针如表1中所示。这相较于前人的同类研究[20,23,25]完全根据GeneBank中的靶标序列设计探针更加准确可靠。
2.2　引物和探针浓度的优化结果
引物和探针的浓度直接影响着mRT-qPCR扩增效果。不同引物和探针用量的RT-qPCR结果如图2A~C所示，在SA中9种用量组合都能扩增出S型曲线，其中组合④（0.4 μL和0.4 μL）荧光信号最强；在LM和STE中，组合⑤（0.6 µL和0.4 µL）和⑥（0.8 µL和0.4 µL）扩增出标准的S型曲线。在mRT-qPCR扩增中，考虑到最大限度提高扩增效率，一般优先选用较低浓度的引物和探针[31]。本试验中，SA引物和探针选用0.4 µL和0.4 µL，LM和STE的引物和探针均选用0.6 µL和0.4 µL，mRT-qPCR扩增的结果如图2D所示，3种目标菌均能扩增出标准的S型曲线，表明选用的3种目标菌的引物和探针浓度可用于后续试验。
2.3　PMA处理浓度优化结果
为了获得合适的PMA处理浓度，对经过300 μmol/L SDS处理的3种菌液分别进行系列浓度（0、5、10、20、30和50 μmol/L）PMA处理后，mRT-qPCR扩增结果如图3所示。在SA菌液中（图3A），对照组（未经热处理）在不同浓度（0~50 μmol/L）PMA处理下的Ct值趋于一致，热处理组（部分菌体细胞被杀死）随着PMA处理浓度增加（0~20 μmol/L），Ct值逐渐升高，且至20 μmol/L趋于平稳，与30 μmol/L和50 μmol/L无显著性差异，表明20 μmol/L PMA处理能有效排除死菌细胞对检测结果的干扰。在LM（图3B）和STE（图3C）的PMA处理试验中也获得与SA的一致结果，表明3种目标菌PMA处理的最佳浓度均为20 μmol/L。这与Liang等[27]对大肠杆菌O157:H7、克罗诺杆菌和沙门氏菌的PMA处理结果一致。
2.4　mRT-qPCR特异性试验结果
图4为混合菌液（3株目标菌和18株非目标菌）同时mRT-qPCR检测的结果，只有3种目标菌株表现出S型扩增曲线，且Ct值小于35，而其他18种非目标菌株均未出现扩增曲线。这表明针对3种目标菌建立的mRT-qPCR方法具有良好的特异性，对其他病原菌不存在交叉反应。杨健等[1]认为在mRT-qPCR反应体系中，靶标序列、引物和探针直接决定着扩增的特异性。因此，本试验针对3种致病菌的特异基因序列设计的引物和探针在一定程度上保证了mRT-qPCR扩增的特异性。
2.5　SDS-PMA-mRT-qPCR标准曲线建立和检出限确定结果
图5A为不同浓度梯度菌液的SDS-PMA-mRT-qPCR扩增结果，SA、LM和STE为102~107 CFU/mL时，均表现出S型扩增曲线，且为有效扩增（Ct值<35）。以菌液浓度的常用对数为横坐标（x）和对应的Ct值为纵坐标（y）绘制标准曲线，结果如图5B所示，SA、LM和STE在102~107 CFU/mL范围内均表现出良好的线性关系，且相关系数（R2）在0.9992~0.9998之间，扩增效率（E）在102.401%~104.893%之间，满足qPCR扩增相关系数不得低于0.99、扩增效率在90%~105%之间的要求[21]。这说明设计的引物和探针与靶基因序列匹配良好，qPCR扩增体系优良。综上所述，3种目标菌的SDS-PMA-mRT-qPCR检出限为102 CFU/mL。
2.6　SDS-PMA-mRT-qPCR重复性试验结果
表2为不同浓度梯度（102~104 CFU/mL）菌液的SDS-PMA-mRT-qPCR检测结果，SA、LM、STE在不同批次内和批次间的Ct值变异系数均在0.12%至0.81%之间，小于1%。在qPCR反应体系中，通常将Ct值变异系数<3%作为重复性良好的标准[28]。可见，本试验建立的SDS-PMA-mRT-qPCR检测方法具有良好的重复稳定性。
2.7　模拟样品检测结果
采用SDS-PMA-mRT-qPCR和国家标准中的培养法分别对人工污染的不同类型的乳品试样（牛奶、酸奶、奶粉和奶酪）进行检测，结果统计如表3所示，两种方法的检测结果完全一致。相较于传统培养法的检测周期5~7 d，SDS-PMA-mRT-qPCR方法缩短至7 h。这表明建立的SDS-PMA-mRT-qPCR方法可以应用于乳品中SA、LM和STE的快速检测。在后续的研究中，需要对该方法进一步优化和完善，以更广泛地应用于其他各类食品的安全检测[20]。
3　结论
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TaqMan定量PCR在PCR反应体系中加入荧光探针，实现了扩增和检测的同时并行，解决了普通PCR需要对扩增产物进行电泳才能获得检测结果的局限性。本研究建立的乳品中SA、LM、STE的SDS-PMA-mRT-qPCR检测方法，即在一个PCR反应管中同时定量检测3种目标菌的活菌，达到了操作过程简便、检测周期短、检测效率高的目的，同时消除了死菌细胞造成检测结果的假阳性的影响。
在本研究中，通过对含有3株目标菌和18株非目标菌的混合菌液进行mRT-qPCR扩增，结果显示3株目标菌呈现出S型扩增曲线，而18株非目标菌均无典型的扩增曲线，说明以SA的侵袭蛋白基因invA、LM的溶血素O基因hly和STE的毒力基因stx的保守区序列为靶标设计引物和探针的SDS-PMA-mRT-qPCR方法具有良好的特异性。此外，本方法对3种目标菌的检出限都达到了102 CFU/mL，这说明本文建立的SDS-PMA-mRT-qPCR检测方法具有更高的灵敏度，在实际乳品安全检测的应用中更加准确可靠。此外，采用本文建立的SDS-PMA-mRT-qPCR方法对SA、LM、STE污染的多种乳品检测的结果与国家标准中的微生物培养法完全一致，且相较于国标培养法5~7 d的检测周期，本研究建立的方法可以将检测周期缩短至7 h，大大节约了时间成本、提高了检测效率。
综上所述，本研究针对SA、LM和STE建立的SDS-PMA-mRT-qPCR检测方法特异性强、灵敏度高、稳定性好、快速高效，可以应用于乳品中食源性致病菌的安全监测。

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2026年第47卷第1期
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doi: 10.13386/j.issn1002-0306.2024120184
  • 接收时间：2024-12-16
  • 首发时间：2026-10-24
  • 出版时间：2026-01-01
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  • 收稿日期：2024-12-16
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    河北科技大学食品与生物学院，河北石家庄 050018

通讯作者:

艾鹏飞（1974−），男，博士，教授，研究方向：生物化学与分子生物学，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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