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Article(id=1153986792152883781, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986777279877909, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20240920002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1726761600000, receivedDateStr=2024-09-20, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1753061491287, onlineDateStr=2025-07-21, pubDate=1736870400000, pubDateStr=2025-01-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1753061491287, onlineIssueDateStr=2025-07-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1753061491287, creator=13701087609, updateTime=1753061491287, updator=13701087609, issue=Issue{id=1153986777279877909, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='1', 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=0, createTime=1753061487741, creator=13701087609, updateTime=1757901302572, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1174286432060453412, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986777279877909, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1174286432060453413, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986777279877909, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=151, endPage=157, ext={EN=ArticleExt(id=1153986793121768019, articleId=1153986792152883781, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Rapid identification of Cronobacter sakazaki by real-time fluorescence polymerase chain reaction, columnId=1153986783114154916, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Detection and Prevention of Foodborne Pathogenic Microorganisms, runingTitle=null, highlight=null, articleAbstract=

Objective To establish subsequently the real-time quantitative polymerase chain reaction (PCR) method that could quickly and accurately identify Cronobacter sakazakii, and design specific primer probes based on the DNA gyrB subunit gene. Methods The target gene sequences were searched and downloaded from National Center for Biotechnology Information (NCBI), sequence comparison was performed using DNAMAN, and primer probes were designed by Primer Express software. This established real-time quantitative PCR method was validated through specificity tests, absolute sensitivity tests, relative sensitivity tests and anti-interference tests. The 40 common pathogenic bacteria standard strains were selected for specificity validation. Results The results of multi-dimensional specificity validation showed that the method was able to specifically detect Cronobacter sakazakii, and there was no non-specific amplification for other closely related Cronobacter and common pathogenic bacteria in food. DNA detection sensitivity was 0.0100 ng/μL, while relative sensitivity was 103 CFU/mL. The anti-interference experiment results showed that mixing interfering bacteria and their DNA with Cronobacter sakazakii DNA and Cronobacter sakazakii did not significantly affect the detection results, indicating that this method had good anti-interference ability. Conclusion The primer probes designed in this study are specific, rapid, sensitive and anti-interference for the detection of Cronobacter sakazakii in food samples under real-time fluorescence PCR method. It can provide technical support for detection of Cronobacter sakazakii in the future.

, correspAuthors=Zhi-Kai HU, 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=Yue-Chuan ZHANG, Qing-Long WANG, Shuang LI, Yu-Ting WANG, Qing-Yao LI, Shuang ZHANG, Lin WU, Xiao-Yu YUAN, Zhi-Kai HU), CN=ArticleExt(id=1153986825283691330, articleId=1153986792152883781, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=实时荧光聚合酶链式反应法快速鉴定阪崎克罗诺杆菌, columnId=1153986783244178342, journalTitle=食品安全质量检测学报, columnName=专题：食源性病原微生物检测与防控, runingTitle=null, highlight=null, articleAbstract=

目的 根据DNA旋转酶B亚基(gyrB)基因设计特异性的引物探针, 建立一种能够快速准确鉴定阪崎克罗诺杆菌的实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)方法。方法 寻找并在美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)中下载目标基因序列, 使用DNAMAN进行序列比对, Primer Express软件设计引物探针。通过特异性实验、绝对灵敏度、相对灵敏性实验、抗干扰实验对所建立方法进行方法验证。选择本实验室保存的36种40株食品中常见致病菌的标准菌株进行特异性验证。结果 多维度特异性验证实验结果显示该方法能够特异性地检测出阪崎克罗诺杆菌, 对亲缘关系较近的其他克罗诺杆菌及食品中较为常见的致病菌均无非特异性扩增。DNA检测灵敏度可以达到0.0100 ng/μL, 相对灵敏度可以达到103 CFU/mL。抗干扰实验结果显示, 将干扰菌和干扰菌DNA分别与阪崎克罗诺杆菌和阪崎克罗诺杆菌DNA进行混合检测, 对检测结果无显著影响, 说明该方法抗干扰能力良好。结论 本研究所设计的引物探针在实时荧光PCR方法下对食品样品中阪崎克罗诺杆菌的检测具有特异、快速、敏感和抗干扰的特点, 可为以后食品中阪崎克罗诺杆菌的检测提供技术支撑。

, correspAuthors=胡智恺, authorNote=null, correspAuthorsNote=
*胡智恺(1991—), 男, 硕士, 高级工程师, 主要研究方向为食品检验检测。E-mail:
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张跃川(1990—), 男, 工程师, 主要研究方向为食品检验检测。E-mail:

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张跃川(1990—), 男, 工程师, 主要研究方向为食品检验检测。E-mail:

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张跃川(1990—), 男, 工程师, 主要研究方向为食品检验检测。E-mail:

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Journal of Food Safety & Quality, 2024, 15 (21): 22-31., articleTitle=Rapid detection of four foodborne pathogenic Vibrio bacteria by real-time fluorescence quantitative polymerase chain reaction, refAbstract=null)], funds=[Fund(id=1174370064485724738, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, awardId=2022YFF1100902, language=CN, fundingSource=国家重点研发计划项目(2022YFF1100902), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1174370058873745855, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, xref=null, ext=[AuthorCompanyExt(id=1174370058877940160, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, companyId=1174370058873745855, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Beijing Institute of Food Inspection and Research (Beijing Municipal Center for Food Safety Monitoring and Risk Assessment), Beijing 100094, China), AuthorCompanyExt(id=1174370058882134465, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, companyId=1174370058873745855, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京市食品检验研究院(北京市食品安全监控和风险评估中心), 北京 100094)])], figs=[ArticleFig(id=1174370061935587839, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Fig.1, caption=Specificity test results of real-time fluorescence PCR, figureFileSmall=ayWXv/7Bczyek3fVA6CrQw==, figureFileBig=quda2r0WHqIAd58gVoUZzA==, tableContent=null), ArticleFig(id=1174370062044639746, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=图1, caption=实时荧光PCR特异性实验测试结果, figureFileSmall=ayWXv/7Bczyek3fVA6CrQw==, figureFileBig=quda2r0WHqIAd58gVoUZzA==, tableContent=null), ArticleFig(id=1174370062115942915, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Fig.2, caption=Absolute sensitivity test result curves of the real-time fluorescence PCR, figureFileSmall=dWj7QXwgRWukpWQC3kCm/g==, figureFileBig=RIH0YP8Rh/chw2yWJBC6Og==, tableContent=null), ArticleFig(id=1174370062191440391, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=图2, caption=实时荧光PCR实验的绝对灵敏度实验结果曲线, figureFileSmall=dWj7QXwgRWukpWQC3kCm/g==, figureFileBig=RIH0YP8Rh/chw2yWJBC6Og==, tableContent=null), ArticleFig(id=1174370062283715083, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Fig.3, caption=Relative sensitivity test result curves of the real-time fluorescence PCR, figureFileSmall=Qx018wWeJt808KHBCIk5Ag==, figureFileBig=UNHoM2Pm7OZejAs7GO1a6A==, tableContent=null), ArticleFig(id=1174370062392766992, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=图3, caption=实时荧光PCR实验的相对灵敏度实验结果曲线, figureFileSmall=Qx018wWeJt808KHBCIk5Ag==, figureFileBig=UNHoM2Pm7OZejAs7GO1a6A==, tableContent=null), ArticleFig(id=1174370062459875858, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Fig.4, caption=Anti-disturbance ability test result curves of the real-time fluorescence PCR method at the level of the genome, figureFileSmall=2FOExUIBYUhdChmyShRCgw==, figureFileBig=I5Pl/YZJCTXdJw5daGoqcQ==, tableContent=null), ArticleFig(id=1174370062573122068, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=图4, caption=实时荧光PCR基因组水平的抗干扰实验结果曲线, figureFileSmall=2FOExUIBYUhdChmyShRCgw==, figureFileBig=I5Pl/YZJCTXdJw5daGoqcQ==, tableContent=null), ArticleFig(id=1174370062740894232, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Fig.5, caption=Anti-disturbance ability test result curves of the real-time fluorescence PCR method at the culturel level, figureFileSmall=H4teqNTkqYAnYm+E4RKiTA==, figureFileBig=s6xdbRdkO3PdDUDdJyxSfg==, tableContent=null), ArticleFig(id=1174370062824780314, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=图5, caption=实时荧光PCR培养物水平的抗干扰实验结果曲线, figureFileSmall=H4teqNTkqYAnYm+E4RKiTA==, figureFileBig=s6xdbRdkO3PdDUDdJyxSfg==, tableContent=null), ArticleFig(id=1174370063076438561, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Table 1, caption=

Names and serial numbers of standard strains

, figureFileSmall=null, figureFileBig=null, tableContent=
菌种名称 菌株编号 菌中名称 菌株编号
阪崎克罗诺杆菌 IQCC10403 大肠埃希氏菌 ATCC25922
阪崎克罗诺杆菌 ATCC29544 大肠杆菌 CMCC44747
穆汀斯克罗诺杆菌 ATCC51329 大肠杆菌 CMCC44752
穆汀斯克罗诺杆菌 DSM21870 弗氏柠檬酸杆菌 ATCC43864
传奇克罗诺杆菌 DSM27963 产气肠杆菌 ATCC13048
苏黎世克罗诺杆菌 DSM18703 产酸克雷伯 ATCC700324
都柏林克罗诺杆菌 DSM18705 弗氏柠檬酸杆菌 CICC10404
丙二酸盐阳性克罗诺杆菌 DSM18702 痢疾志贺氏菌 CMCC(B)51105
调味品克罗诺杆菌 DSM27966 福氏志贺氏菌 CICC21534
鼠伤寒沙门氏菌 CMCC50013 粪肠球菌 ATCC29212
丙型副伤寒沙门氏菌 CMCC50017 铜绿假单胞菌 ATCC27853
乙型副伤寒沙门氏菌 CMCC50004 荧光假单胞菌 CICC20066
甲型副伤寒沙门氏菌 CMCC50001 恶臭假单胞 CGMCC1.2309
亚利桑纳沙门氏菌 CMCC47001 杀鲑气单胞菌 CICC23564
甲型副伤寒沙门 ATCC9150 洋葱伯克霍尔德氏菌 CICC21896
鸭沙门氏菌 ATCC9270 普通变形菌 CMCC(B)49027
肠沙门氏菌肠亚种肠炎血清型 ATCC13076 单核增生李斯特氏菌 ATCC19115
布伦登卢普沙门氏菌 ATCCH9812 肠道出血性大肠埃希氏菌 CICC21530
肠沙门氏菌肠亚种伤寒血清型 CMCC(B)50071 肠道集聚性大肠埃希氏菌 CICC24186
肠沙门氏菌肠亚种鼠伤寒血清型 ATCC14028 肠道致病性大肠埃希氏菌 CICC24189
(Ituri)伊图里沙门氏菌 ATCC15611 产肠毒素大肠埃希氏菌 CICC10667
亚利桑纳沙门氏菌 CMCC47001 阴沟肠杆菌 CICC10450
大肠埃希氏菌 ATCC8739 产志贺毒素大肠埃希氏菌 CICC10670
大肠埃希氏菌 CICC10354 肠道侵袭性大肠埃希氏菌 CICC24188
大肠埃希氏菌 AS1.3373
), ArticleFig(id=1174370063164518946, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=表1, caption=

标准菌株的名称及编号

, figureFileSmall=null, figureFileBig=null, tableContent=
菌种名称 菌株编号 菌中名称 菌株编号
阪崎克罗诺杆菌 IQCC10403 大肠埃希氏菌 ATCC25922
阪崎克罗诺杆菌 ATCC29544 大肠杆菌 CMCC44747
穆汀斯克罗诺杆菌 ATCC51329 大肠杆菌 CMCC44752
穆汀斯克罗诺杆菌 DSM21870 弗氏柠檬酸杆菌 ATCC43864
传奇克罗诺杆菌 DSM27963 产气肠杆菌 ATCC13048
苏黎世克罗诺杆菌 DSM18703 产酸克雷伯 ATCC700324
都柏林克罗诺杆菌 DSM18705 弗氏柠檬酸杆菌 CICC10404
丙二酸盐阳性克罗诺杆菌 DSM18702 痢疾志贺氏菌 CMCC(B)51105
调味品克罗诺杆菌 DSM27966 福氏志贺氏菌 CICC21534
鼠伤寒沙门氏菌 CMCC50013 粪肠球菌 ATCC29212
丙型副伤寒沙门氏菌 CMCC50017 铜绿假单胞菌 ATCC27853
乙型副伤寒沙门氏菌 CMCC50004 荧光假单胞菌 CICC20066
甲型副伤寒沙门氏菌 CMCC50001 恶臭假单胞 CGMCC1.2309
亚利桑纳沙门氏菌 CMCC47001 杀鲑气单胞菌 CICC23564
甲型副伤寒沙门 ATCC9150 洋葱伯克霍尔德氏菌 CICC21896
鸭沙门氏菌 ATCC9270 普通变形菌 CMCC(B)49027
肠沙门氏菌肠亚种肠炎血清型 ATCC13076 单核增生李斯特氏菌 ATCC19115
布伦登卢普沙门氏菌 ATCCH9812 肠道出血性大肠埃希氏菌 CICC21530
肠沙门氏菌肠亚种伤寒血清型 CMCC(B)50071 肠道集聚性大肠埃希氏菌 CICC24186
肠沙门氏菌肠亚种鼠伤寒血清型 ATCC14028 肠道致病性大肠埃希氏菌 CICC24189
(Ituri)伊图里沙门氏菌 ATCC15611 产肠毒素大肠埃希氏菌 CICC10667
亚利桑纳沙门氏菌 CMCC47001 阴沟肠杆菌 CICC10450
大肠埃希氏菌 ATCC8739 产志贺毒素大肠埃希氏菌 CICC10670
大肠埃希氏菌 CICC10354 肠道侵袭性大肠埃希氏菌 CICC24188
大肠埃希氏菌 AS1.3373
), ArticleFig(id=1174370063281959461, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Table 2, caption=

Primers and the probe sequence of real-time fluorescence PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
目标菌名称 目标基因 引物探针序列 Ganbank号
阪崎克罗诺杆菌 gyrB StF: 5’-ACGCTGAACGCCTATATGGA-3’
StR: 5’-TAACGGACACAACGGCAATC-3’
StP: 5’-FAM-CGCGCGTCATCGCCGGTAGC-TAMRA-3’		 JX983606.1
), ArticleFig(id=1174370063374234152, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=表2, caption=

实时荧光PCR引物和探针序列

, figureFileSmall=null, figureFileBig=null, tableContent=
目标菌名称 目标基因 引物探针序列 Ganbank号
阪崎克罗诺杆菌 gyrB StF: 5’-ACGCTGAACGCCTATATGGA-3’
StR: 5’-TAACGGACACAACGGCAATC-3’
StP: 5’-FAM-CGCGCGTCATCGCCGGTAGC-TAMRA-3’		 JX983606.1
), ArticleFig(id=1174370063504257578, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Table 3, caption=

Absolute sensitivity test results of the real-time fluorescence PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
菌名 质量浓度/(ng/μL)
10.0000 1.0000 0.1000 0.0100 0.0010 0.0001
阪崎克罗诺杆菌ATCC29544 20/20 20/20 20/20 20/20 18/20 0/20
), ArticleFig(id=1174370063655252526, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=表3, caption=

实时荧光PCR实验的绝对灵敏度实验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
菌名 质量浓度/(ng/μL)
10.0000 1.0000 0.1000 0.0100 0.0010 0.0001
阪崎克罗诺杆菌ATCC29544 20/20 20/20 20/20 20/20 18/20 0/20
), ArticleFig(id=1174370063747527216, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Table 4, caption=

Relative sensitivity test results of the real-time fluorescence PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
基质 菌名 菌浓度/(CFU/mL)
107 106 105 104 103 102 101
BPW 1:10溶解的婴幼儿配方奶粉 阪崎克罗诺杆菌ATCC29544 20/20 20/20 20/20 20/20 20/20 18/20 14/20
), ArticleFig(id=1174370063923687987, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=表4, caption=

实时荧光PCR实验的相对灵敏度实验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
基质 菌名 菌浓度/(CFU/mL)
107 106 105 104 103 102 101
BPW 1:10溶解的婴幼儿配方奶粉 阪崎克罗诺杆菌ATCC29544 20/20 20/20 20/20 20/20 20/20 18/20 14/20
), ArticleFig(id=1174370064011768373, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Table 5, caption=

Anti-disturbance ability of the real-time fluorescence PCR method at the level of the genome

, figureFileSmall=null, figureFileBig=null, tableContent=
质量浓度
/(ng/μL)		 Ct值
1:1 (V:V)
添加TE*		 1:1 (V:V)添加
大肠杆菌DNA
100.000 18.8 18.6
10.000 22.0 22.0
1.000 25.4 25.4
0.100 28.7 28.8
0.010 32.3 32.0
0.001 35.8 35.9
阳性对照 17.5 17.5
阴性对照 — —
空白对照 — —
), ArticleFig(id=1174370064112431671, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=表5, caption=

实时荧光PCR基因组水平的抗干扰实验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
质量浓度
/(ng/μL)		 Ct值
1:1 (V:V)
添加TE*		 1:1 (V:V)添加
大肠杆菌DNA
100.000 18.8 18.6
10.000 22.0 22.0
1.000 25.4 25.4
0.100 28.7 28.8
0.010 32.3 32.0
0.001 35.8 35.9
阳性对照 17.5 17.5
阴性对照 — —
空白对照 — —
), ArticleFig(id=1174370064208900665, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=EN, label=Table 6, caption=

Anti-disturbance ability of the real-time fluorescence PCR method at the culture level

, figureFileSmall=null, figureFileBig=null, tableContent=
阪崎肠杆菌/(CFU/mL) Ct值
添加1 mL
无菌水*		 添加1 mL 107 CFU/mL
大肠杆菌
108 18.9 19.1
107 22.5 22.8
106 26.2 26.2
105 29.3 29.2
104 32.6 33.0
103 35.6 36.2
阳性对照 17.5 17.5
阴性对照 — —
空白对照 — —
), ArticleFig(id=1174370064343118396, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986792152883781, language=CN, label=表6, caption=

实时荧光PCR培养物水平的抗干扰实验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
阪崎肠杆菌/(CFU/mL) Ct值
添加1 mL
无菌水*		 添加1 mL 107 CFU/mL
大肠杆菌
108 18.9 19.1
107 22.5 22.8
106 26.2 26.2
105 29.3 29.2
104 32.6 33.0
103 35.6 36.2
阳性对照 17.5 17.5
阴性对照 — —
空白对照 — —
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实时荧光聚合酶链式反应法快速鉴定阪崎克罗诺杆菌
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张跃川 , 王青龙 , 李爽 , 王雨婷 , 李庆尧 , 张爽 , 吴林 , 袁晓雨 , 胡智恺 *
食品安全质量检测学报 | 专题：食源性病原微生物检测与防控 2025,16(1): 151-157
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食品安全质量检测学报 | 专题：食源性病原微生物检测与防控 2025, 16(1): 151-157
实时荧光聚合酶链式反应法快速鉴定阪崎克罗诺杆菌
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张跃川 , 王青龙, 李爽, 王雨婷, 李庆尧, 张爽, 吴林, 袁晓雨, 胡智恺*
作者信息
  • 北京市食品检验研究院(北京市食品安全监控和风险评估中心), 北京 100094

    • 张跃川(1990—), 男, 工程师, 主要研究方向为食品检验检测。E-mail:

通讯作者:

*胡智恺(1991—), 男, 硕士, 高级工程师, 主要研究方向为食品检验检测。E-mail:
Rapid identification of Cronobacter sakazaki by real-time fluorescence polymerase chain reaction
Yue-Chuan ZHANG , Qing-Long WANG, Shuang LI, Yu-Ting WANG, Qing-Yao LI, Shuang ZHANG, Lin WU, Xiao-Yu YUAN, Zhi-Kai HU*
Affiliations
  • Beijing Institute of Food Inspection and Research (Beijing Municipal Center for Food Safety Monitoring and Risk Assessment), Beijing 100094, China
出版时间: 2025-01-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20240920002
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摘要
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目的 根据DNA旋转酶B亚基(gyrB)基因设计特异性的引物探针, 建立一种能够快速准确鉴定阪崎克罗诺杆菌的实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)方法。方法 寻找并在美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)中下载目标基因序列, 使用DNAMAN进行序列比对, Primer Express软件设计引物探针。通过特异性实验、绝对灵敏度、相对灵敏性实验、抗干扰实验对所建立方法进行方法验证。选择本实验室保存的36种40株食品中常见致病菌的标准菌株进行特异性验证。结果 多维度特异性验证实验结果显示该方法能够特异性地检测出阪崎克罗诺杆菌, 对亲缘关系较近的其他克罗诺杆菌及食品中较为常见的致病菌均无非特异性扩增。DNA检测灵敏度可以达到0.0100 ng/μL, 相对灵敏度可以达到103 CFU/mL。抗干扰实验结果显示, 将干扰菌和干扰菌DNA分别与阪崎克罗诺杆菌和阪崎克罗诺杆菌DNA进行混合检测, 对检测结果无显著影响, 说明该方法抗干扰能力良好。结论 本研究所设计的引物探针在实时荧光PCR方法下对食品样品中阪崎克罗诺杆菌的检测具有特异、快速、敏感和抗干扰的特点, 可为以后食品中阪崎克罗诺杆菌的检测提供技术支撑。

实时荧光聚合酶链式反应  /  阪崎克罗诺杆菌  /  gyrB基因

Objective To establish subsequently the real-time quantitative polymerase chain reaction (PCR) method that could quickly and accurately identify Cronobacter sakazakii, and design specific primer probes based on the DNA gyrB subunit gene. Methods The target gene sequences were searched and downloaded from National Center for Biotechnology Information (NCBI), sequence comparison was performed using DNAMAN, and primer probes were designed by Primer Express software. This established real-time quantitative PCR method was validated through specificity tests, absolute sensitivity tests, relative sensitivity tests and anti-interference tests. The 40 common pathogenic bacteria standard strains were selected for specificity validation. Results The results of multi-dimensional specificity validation showed that the method was able to specifically detect Cronobacter sakazakii, and there was no non-specific amplification for other closely related Cronobacter and common pathogenic bacteria in food. DNA detection sensitivity was 0.0100 ng/μL, while relative sensitivity was 103 CFU/mL. The anti-interference experiment results showed that mixing interfering bacteria and their DNA with Cronobacter sakazakii DNA and Cronobacter sakazakii did not significantly affect the detection results, indicating that this method had good anti-interference ability. Conclusion The primer probes designed in this study are specific, rapid, sensitive and anti-interference for the detection of Cronobacter sakazakii in food samples under real-time fluorescence PCR method. It can provide technical support for detection of Cronobacter sakazakii in the future.

real-time quantitative polymerase chain reaction  /  Cronobacter sakazakii  /  gyrB gene
张跃川, 王青龙, 李爽, 王雨婷, 李庆尧, 张爽, 吴林, 袁晓雨, 胡智恺. 实时荧光聚合酶链式反应法快速鉴定阪崎克罗诺杆菌. 食品安全质量检测学报, 2025 , 16 (1) : 151 -157 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240920002
Yue-Chuan ZHANG, Qing-Long WANG, Shuang LI, Yu-Ting WANG, Qing-Yao LI, Shuang ZHANG, Lin WU, Xiao-Yu YUAN, Zhi-Kai HU. Rapid identification of Cronobacter sakazaki by real-time fluorescence polymerase chain reaction[J]. Journal of Food Safety & Quality, 2025 , 16 (1) : 151 -157 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240920002
正文
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0 引言
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克罗诺杆菌属(Cronobacter spp.)为革兰氏阴性杆菌, 属于肠杆菌科, 是一种有鞭毛、兼性厌氧、且能运动的食源性条件致病菌, 常寄生在动物和人肠道内[1-2]。公开资料显示, 克罗诺杆菌属有7个种和3个亚种。种包括阪崎克罗诺杆菌(Cronobacter sakazakii)、丙二酸盐阳性克罗诺杆菌(Cronobacter malonaticus)、穆汀斯克罗诺杆菌(Cronobacter muytjensii)、都柏林克罗诺杆菌(Cronobacter dublinensis)、传奇克罗诺杆菌(Cronobacter universalis)、苏黎世克罗诺杆菌(C. turicensisi)、调味品克罗诺杆菌(Cronobacter condimenti); 亚种包括都柏林克罗诺杆菌洛桑亚种(Cronobacter dublinensis subsp. lausannensis)、都柏林克罗诺杆菌粉乳亚种(Cronobacter dublinensis subsp. lactaridi)、都柏林克罗诺杆菌都柏林亚种(Cronobacter dublinensis subsp. Dublinensis)[3-4]。
克罗诺杆菌是一种耐热菌, 该菌可以在最高47 ℃的条件下生长, 具有耐高渗透压和抗干燥的特点, 特别是能够在水活度非常低的乳粉中存活两年以上。作为一种食源性条件致病菌, 主要感染免疫力低的老人和儿童, 其能够引起新生儿以及抵抗力不足的幼儿患有脑膜炎、坏死性小肠结肠炎甚至可以引起败血症等能够致人死亡的疾病[5-7]。新生儿或者早产儿在食用被克罗诺杆菌污染的婴幼儿配方乳粉时均存在感染的风险, 而一旦感染克罗诺杆菌其致死率高达40%~80%[8]。根据目前报道可以发现, 克罗诺杆菌在自然界中分布广泛, 可以在乳粉、土壤、蔬菜、污水、谷物制品、肉制品以及动物和人的粪便中生存[9-11]。其分布虽然广泛但并不是所有的克罗诺杆菌对人类都具有致病性, 7种不同的克罗诺杆菌的致病能力也有比较大的差异[12-13]。阪崎克罗诺杆菌已被世界卫生组织列为婴幼儿配方乳粉的A类致病菌。近年来, 国内很多省份报道从婴幼儿配方乳粉和谷物及相关制品等食品中检出阪崎克罗诺杆菌。因此, 对克罗诺杆菌属内进行种水平的快速鉴定具有非常重要的实际意义。
常规生化方法对克罗诺杆菌的检测耗时耗力还经常导致结果的不准确, 比如FDA(2002)、ISO(2006)、GB 4789.40—2016《食品安全国家标准 食品微生物学检验 克罗诺杆菌属(阪崎肠杆菌)检验》等方法[14-15]。核酸检测是在DNA水平进行检测的技术, 常用的核酸检测技术有探针杂交法、聚合酶链式反应(polymerase chain reaction, PCR)法、实时荧光PCR方法、脉冲场凝胶电泳(pulsed-field gel electrophoresis, PFGE)、多位点序列分型(multilocus sequence typing, MLST)和全基因组测序(whole genome sequencing, WGS)等[16-18], 实时荧光PCR方法具有检测周期短、灵敏度高、特异性强等优点, 能从基因层面对对克罗诺杆菌进行检测。2024年我国发布了克罗诺杆菌的检验国家标准GB 4789.40—2024《食品安全国家标准 食品微生物学检验 克罗诺杆菌检验》, 新标准增加了克罗诺杆菌属鉴定的PCR方法, 此次修订大大减轻了对克罗诺杆菌的检测压力并提高了克罗诺杆菌的准确度, 但该方法只是在属水平对克罗诺杆菌进行鉴定。CHEN等[19]采用PCR检测技术对苏黎世克罗诺杆菌进行检测, WANG等[20]采用多重PCR方法对阪崎克罗诺杆菌、丙二酸盐克罗诺杆菌、苏黎世克罗诺杆菌进行检测, 以上是采用普通PCR方法对克罗诺杆菌进行鉴定。此外, 国内外有关实时荧光PCR方法在属水平对克罗诺杆菌鉴定的方法也多有报道, 选择的目标基因一般有: 大分子合成(MMS)操纵子序列、16S rRNA基因、外膜蛋白A基因(OmpA)、16S-23S rRNA内部转录间隔区(ITS)等[21-26]。本研究选择促旋酶(gyrase) B亚单位基因gyrB作为目标基因进行实验, 根据文献报道和基因序列比对发现, 该基因在亲缘关系较近的菌种鉴定中明显好于16S rRNA基因[27-31]。
本研究设计了引物探针, 并构建了实时荧光PCR方法, 此方法可为食品中阪崎克罗诺杆菌种检测工作提供技术支撑。
1 材料与方法
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1.1 实验菌株
阪崎克罗诺杆菌标准菌2株、克罗诺杆菌属内其克罗诺杆菌共6种7株、阴沟肠杆菌、大肠杆菌、沙门氏菌等其他细菌的标准菌株共36种40株。这些菌株分别来自中国医学微生物菌种保藏管理中心(National Center for Medical Culture Collection, CMCC)、美国典型菌种保藏中心(American Type Culture Collection, ATCC)、中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center, CGMCC)以及中国工业微生物菌种保藏管理中心(China Center Of Industrial Culture Collection, CICC), 具体的菌株信息见表1。
1.2 试剂与仪器
缓冲蛋白胨水(buffered peptone water, BPW)干粉培养基(美国BD公司); TSA干粉培养基、血琼脂平板即用培养基(北京陆桥技术有限公司)。
Taqman master mix、细菌基因组盒(编号DP302-02)、ABI QuantStudio 7实时荧光PCR仪(美国ABI公司); VITEK® Compact微生物分析系统(法国生物梅里埃公司)。
1.3 方法
1.3.1 菌株活化及处理
首先把冷冻保存于-80 ℃的标准菌株直接转接于血琼脂培养基上, 37 ℃需氧培养24 h±2 h, 然后调取单个菌落转接到BPW中进行传代, 传代两代后充分恢复菌种活力, 用于后续实验。
1.3.2 DNA提取
依据细菌DNA提取试剂盒说明书开展DNA提取工作。
1.3.3 引物探针设计
从GenBank网站下载已公布的阪崎克罗诺杆菌及克罗诺杆菌属内其他菌株的gyrB基因序列, 利用DNAMAN软件对这些序列进行比对, 从中挑选出一段特异性的基因目标序列片段, 以此作为引物探针设计目标片段。随后用Primer Express 3.0.1软件设计相应的引物和探针。设计完成后, 将所获引物和探针序列在NCBI网站上进行primerBlast比对, 筛选出特异性良好的引物探针序列, 并送往上海生物工程有限公司进行合成。引物探针基因序列详见表2。
1.3.4 实时荧光PCR的反应体系与参数详情
实时荧光PCR的反应体系总体积为25 μL, 其具体组成如下: 上下游引物对(浓度为10 µmol/L)各1 µL、探针(浓度10 µmol/L) 0.5 µL、模板DNA 1 µL、2×master mix 12.5 µL、ddH2O 9 µL。
实时荧光PCR的反应参数设定为: 首先在50 ℃保持2 min, 接着在95 ℃进行预变性, 时长为10 min。之后进入循环阶段, 95 ℃变性持续5 s, 66 ℃退火延伸持续40 s, 此循环共进行40次, 同时收集FAM荧光信号, 反应结束后在4 ℃保存反应产物。
结果判读规则如下: 若实时荧光PCR的扩增曲线呈现典型的S型曲线, 且Ct值小于35, 则判定为阳性; 若Ct值处于35~40之间, 则判定为可疑, 此时需要重复实验, 若重复实验后的Ct值仍然在35~40这个区间, 也可判定为阳性; 若Ct值大于40, 则判定为阴性。
1.3.5 特异性、灵敏性实验
特异性验证实验包括阪崎克罗诺杆菌标准菌2株、克罗诺杆菌属内其克罗诺杆菌共7株、阴沟肠杆菌、大肠杆菌、沙门氏菌等其他细菌的标准菌株共36种40株。按照1.3.4中的反应体系和反应参数进行PCR扩增。
灵敏性实验分为两个部分, 即绝对灵敏性实验和相对灵敏性实验。
绝对灵敏性实验: 运用DNA提取试剂盒提取阪崎克罗诺杆菌(ATCC29544)的DNA, 随后将提取出的DNA溶液按照梯度稀释, 质量浓度分别为10.0000、1.0000、0.1000、0.0100、0.0010、0.0001 ng/μL。所有这些不同浓度的DNA稀释液在相同条件下进行实时荧光PCR扩增, 每次实验设置5个平行实验, 并且重复实验4次。依据1.3.4中的结果判读方法对实时荧光PCR的结果进行判读, 再运用统计学方法对结果进行分析, 以此确定该方法的绝对灵敏性下限。
相对灵敏性实验: 使用BPW溶液将婴幼儿配方奶粉按10倍比例溶解, 然后向奶粉溶液中添加阪崎克罗诺杆菌, 使其添加后的菌落终浓度分别为107、106、105、104、103、102、101 CFU/mL, 每个浓度设置5个平行实验, 重复实验4次。采用细菌提取试剂盒提取DNA作为模板, 按照1.3.4中的反应方法进行实时荧光PCR扩增, 并对扩增结果进行判读, 利用统计学方法分析结果以确定该方法的相对灵敏性下限。按照1.3.4中的反应体系和反应参数进行实时荧光PCR扩增, 此过程重复20次, 记录阳性结果出现的次数。
1.3.6 抗干扰实验
本研究设计的抗干扰实验包括基因组水平和培养物水平两个层面的抗干扰实验。
基因组水平的抗干扰实验: 将提取好的阪崎克罗诺杆菌DNA的质量浓度分别稀释至100.000、10.000、1.000、0.100、0.010、0.001 ng/μL, 然后分别按照1:1的体积比例添加质量浓度为100 ng/μL的大肠杆菌(ATCC25922)DNA, 将混合后的DNA作为扩增模板进行实时荧光PCR反应, 每次实验设置5个平行, 重复4次, 记录混合干扰DNA后实验结果的变化情况。
培养物水平的抗干扰实验: 将活性良好的阪崎克罗诺杆菌制成菌悬液, 并将菌悬液浓度依次稀释至108、107、106、105、104 CFU/mL, 之后分别添加等体积的108 CFU/mL大肠杆菌(ATCC25922)菌悬液, 取充分混合后的菌悬液1 mL提取DNA, 将提取后的DNA作为扩增模板进行实时荧光PCR扩增, 每个梯度设置5个平行, 重复4次, 记录添加干扰菌后对实验结果产生的影响。
对照设置: 阳性对照为阪崎克罗诺DNA; 阴性对照为大肠杆菌DNA; 空白对照为无菌水。
1.4 数据处理
使用SPSS 24.0软件通过方差分析的方法对实验结果进行验证, 采用Excel 2019软件来制作表格。
2 结果与分析
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2.1 特异性验证结果分析
从图1可以看出, 以阪崎克罗诺杆菌DNA为模板时, 本研究所设计的引物探针对应的反应呈现出典型的S型扩增曲线。然而, 当以其他克罗诺杆菌属内的克罗诺杆菌以及食品中常见致病菌DNA为模板进行反应时, 却并无明显的S型扩增曲线。这一结果充分表明, 本研究所构建的方法对阪崎克罗诺杆菌具有特异性扩增能力, 对于表1中的非克罗诺杆菌和其他常见细菌则无法进行扩增, 由此可见该引物探针的特异性良好。
2.2 灵敏性验证结果分析
2.2.1 绝对灵敏度验证
对提取自阪崎克罗诺杆菌(ATCC29544)的DNA溶液进行梯度稀释, 使其质量浓度依次为10.0000、1.0000、0.1000、0.0100、0.0010、0.0001 ng/μL。在相同的反应条件下, 对所有梯度的稀释液开展扩增操作, 每次实验均设置5个平行样, 且重复实验4次。由表3与图2的结果可知, 本研究设计的阪崎克罗诺杆菌(ATCC29544)引物探针能够稳定检出的DNA质量浓度为0.0100 ng/μL, 其检测下限可达0.0100 ng/μL。依据绝对灵敏度的实验结果, 能够确定本研究建立的实时荧光PCR方法的绝对灵敏度为0.0100 ng/μL。
2.2.2 相对灵敏度验证
取灭菌后的BPW溶液, 按照1:10 (V:V)的比例对婴幼儿配方奶粉进行溶解。接着, 将处于充分活性状态的阪崎克罗诺杆菌添加至奶粉溶液中, 添加后使菌落浓度分别为107、106、105、104、103、102、101 CFU/mL。充分混匀后, 分别取不同菌落浓度的1 mL奶粉溶液用于DNA提取。以提取得到的DNA为模板开展实时荧光PCR实验, 每个浓度的DNA模板均设置5个平行样, 且重复实验4次。依据1.3.4的结果判读方式对该方法的相对灵敏度予以验证。从表4和图3所呈现的结果能够看出, 当菌落浓度为103 CFU/mL时, 本方法能够稳定地检测出目标。据此可以推断, 本研究所涉及的引物探针相对灵敏度可达到103 CFU/mL。
2.3 抗干扰能力的验证结果分析
2.3.1 基因组水平的抗干扰实验结果
首先, 把提取好的阪崎克罗诺杆菌DNA的质量浓度依次稀释至100.000、10.000、1.000、0.100、0.010 ng/μL、0.001 ng/μL。随后, 针对每一个稀释浓度的阪崎克罗诺杆菌DNA, 均按照1:1的体积比例添加质量浓度为100.000 ng/μL的大肠杆菌(ATCC25922) DNA。将这些混合后的DNA当作扩增模板, 用于开展实时荧光PCR反应。每次实验都设置5个平行样, 并且重复进行4次。
实验结果见表5和图4, 发现即使干扰基因(大肠杆菌DNA)的浓度达到目标基因(阪崎克罗诺杆菌DNA)浓度的100000倍时, Ct值仍未出现显著性变化。这一现象充分表明本研究设计的引物探针在基因组水平展现出了出色的抗干扰能力。
2.3.2 培养物水平的抗干扰实验结果
把活性优良的阪崎克罗诺杆菌制成菌悬液, 将该菌悬液浓度依次稀释至108、107、106、105、104、103 CFU/mL。之后, 分别向其中添加等体积的108 CFU/mL大肠杆菌(ATCC25922)菌悬液。取充分混合后的菌悬液1 mL用于提取DNA, 把提取得到的DNA当作扩增模板来开展实时荧光PCR扩增。每个梯度设置5个平行实验, 且重复操作4次。实验结果展示于表6和图5, 从这些结果能够看出, 即便干扰菌大肠杆菌(ATCC25922)的浓度达到目标菌浓度的100000倍, Ct值也未受到显著影响。由此可知, 本研究设计的引物探针在培养物水平具备出色的抗干扰能力。
3 讨论与结论
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传统的阪崎克罗诺杆菌检测主要依赖生化和血清学方法, 这种方式属于劳动密集型, 不仅耗费大量人力物力, 而且准确性不太稳定。已有研究表明, 使用TaqMan探针法的实时荧光PCR法在检测食品样品中的致病菌是可靠的。因此, 在众多更快速、更准确的检测方法中, 实时荧光定量PCR方法最具发展潜力。与常规方法相比, 实时荧光定量PCR方法检测周期短、操作简便, 特异性和灵敏性均较高。由于该方法中活菌存在的步骤较少, 能有效避免活菌交叉污染增殖, 从而防止对结果判断产生不良影响。
在此背景下, 本研究在查阅文献并进行序列比对后, 选取了阪崎克罗诺杆菌中具有高区分度的DNA旋转酶B亚基(gyrB)基因作为目标基因, 设计出可特异性扩增阪崎克罗诺杆菌的引物探针, 以此构建了阪崎克罗诺杆菌的实时荧光PCR检测方法。该方法可用于食品样品中阪崎克罗诺杆菌的检测, 具有出色的灵敏性和稳定性。其绝对灵敏度可达10~100 pg/μL, 相对灵敏度可达10³ CFU/mL, 在菌种和基因层面都有很强的抗干扰能力。同时, 本研究还使用了36种共40株常见致病菌标准菌株对该方法进行特异性验证, 也获得了非常理想的特异性结果。
综上所述, 本研究所阐述的实时荧光定量PCR方法在检测食品中的阪崎克罗诺杆菌时, 具有快速、特异、灵敏的特性, 能够为日后阪崎克罗诺杆菌的日常检测提供有力的技术支持, 不但能提升检测的灵敏性和准确性, 而且能大幅缩短常见检测方法的检测时长。
基金
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  • 国家重点研发计划项目(2022YFF1100902)
文献
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2025年第16卷第1期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20240920002
  • 接收时间：2024-09-20
  • 首发时间：2025-07-21
  • 出版时间：2025-01-15
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  • 收稿日期：2024-09-20
基金
国家重点研发计划项目(2022YFF1100902)
作者信息
    北京市食品检验研究院(北京市食品安全监控和风险评估中心), 北京 100094

通讯作者:

*胡智恺(1991—), 男, 硕士, 高级工程师, 主要研究方向为食品检验检测。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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