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Article(id=1241342456269173739, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241342451043061769, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202308063, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1691078400000, receivedDateStr=2023-08-04, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773888704602, onlineDateStr=2026-03-19, pubDate=1708790400000, pubDateStr=2024-02-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773888704602, onlineIssueDateStr=2026-03-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773888704602, creator=13701087609, updateTime=1773888704602, updator=13701087609, issue=Issue{id=1241342451043061769, tenantId=1146029695717560320, journalId=1227665162245664772, year='2024', volume='51', issue='4', pageStart='577', pageEnd='768', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773888703356, creator=13701087609, updateTime=1773893026321, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241360582939562716, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241342451043061769, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241360582939562717, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241342451043061769, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=705, endPage=712, ext={EN=ArticleExt(id=1241342456843792388, articleId=1241342456269173739, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Establishment of multienzyme isothermal rapid amplification method for detection of Vibrio cholerae O1 and O139 toxigenic strains, columnId=1228016572065837304, journalTitle=Modern Preventive Medicine, columnName=Experimental Technology and Applications, runingTitle=null, highlight=null, articleAbstract=
Objective

To establish a rapid and accurate method for the detection of Vibrio cholerae O1 and O139 toxigenic strains.

Methods

On the basis of multienzyme isothermal rapid amplification technology, primers and probes were designed according to the hemolysin coding gene, cholera toxin coding gene and O antigen coding genes of Vibrio cholerae O1 and O139, and the detection method was preliminary established. With DNA of different Serotypes of Vibrio cholerae and other bacteria used as templates, the specificity and sensitivity of the detection method was verified.

Results

When the probe addition amount of the reaction system was 0.6μL as recommended by kit instructions, it could successfully amplify four target genes including hemolysin coding gene, cholera toxin coding gene, and O antigen coding genes of Vibrio cholerae O1 and O139. When the probe addition amount was respectively optimized to 1.0 μL, 1.0 μL, 1.0 μL, 0.8 μL of the above-mentioned four genes, the amplification efficiency was higher. After optimizing probe addition, the specificity of the detection method was verified and each primer probe combination did not cross react with non target bacterial DNA. When conducting sensitivity analysis of the detection method, the genomic detection sensitivity of different target genes was 70fg or 290fg. The above results showed that the sensitivity and specificity of the detection method meet the design requirements.

Conclusion

A new method for the detection of Vibrio cholerae O1 and O139 toxigenic strains has been established. This method has characteristics of strong specificity, high sensitivity, and short detection time. It can be applied to the routine detection and rapid detection of Vibrio cholerae, and has a positive role in the prevention of cholera.

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目的

建立一种霍乱弧菌O1群与O139群产毒株快速准确的检测方法。

方法

基于多酶恒温快速扩增技术，根据霍乱弧菌溶血素编码基因、霍乱毒素编码基因、O1群与O139群O抗原编码基因设计引物与探针，初步建立检测方法。以不同血清型霍乱弧菌及其它细菌DNA为模板，验证检测方法的特异性与灵敏度。

结果

配制反应体系时，按试剂盒说明书推荐加入量0.6 μL加入探针时可成功扩增溶血素编码基因、霍乱毒素编码基因及O1群与O139群O抗原编码基因4个目的基因，针对上述4个基因分别优化探针加入量为1.0 μL、1.0 μL、1.0 μL、0.8 μL时扩增效率更高。对优化探针加入量的检测方法进行方法特异性验证时，各引物探针组合不与非目标菌DNA产生交叉反应。进行检测方法的灵敏度分析时，不同目的基因的基因组检测灵敏度为70 fg或290 fg。上述实验结果证明检测方法的灵敏度及特异性满足设计要求。

结论

建立了一种霍乱弧菌O1群与O139群产毒株检测的新方法，该方法特异性强、灵敏度高、检测时间短，可适用于霍乱弧菌的常规检测及快检，对霍乱的预防具有一定的积极作用。

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蔡磊，E-mail：
, copyrightStatement=本刊刊出的所有文章不代表中华预防医学会和本刊编委会的观点，除非特别声明。, copyrightOwner=中华预防医学会和四川大学华西公共卫生学院, extLink=null, articleAbsUrl=null, sourceXml=cYwnjWgUWmNVcuMI7fqmGA==, magXml=ytzBSRafFgHvetnKS3MiDQ==, pdfUrl=null, pdf=9dNjTkvMHDMMguE5RA9zDA==, pdfFileSize=3624727, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=CkNJIaYhUi+zWgrqQvcWhg==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=Rppdqw9xmMRJCfB952bqpw==, mapNumber=null, authorCompany=null, fund=null, authors=

丁卫平（1983－），男，硕士，高级工程师，研究方向：食品药品检测

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丁卫平（1983－），男，硕士，高级工程师，研究方向：食品药品检测

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丁卫平（1983－），男，硕士，高级工程师，研究方向：食品药品检测

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注：A为 hlyA基因引物探针初步验证; B为O1群rfb基因引物探针初步验证;C为O139群rfb基因引物探针初步验证; D为ctxA基因引物探针初步验证。

, figureFileSmall=+Xgj7R81/nQ5RbWEuA4U2Q==, figureFileBig=Xzj5GIh6Fxs2VhyJQ0Taow==, tableContent=null), ArticleFig(id=1241359969669411799, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=EN, label=Fig.2, caption=Optimization of probe dosage for detection of Vibrio cholerae O1 and O139 toxigenic strains by MIRA method, figureFileSmall=ChuFthFbkrnP8gh0l1sQyw==, figureFileBig=KJMmVr8Jv5dzs16Eatfk3A==, tableContent=null), ArticleFig(id=1241359969770075098, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=CN, label=图2, caption=MIRA方法检测霍乱弧菌O1群与O139群产毒株的探针量的优化

注：A为hlyA基因扩增效率与探针加入量关系; B为O1群rfb基因扩增效率与探针加入量关系；C为O139群rfb基因扩增效率与探针加入量关系；D为ctxA基因扩增效率与探针加入量关系。

, figureFileSmall=ChuFthFbkrnP8gh0l1sQyw==, figureFileBig=KJMmVr8Jv5dzs16Eatfk3A==, tableContent=null), ArticleFig(id=1241359969916875742, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=EN, label=Fig.3, caption=Detection sensitivity of the genome of Vibrio cholerae O1 and O139 toxigenic strains by MIRA method, figureFileSmall=uSlfZxDLrOagKQmlB2MlCA==, figureFileBig=Jn1ouAsqrGnYuUQvZPeW9Q==, tableContent=null), ArticleFig(id=1241359970009150433, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=CN, label=图3, caption=MIRA 方法检测霍乱弧菌O1群与O139群产毒株基因组的灵敏度

注：A为核酸质量浓度与hlyA基因检测效率之间的关系；B为核酸质量浓度与O1群rfb基因检测效率之间的关系；C为核酸质量浓度与O139群rfb基因检测效率之间的关系；D为核酸质量浓度与ctxA基因检测效率之间的关系。

, figureFileSmall=uSlfZxDLrOagKQmlB2MlCA==, figureFileBig=Jn1ouAsqrGnYuUQvZPeW9Q==, tableContent=null), ArticleFig(id=1241359970105619428, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=EN, label=Table 1, caption=

Sequences of MIRA primers and probes

, figureFileSmall=null, figureFileBig=null, tableContent=
引物与探针名称引物序列(5’-3’)
hlyA-F5’-ATTTATCGAGTTAACCTAGAGCGTTCATTG-3’
hlyA-R5’-GTTAAACACGAAGCGATAATCTTGGGCAATC-3’
hlyA-P5’-TGATCAACTCGGTTATCGTCAGTTTGGAGCCAG[FAM-dT]T[THF][BHQ1-dT]ACGACGTTAGATGCC-3’C3-spacer
ctxA-F5’-ACTCCCAAATATATGGATGGTATCGAGTTCA-3’
ctxA-R5’-TCGCAAGTATTACTCATCGATGATCTTGGA-3’
ctxA-P5’-ACAATTACATCGTAATAGGGGCTACAGAGA[FAM-dT]]AG[THF][BHQ1-dT]ATTACAGTAACTTAGA-C3-spacer
rfbO1-F5’-TGGATATCTATGGTTTCACTGAACAGATGGG-3’
rfbO1-R5’-TCAATCACACCAAGGTCATCTGTAAGTACAAC-3
rfbO1-P5’-TGTGTGGGCCAGGTAAAGTAGGCTTACTTGAGT[FAM-dT][THF]G[BHQ1-dT]AAGCCCACTACCGCAT-C3-spacer
rfbO139-F5’-GATTTGGTGAGAAGCCTCTTTATTACGGGTGG-3’
rfbO139-R5’-AGAGAAACAGCATGACTGGCATCCCAAAAT-3’
rfbO139-P5’-ATGAAGGTATCTTCAAGTTAGAGCCGGGTGT[FAM-dT]A[THF][BHQ1-dT]GCTGTCTTTTCTCACGA-3’C3-spacer
), ArticleFig(id=1241359970185311207, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=CN, label=表1, caption=

MIRA引物和探针序列

, figureFileSmall=null, figureFileBig=null, tableContent=
引物与探针名称引物序列(5’-3’)
hlyA-F5’-ATTTATCGAGTTAACCTAGAGCGTTCATTG-3’
hlyA-R5’-GTTAAACACGAAGCGATAATCTTGGGCAATC-3’
hlyA-P5’-TGATCAACTCGGTTATCGTCAGTTTGGAGCCAG[FAM-dT]T[THF][BHQ1-dT]ACGACGTTAGATGCC-3’C3-spacer
ctxA-F5’-ACTCCCAAATATATGGATGGTATCGAGTTCA-3’
ctxA-R5’-TCGCAAGTATTACTCATCGATGATCTTGGA-3’
ctxA-P5’-ACAATTACATCGTAATAGGGGCTACAGAGA[FAM-dT]]AG[THF][BHQ1-dT]ATTACAGTAACTTAGA-C3-spacer
rfbO1-F5’-TGGATATCTATGGTTTCACTGAACAGATGGG-3’
rfbO1-R5’-TCAATCACACCAAGGTCATCTGTAAGTACAAC-3
rfbO1-P5’-TGTGTGGGCCAGGTAAAGTAGGCTTACTTGAGT[FAM-dT][THF]G[BHQ1-dT]AAGCCCACTACCGCAT-C3-spacer
rfbO139-F5’-GATTTGGTGAGAAGCCTCTTTATTACGGGTGG-3’
rfbO139-R5’-AGAGAAACAGCATGACTGGCATCCCAAAAT-3’
rfbO139-P5’-ATGAAGGTATCTTCAAGTTAGAGCCGGGTGT[FAM-dT]A[THF][BHQ1-dT]GCTGTCTTTTCTCACGA-3’C3-spacer
), ArticleFig(id=1241359970273391595, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=EN, label=Table 2, caption=

Specificity of MIRA detection method

, figureFileSmall=null, figureFileBig=null, tableContent=
序号菌种名称hlyA基因检测结果O1群rfb基因检测结果O139群rfb基因检测结果ctxA基因检测结果
1霍乱弧菌O1群产毒株++－+
2霍乱弧菌O139群产毒株+－++
3霍乱弧菌VBO+－－－
4鼠伤寒沙门氏菌ATCC 14028－－－－
5金黄色葡萄球菌ATCC6538－－－－
6副溶血性弧菌ATCC 17802－－－－
7单核细胞增生李斯特氏菌ATCC19115－－－－
8铜绿假单胞菌ATCC9027－－－－
9产气荚膜梭菌 ATCC13124－－－－
10大肠埃希氏菌O157:H7/NM ATCC43888－－－－
), ArticleFig(id=1241359970369860592, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=CN, label=表2, caption=

MIRA检测方法的特异性

, figureFileSmall=null, figureFileBig=null, tableContent=
序号菌种名称hlyA基因检测结果O1群rfb基因检测结果O139群rfb基因检测结果ctxA基因检测结果
1霍乱弧菌O1群产毒株++－+
2霍乱弧菌O139群产毒株+－++
3霍乱弧菌VBO+－－－
4鼠伤寒沙门氏菌ATCC 14028－－－－
5金黄色葡萄球菌ATCC6538－－－－
6副溶血性弧菌ATCC 17802－－－－
7单核细胞增生李斯特氏菌ATCC19115－－－－
8铜绿假单胞菌ATCC9027－－－－
9产气荚膜梭菌 ATCC13124－－－－
10大肠埃希氏菌O157:H7/NM ATCC43888－－－－
), ArticleFig(id=1241359970457940978, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=EN, label=Table 3, caption=

Judgment of MIRA detection results

, figureFileSmall=null, figureFileBig=null, tableContent=
序号hlyA检测结果O1-rfb检测结果O139-rfb检测结果ctxA检测结果结果判定
1－－－－非霍乱弧菌
2＋－－－不产毒非O1非O139群霍乱弧菌
3＋＋－－不产毒O1群霍乱弧菌
4＋＋－＋产毒O1群霍乱弧菌
5＋－＋－不产毒O139群霍乱弧菌
6＋－＋＋产毒O139群霍乱弧菌
7＋－－＋产毒非O1群非O139群霍乱弧菌
), ArticleFig(id=1241359970579575798, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241342456269173739, language=CN, label=表3, caption=

MIRA检测结果的判定

, figureFileSmall=null, figureFileBig=null, tableContent=
序号hlyA检测结果O1-rfb检测结果O139-rfb检测结果ctxA检测结果结果判定
1－－－－非霍乱弧菌
2＋－－－不产毒非O1非O139群霍乱弧菌
3＋＋－－不产毒O1群霍乱弧菌
4＋＋－＋产毒O1群霍乱弧菌
5＋－＋－不产毒O139群霍乱弧菌
6＋－＋＋产毒O139群霍乱弧菌
7＋－－＋产毒非O1群非O139群霍乱弧菌
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霍乱弧菌O1群与O139群产毒株多酶恒温快速扩增检测方法的建立
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丁卫平 1 , 张士财 1 , 伊廷存 2 , 刘凤莲 1 , 刘晓鹏 1 , 赵志强 1 , 霍胜楠 2 , 钟棋宝 3 , 蔡磊 3
现代预防医学 | 实验技术及其应用 2024,51(4): 705-712
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现代预防医学 | 实验技术及其应用 2024, 51(4): 705-712
霍乱弧菌O1群与O139群产毒株多酶恒温快速扩增检测方法的建立
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丁卫平1, 张士财1, 伊廷存2, 刘凤莲1, 刘晓鹏1, 赵志强1, 霍胜楠2, 钟棋宝3, 蔡磊3
作者信息
  • 1.滨州市检验检测中心，山东 滨州 256603
  • 2.山东省食品药品检验研究院
  • 3.浙江工商大学食品与生物工程学院

    • 丁卫平（1983－），男，硕士，高级工程师，研究方向：食品药品检测

通讯作者:

蔡磊，E-mail：
Establishment of multienzyme isothermal rapid amplification method for detection of Vibrio cholerae O1 and O139 toxigenic strains
Wei-ping DING1, Shi-cai ZHANG1, Ting-cun YI2, Feng-lian LIU1, Xiao-peng LIU1, Zhi-qiang ZHAO1, Sheng-nan HUO2, Qi-bao ZHONG3, Lei CAI3
Affiliations
  • Binzhou Testing Center, Binzhou, Shandong 256603, China
出版时间: 2024-02-25 doi: 10.20043/j.cnki.MPM.202308063
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摘要
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目的

建立一种霍乱弧菌O1群与O139群产毒株快速准确的检测方法。

方法

基于多酶恒温快速扩增技术，根据霍乱弧菌溶血素编码基因、霍乱毒素编码基因、O1群与O139群O抗原编码基因设计引物与探针，初步建立检测方法。以不同血清型霍乱弧菌及其它细菌DNA为模板，验证检测方法的特异性与灵敏度。

结果

配制反应体系时，按试剂盒说明书推荐加入量0.6 μL加入探针时可成功扩增溶血素编码基因、霍乱毒素编码基因及O1群与O139群O抗原编码基因4个目的基因，针对上述4个基因分别优化探针加入量为1.0 μL、1.0 μL、1.0 μL、0.8 μL时扩增效率更高。对优化探针加入量的检测方法进行方法特异性验证时，各引物探针组合不与非目标菌DNA产生交叉反应。进行检测方法的灵敏度分析时，不同目的基因的基因组检测灵敏度为70 fg或290 fg。上述实验结果证明检测方法的灵敏度及特异性满足设计要求。

结论

建立了一种霍乱弧菌O1群与O139群产毒株检测的新方法，该方法特异性强、灵敏度高、检测时间短，可适用于霍乱弧菌的常规检测及快检，对霍乱的预防具有一定的积极作用。

多酶恒温快速扩增  /  霍乱弧菌  /  产毒株  /  检测
Objective

To establish a rapid and accurate method for the detection of Vibrio cholerae O1 and O139 toxigenic strains.

Methods

On the basis of multienzyme isothermal rapid amplification technology, primers and probes were designed according to the hemolysin coding gene, cholera toxin coding gene and O antigen coding genes of Vibrio cholerae O1 and O139, and the detection method was preliminary established. With DNA of different Serotypes of Vibrio cholerae and other bacteria used as templates, the specificity and sensitivity of the detection method was verified.

Results

When the probe addition amount of the reaction system was 0.6μL as recommended by kit instructions, it could successfully amplify four target genes including hemolysin coding gene, cholera toxin coding gene, and O antigen coding genes of Vibrio cholerae O1 and O139. When the probe addition amount was respectively optimized to 1.0 μL, 1.0 μL, 1.0 μL, 0.8 μL of the above-mentioned four genes, the amplification efficiency was higher. After optimizing probe addition, the specificity of the detection method was verified and each primer probe combination did not cross react with non target bacterial DNA. When conducting sensitivity analysis of the detection method, the genomic detection sensitivity of different target genes was 70fg or 290fg. The above results showed that the sensitivity and specificity of the detection method meet the design requirements.

Conclusion

A new method for the detection of Vibrio cholerae O1 and O139 toxigenic strains has been established. This method has characteristics of strong specificity, high sensitivity, and short detection time. It can be applied to the routine detection and rapid detection of Vibrio cholerae, and has a positive role in the prevention of cholera.

Multienzyme isothermal rapid amplification  /  Vibrio cholerae  /  Toxigenic strain  /  Detection
丁卫平, 张士财, 伊廷存, 刘凤莲, 刘晓鹏, 赵志强, 霍胜楠, 钟棋宝, 蔡磊. 霍乱弧菌O1群与O139群产毒株多酶恒温快速扩增检测方法的建立. 现代预防医学, 2024 , 51 (4) : 705 -712 . DOI: 10.20043/j.cnki.MPM.202308063
Wei-ping DING, Shi-cai ZHANG, Ting-cun YI, Feng-lian LIU, Xiao-peng LIU, Zhi-qiang ZHAO, Sheng-nan HUO, Qi-bao ZHONG, Lei CAI. Establishment of multienzyme isothermal rapid amplification method for detection of Vibrio cholerae O1 and O139 toxigenic strains[J]. Modern Preventive Medicine, 2024 , 51 (4) : 705 -712 . DOI: 10.20043/j.cnki.MPM.202308063
正文
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霍乱是一种由霍乱弧菌感染所引起的强烈肠道传染病[1]，主要由食入霍乱弧菌污染的食物或饮用污染的水源引发[2-4]，传播性极强，危害性极大[5]，已经肆虐人类几个世纪，使很多人因此而丧失生命，是《中华人民共和国传染病防治法》规定的两种甲类传染病之一，也是《中华人民共和国国境卫生检疫法》规定需要检疫的三种传染病之一。随着科学技术的进步，口服补液疗法、抗生素/抗菌剂和疫苗[6]等干预措施挽救了无数霍乱患者，显著降低了霍乱患者的死亡率，益生菌和噬菌体疗法等新的干预措施正在被开发出来，但即使到现在，在一些公共卫生条件较差的国家和地区比如非洲、印度、也门、海地，每年仍有很多人因霍乱而丧命[7-11]，从世界范围内来看，霍乱的整体防控形势仍然非常严峻。我国当前霍乱疫情形势相对较为平稳，但不时仍会有一些散发及聚餐暴发的病例被报道[12-14]。我国霍乱防控良好形势的取得有赖于我国公共卫生条件的不断进步及多年来持续的严格防控，在当前形势下，必须更加重视，防止霍乱在我国大规模暴发流行。
并非所有的霍乱弧菌都可引发霍乱[1]。根据菌体表面脂多糖[15]抗原（O抗原）的不同，霍乱弧菌目前已鉴定到210余个血清群；根据能否产生霍乱肠毒素[16]，霍乱弧菌又可分为产毒株与非产毒株。根据目前的研究，仅霍乱弧菌O1群、O139 群产毒株会导致霍乱持续流行，而O1群与O139群非产毒株以及其它血清群霍乱弧菌只引起偶然的散发病例或导致很小规模的暴发[17]。因此，对霍乱弧菌O1群与O139群产毒株进行准确快速地检测就显得特别重要，已成为霍乱领域的研究热点之一[18-19]。
霍乱发病急且传播快，控制霍乱救治霍乱患者的关键在于早预防、早发现、早治疗，而早发现的前提在于对霍乱弧菌O1群及O139群产毒株进行快速准确地检测。同时，与城市相比，农村地区水源及公共卫生整体状况仍相对较差，发生霍乱感染的几率也相对更大，因此开发一种成本低、检测速度快、可现场部署、适合中国及世界广大地区特别是农村地区的霍乱弧菌O1群及O139群产毒株检测方法就显得尤为重要了。对于霍乱弧菌的检测，我国目前尚未制定国家标准，仅原国家质量监督检验检疫总局及中华人民共和国海关总署颁布了一些霍乱弧菌检验的行业标准。权威的《霍乱防治手册》第6版规定对于霍乱弧菌检测时首先对标本进行增菌分离，然后采用诊断血清凝集、生化特征、形态学等方法进行鉴定。这些行业标准及《霍乱防治手册》第6版规定的鉴定方法或耗时较长、步骤繁琐，或对检测仪器、检测人员、检测环境要求较高，无法适用于霍乱弧菌的便携式快速鉴定。
恒温核酸快速扩增技术具有操作简单、灵敏度高、检测时间短、设备要求低、抗干扰能力强的特点，被视为致病菌较为理想的便携式快速检测方法[20]，但最早发明的恒温核酸快速扩增技术-重组酶聚合酶扩增技术[21] (Recombinase Polymerase Amplification, RPA)知识产权被国外所垄断，试剂依赖进口、价格昂贵[22]，因此国内近年来致力于研发具有自主知识产权的恒温核酸快速扩增技术。多酶恒温快速扩增技术（Multienzyme Isothermal Rapid Amplification，MIRA，以下均用简称）是国内研发的一种具有完整的自主知识产权的新型恒温核酸快速扩增技术，这种技术依靠多种功能蛋白（解旋酶、重组酶、单链结合蛋白、DNA聚合酶等）协同作用，在常温下实现核酸快速扩增，扩增效果与RPA相当并已在致病菌检测领域实现了广泛的应用[23-25]。
鉴于霍乱弧菌O1群与O139群产毒株快速检测的需求及MIRA技术知识产权的自主性，本研究拟采用MIRA技术，以霍乱弧菌为研究材料，探究其不同血清型及产毒株与非产毒株基因之间的的差异，旨在建立一种对霍乱弧菌O1群与O139群产毒株进行快速鉴定的方法，为霍乱弧菌防治提供支持。
1　材料与方法
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1.1　材料与试剂
1.1.1　菌株及DNA来源
霍乱弧菌（Vibrio cholerae）O139群产毒株DNA由山东省疾病预防控制中心惠赠；霍乱弧菌（Vibrio cholerae）O1群产毒株DNA由中国疾病预防控制中心传染病预防控制所惠赠；产气荚膜梭菌（C. perfringens）ATCC13124、鼠伤寒沙门氏菌（Salmonella typhimurium）ATCC14028、金黄色葡萄球菌（Staphyloccocus aurous）ATCC 6538、副溶血性弧菌（Vibrio parahaemolyticus）ATCC17802、单核细胞增生李斯特氏菌（Listeria monocytogenes）ATCC19115、铜绿假单胞菌（P. aeruginosa）ATCC9027、霍乱弧菌VBO（Vibrio cholerae VBO），购自于广东环凯微生物科技有限公司；大肠埃希氏菌O157:H7/NM（Escherichia coli O157:H7）ATCC43888由山东省食品药品检验研究院惠赠。
1.1.2　试剂
DNA恒温快速扩增试剂盒（荧光型）：潍坊安普未来生物科技有限公司；霍乱弧菌通用引物、探针，霍乱弧菌O1群与O139群特异性引物、探针，均购自生工生物工程（上海）股份有限公司；TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0：宝日医生物技术（北京）有限公司；营养肉汤培养基：北京陆桥技术股份有限公司。
1.2　仪器与设备
SpectraMax QuickDrop超微量全波长分光光度计（美谷分子仪器（上海）有限公司）；LightCycler© 480 II实时荧光定量PCR仪（瑞士罗氏）；Sorvall Legend Micro 21R冷冻型微量台式离心机（美国赛默飞世尔）；SPL-350生化培养箱（天津市莱玻特瑞仪器设备有限公司）；BSC-1500IIA2-X生物安全柜（博科控股集团有限公司）；DK-98-Ⅱ智能电热恒温水浴锅（天津市泰斯特仪器有限公司）。
1.3　方法
1.3.1　引物与探针设计
在研究霍乱弧菌相关检验标准与研究文献的基础上，遵循潍坊安普未来生物科技有限公司提供的MIRA技术引物与荧光探针设计原则，本研究选择霍乱弧菌的溶血素编码基因（hlyA）、霍乱毒素编码基因（ctxA）、O1群与O139群O抗原编码基因（rfb）设计了四对引物与探针。
1.3.2　细菌培养与核酸提取
将本研究中所用菌株按照所购买菌株说明书要求或国家标准要求进行复苏增菌，增菌液按照宝日医生物（北京）有限公司细菌DNA提取试剂盒（TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0）的产品说明书要求，提取DNA。提取后的DNA采用超微量全波长分光光度计测定质量浓度与纯度。
1.3.3　MIRA扩增体系及优化
按照潍坊安普未来生物科技有限公司DNA 恒温快速扩增试剂盒（荧光型）说明书的规定，常用的MIRA扩增体系为50 μL，其中各组分的量如下：A buffer，29.4 μL；上游引物（10 μM），2 μL；下游引物（10 μM），2 μL；探针（10 μM），0.6 μL；ddH2O和DNA模板，13.5 μL；B buffer，2.5 μL。在本研究中，DNA模板的加入量为2 μL。在对扩增体系进行优化时，根据王淑娟等人的研究[26]，只针对探针加入量（0.4、0.6、0.8、1.0、1.2 μL）进行优化，根据探针加入量调整ddH2O的加入量，其余试剂加入量不变，扩增体系总体积仍保持50 μL。本研究所用扩增程序为：39 ℃ 1 min；39 ℃ 20 min，每30 s采集一次荧光信号。
1.3.4　MIRA体系的特异性
在1.3.3优化后的体系中加入2μL目标菌DNA作为阳性对照，2μL ddH2O替代DNA作为空白对照，加入常见非目标菌DNA进行扩增检测，对该方法的特异性进行验证。
1.3.5　MIRA检测方法的灵敏度
对霍乱弧菌O1群与O139群产毒株DNA进行10倍梯度稀释。稀释完成后，分别取2 μL不同浓度的DNA稀释液作为模板，加入探针优化后的MIRA体系中，根据扩增结果判断霍乱弧菌的检测灵敏度。
1.3.6　结果判断
阳性结果有类似“S”形扩增曲线，且Ct≤35；阴性结果为无扩增曲线，且Ct>40或未检出；若35<Ct<40，需复检一次。如再次扩增后Ct值仍为<40.0，则判定检验结果阳性；如再次扩增后Ct值≥40.0，则判定检验结果阴性。
1.3.7　数据处理及图片绘制
导出荧光定量PCR仪运行数据，通过软件OriginPro 2022进行数据处理及绘制图像。
2　结果
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2.1　MIRA检测方法引物探针序列
本研究设计的MIRA引物与探针序列见表1。
2.2　MIRA检测方法中引物与探针的适用性
首先按照常规探针加入量0.6 μL配制MIRA扩增体系进行扩增，以探究设计的引物探针能否扩增目的基因。从扩增结果来看，以所有霍乱弧菌血清群中提取的DNA为模板，根据溶血素基因设计的引物探针都能成功扩增目的基因；以霍乱弧菌O1群与O139群产毒株DNA为模板时，根据霍乱弧菌肠毒素基因设计的引物探针能扩增目的基因，以霍乱弧菌非产毒株DNA为模板时，不能扩增目的基因；以霍乱弧菌O1群与O139群O抗原编码基因设计的引物探针进行扩增时，以对应血清型DNA为模板时能成功扩增，以非对应血清型DNA为模板时，不能扩增。初步说明设计的引物探针能正常用于扩增，以不同血清型霍乱弧菌DNA为模板时特异性较好（见图1）。
2.3　MIRA法检测霍乱弧菌时探针的量的优化
调整探针加入量，研究探针加入量与扩增效率之间的关系。对于hlyA基因，以霍乱弧菌O1群与O139群DNA为模板时，荧光强度与探针加入量的关系是0.6<0.4<0.8<1.0<1.2；对于ctxA基因，以霍乱弧菌O1群与O139群DNA为模板时，荧光强度与探针加入量的关系是0.6<0.4<0.8<1.2<1.0；对于O1群rfb基因，荧光强度与探针加入量的关系是0.6=0.4<0.8<1.0<1.2；对于O139群rfb基因，荧光强度与探针加入量的关系是0.4<0.6=1.0<0.8=1.2（见图2）。基于保证检测灵敏度与节约试剂兼顾的原则，hlyA基因、ctxA基因、O1群rfb基因MIRA扩增体系探针加入量选择1.0 μL，O139群rfb基因MIRA扩增体系探针加入量选择0.8 μL。
2.4　MIRA体系的特异性
在对MIRA扩增体系进行优化后，分别提取多种常见菌的DNA与原先提取及获得的霍乱弧菌DNA进行检测方法特异性验证。结果显示，根据溶血素基因设计的引物探针，对所有种类的霍乱弧菌DNA都能成功扩增，对非霍乱弧菌不能进行扩增；根据霍乱弧菌肠毒素基因设计的引物探针，仅对霍乱弧菌产毒株DNA成功扩增，对霍乱弧菌非产毒株与非霍乱弧菌不能进行扩增；根据霍乱弧菌O1群与O139群rfb基因设计的引物探针，则仅能针对相应血清型霍乱弧菌DNA进行扩增，对其它血清型的霍乱弧菌与非霍乱弧菌则不能扩增（见表2）。
2.5　MIRA体系的灵敏度
从检测霍乱弧菌的最低DNA质量浓度来考察该方法的灵敏度。经超微量全波长分光光度计测定，提取的霍乱弧菌O1菌株DNA质量浓度为7 ng/μL，O139菌株DNA质量浓度为29 ng /μL，将霍乱弧菌O1与O139菌株提取DNA经梯度稀释后取2 μL作为模板加入MIRA检测体系中。在研究该方法的灵敏度时，扩增霍乱弧菌O1群rfb基因所采用的O1群DNA稀释梯度为7 ng~0.7 fg，扩增霍乱弧菌O139群rfb基因所采用的O139群DNA稀释梯度为2.9 ng~0.29 fg；扩增hlyA基因与ctxA基因时，先进行了预实验，在预实验中，发现MIRA检测这两种基因的灵敏度非常高，为了图形呈现的美观性，采用的霍乱弧菌O1用于扩增的DNA稀释梯度为7 pg~0.7 fg，霍乱弧菌O139用于扩增的DNA梯度为2.9 pg~0.29 fg。以霍乱弧菌O1菌株提取DNA为模板，采用针对其rfb基因设计的引物探针扩增时，检出限为70 fg；以霍乱弧菌O139菌株提取DNA为模板，采用针对其rfb基因专门设计的引物探针扩增时，检出限为290 fg；以霍乱弧菌溶血素基因与肠毒素基因引物探针为模板，采用霍乱弧菌O1与O139菌株提取DNA为模板，检出限分别为70 fg与290 fg（见图3）。
2.6　MIRA检测结果的判定
MIRA检测结果的判定可按表3进行。
3　讨论
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选取合适的基因是采用多酶恒温快速扩增法鉴定霍乱弧菌O1群与O139群产毒株的基础与前提。本研究确定的4种鉴定基因中，溶血素编码基因为霍乱弧菌所共有，针对溶血素编码基因设计的引物与探针可用于所有血清型霍乱弧菌的扩增；rfb基因用于霍乱弧菌O抗原的编码，不同O血清型的霍乱弧菌此基因序列有所差异，这也是霍乱弧菌O血清型分型的分子生物学基础，针对霍乱弧菌O1群与O139群rfb基因设计的引物和探针具有特异性，仅可扩增对应血清型的霍乱弧菌；霍乱毒素是霍乱弧菌的主要毒力因子，为霍乱弧菌产毒株所共有，由一个A亚基及5个B亚基所构成，ctxA基因是霍乱毒素A亚基的编码基因，因此ctxA基因可以作为区分霍乱弧菌产毒株与非产毒株的基因。从上述分析中可以看出，选取的4种基因从功能上可以满足霍乱弧菌O1群与O139群产毒株的鉴定需求。在进行实际检测时，可以先用扩增溶血素编码基因的引物与探针去进行检测，对初步检测结果为霍乱弧菌的再进行进一步检测，以确定霍乱弧菌类型。对于非霍乱弧菌，只需要1个反应管即能完成检测；对于初步检测结果为霍乱弧菌的菌株，总计需要4个反应管才能最终确定样品的血清型与产毒情况。用溶血素编码基因进行初步筛选的意义在于可以节约检验成本。
在对基因进行深入分析的基础上，本研究基于MIRA技术，建立了霍乱弧菌O1群与O139群产毒株快速准确的检测方法。该方法特异性强，总检测时长仅为21分钟左右，不同目的基因的基因组检测灵敏度为70fg或290fg，且只需要价格低方便携带的恒温荧光检测仪即可完成扩增检测过程，能充分满足我国及世界广大地区特别是欠发达地区霍乱弧菌快速检测的需求。与近几年开发的霍乱弧菌检测方法相比，本研究建立的检测方法具有独特的优势。李涛等建立的检测方法[27]与凌娇等建立的检测方法[28]仅选取霍乱弧菌的1个基因进行检测，其检验结果仅能证明样品是否为霍乱弧菌。万莹等建立的检测方法[29]选取了霍乱弧菌的3个基因，包括1个种特异性基因与2个毒力基因，但未包含血清型鉴定相关基因，不能对霍乱弧菌进行分型。李盛杰建立的霍乱弧菌检测方法[30]使用了多酶恒温快速扩增技术，但仅选取了霍乱弧菌的1个基因进行检测，其检验结果也仅能证明样品是否为霍乱弧菌。传染病防控制国家重点实验室的Lu等人认识到了用快检技术对霍乱弧菌进行分型检测的重要性，联用恒温扩增技术与基因编辑技术开发了霍乱弧菌O1群及O139群产毒株快速检测的方法[19]，此方法需要先进行扩增再对扩增产物进行进一步处理后才能观察到检测结果，步骤相对繁琐，检测成本也相对较高，且扩增完成后再打开反应管盖子进行检测易造成气溶胶污染从而影响检验结果。
综上所述，本研究成果为霍乱弧菌的快速检测提供了一条新的思路，研究成果的应用推广不仅会对霍乱弧菌防治及食品安全监管产生积极作用，对于人民生命健康的保护也具有重大意义。
基金
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  • 山东省市场监督管理局科研项目(202108)
  • 浙江省基础公益研究计划项目(LY23C010002)
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2024年第51卷第4期
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文章信息
doi: 10.20043/j.cnki.MPM.202308063
  • 接收时间：2023-08-04
  • 首发时间：2026-03-19
  • 出版时间：2024-02-25
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  • 收稿日期：2023-08-04
基金
山东省市场监督管理局科研项目(202108)
浙江省基础公益研究计划项目(LY23C010002)
作者信息
    1.滨州市检验检测中心，山东 滨州 256603
    2.山东省食品药品检验研究院
    3.浙江工商大学食品与生物工程学院

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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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