Article(id=1324050969033585002, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1324050876230415089, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202511316, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1763740800000, receivedDateStr=2025-11-22, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1793607950694, onlineDateStr=2026-11-02, pubDate=1781020800000, pubDateStr=2026-06-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793607950694, onlineIssueDateStr=2026-11-02, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793607950694, creator=13701087609, updateTime=1793607950694, updator=13701087609, issue=Issue{id=1324050876230415089, tenantId=1146029695717560320, journalId=1227665162245664772, year='2026', volume='53', issue='11', pageStart='1921', pageEnd='2112', issueExtLink='null', onlineDate='null', pubDate='1781020800000', pubDateStr='2026-06-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1793607928569, creator='13701087609', updateTime=1793670049253, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1324311429322507008, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1324050876230415089, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1324311429322507009, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1324050876230415089, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2068, endPage=2073, ext={EN=ArticleExt(id=1324050969222328683, articleId=1324050969033585002, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Comparative study on Salmonella serotyping based on multiplex qPCR and whole-genome sequencing, columnId=1228016572065837304, journalTitle=Modern Preventive Medicine, columnName=Experimental Technology and Applications, runingTitle=null, highlight=null, articleAbstract=
Objective

To analyze and compare the Salmonella serotyping methods based on multiplex quantitative polymerase chain reaction (multiplex qPCR) and whole-genome sequencing (WGS), and to evaluate the serotyping capability and application scenarios of each method.

Methods

A total of 242 Salmonella strains isolated in our province, covering 96 serotypes, were subjected to whole-genome sequencing. The serotypes of these strains were predicted using SeqSero2 and SISTR, followed by a concordance analysis of the results. Meanwhile, multiplex qPCR was employed for the identification of each serotype, so as to assess the accuracy and applicability of this method.

Results

Three methods were used to detect 96 Salmonella serovars. The overall concordance rates of SeqSero2 and SISTR were both 87.50% (84/96), which were significantly higher than that of multiplex qPCR (52.08%, 50/96). For the 242 Salmonella strains, the discordance rates of SeqSero2 and SISTR were 2.48% (6/242) and 7.85% (19/242), respectively. The multiplex qPCR method could only accurately distinguish common serotypes. The main reasons for the discordance of the SeqSero2 method included the deletion or error of O antigen genes. The main reasons for partial concordance were the interference from the similarity of H antigen alleles and the inability to distinguish some serovars in the C2 serogroup that differ only in the O6 antigen. The main reasons for the discordance of the SISTR method were cgMLST cluster misclassification, O antigen gene deletion, and the inability to distinguish monophasic variant strains.

Conclusion

The WGS-based serotyping method exhibits high accuracy and can replace the traditional serum agglutination test for Salmonella serotyping in numerous application scenarios. Although the multiplex qPCR method has limitations with a lower concordance rate than WGS-based methods, it features a short detection time and can accurately identify common serotypes, thus being suitable for emergency rapid detection.

, authors=Wei-feng HUANG, Gao-peng LEI, Yu-lan HUANG, Lin ZHANG, Tao XIAO, Li LIU, Hong LV, Wen-xia ZHAO, Xiao-rong YANG, authorsList=Wei-feng HUANG, Gao-peng LEI, Yu-lan HUANG, Lin ZHANG, Tao XIAO, Li LIU, Hong LV, Wen-xia ZHAO, Xiao-rong YANG, authorCompany=null, correspAuthors=Xiao-rong YANG, 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, fund=null), CN=ArticleExt(id=1324050969675313516, articleId=1324050969033585002, tenantId=1146029695717560320, journalId=1227665162245664772, language=CN, title=多重荧光PCR和基于全基因组测序的沙门菌血清分型方法比较研究, columnId=1228016572288135432, journalTitle=现代预防医学, columnName=实验技术及其应用, runingTitle=null, highlight=null, articleAbstract=
目的

分析比较多重荧光聚合酶链式反应(polymerase chain reaction,PCR)和基于全基因组测序的沙门菌血清分型方法,评估每种方法的分型能力和应用场景。

方法

对四川省覆盖96个血清型的242株沙门菌进行全基因组测序,利用SeqSero2以及SISTR预测血清型,进行一致性比较分析。并运用多重荧光PCR对每种血清型进行鉴定,评估其准确性及适用性。

结果

三种方法检测96种沙门菌血清型,SeqSero2与SISTR的完全符合率均为87.50%(84/96),显著高于多重荧光PCR的52.08%(50/96)。242株沙门菌株中SeqSero2和SISTR鉴定的不符合率分别为2.48%(6/242)、7.85%(19/242)。多重荧光PCR方法仅能准确区分常见血清型;SeqSero2方法不符合主要原因为O抗原基因缺失或错误,部分符合主要原因为H 抗原等位基因相似性干扰,以及不能区分C2血清群中仅O6不同的部分血清型;SISTR方法不符合主要原因是cgMLST聚类误判、O 抗原基因缺失以及不能区分单相变异菌株。

结论

基于基因组测序的血清型预测方法具有较高准确度,在许多应用场景中可替代传统血清凝集试验进行沙门菌血清分型。多重荧光PCR方法虽有局限性,符合率低于基因组测序方法,但其检测时限短,能准确区分常见血清型,可应用于应急快速检测。

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黄伟峰与雷高鹏为共同第一作者

黄伟峰(1987—),男,硕士,副主任技师,研究方向:微生物检验;

雷高鹏(1982—),男,博士,主任技师,研究方向:微生物检验;

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杨小蓉,E-mail:
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Disease Surveillance, 2020, 35(9): 827-830.(In Chinese), articleTitle=Comparison of salmonella serotyping methods of slide agglutination and multiple fluorescence PCR serotyping kit, refAbstract=null)], funds=[Fund(id=1324050980198822318, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, awardId=WSJK2024QN083, language=CN, fundingSource=海南省科技厅卫生健康科技创新联合青年项目计划(WSJK2024QN083), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1324050969910194541, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, xref=null, ext=[AuthorCompanyExt(id=1324050969922777454, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, companyId=1324050969910194541, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Sichuan Provincial Center for Disease Control and Prevention, Chengdu, Sichuan 610041, China), AuthorCompanyExt(id=1324050969926971759, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, companyId=1324050969910194541, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=四川省疾病预防控制中心,四川 成都 610041)])], figs=[ArticleFig(id=1324050979443847592, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, language=EN, label=Table 1, caption=

Comparison of serotyping results from SeqSero2, SISTR, and multiplex qPCR [n=96,n(%)]

, figureFileSmall=null, figureFileBig=null, tableContent=
鉴定方法完全符合部分符合不符合
SeqSero284(87.50)7(7.29)5(5.21)
SISTR84(87.50)0(0)12(12.50)
多重荧光PCR50(52.08)19(19.79)27(28.13)
), ArticleFig(id=1324050979557093801, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, language=CN, label=表1, caption=

SeqSero2、SISTR以及多重荧光PCR预测血清型结果比较[n=96,n(%)]

, figureFileSmall=null, figureFileBig=null, tableContent=
鉴定方法完全符合部分符合不符合
SeqSero284(87.50)7(7.29)5(5.21)
SISTR84(87.50)0(0)12(12.50)
多重荧光PCR50(52.08)19(19.79)27(28.13)
), ArticleFig(id=1324050979620008362, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, language=EN, label=Table 2, caption=

Partially concordant and discordant serotypes predicted by SeqSero2

, figureFileSmall=null, figureFileBig=null, tableContent=
血清型抗原式菌株数符合菌株数部分符合菌株数不符合菌株数部分符合或不符合情况
Indiana[1],4,12:z:1,76402O抗原基因缺失:预测结果为I -:z:1,7
Albany8,[20]:z4,z24:-4040Albany or Duesseldorf(6,8:z4,z24:-)
Enteritidis[1],9,12:g,m:-4310Enteritidis or Gallinarum([1],9,12:-:-)
Infantis6,7,[14]:r:1,54301O抗原基因缺失:预测结果为I -:r:1,5
Poona[1],13,22:z:1,64310Poona or Farmsen(13,23:z:1,6)
Typhimurium[1],4,[5],12:i:1,24301O抗原基因缺失:预测结果为I -:i:1,2
Weltevreden3,10[15]:r:z64301O抗原基因错误:Sokode(9,46:r:z6)
Goldcoast6,8:r:l,w3030Goldcoast or Brikama(8,[20]:r,[i]:l,w)
IV 43:z4,z23:-43:z4,z23:-3201O抗原基因缺失:预测结果为IV -:z4,z23:-
Molade8,[20]:z10:z63030Molade or Wippra(6,8;z10;z6)
Chincol6,8:g,m,[s]:[e,n,x]1010Chincol or Emek(8,20;g,m,s;-)
Choleraesuis6,7:c:1,51010Choleraesuis or Paratyphi C(6,7,[Vi]:c:1,5)or Typhisuis(6,7:c:1,5)
总计4121146
), ArticleFig(id=1324050979716477355, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, language=CN, label=表2, caption=

SeqSero2预测部分符合及不符合血清型

, figureFileSmall=null, figureFileBig=null, tableContent=
血清型抗原式菌株数符合菌株数部分符合菌株数不符合菌株数部分符合或不符合情况
Indiana[1],4,12:z:1,76402O抗原基因缺失:预测结果为I -:z:1,7
Albany8,[20]:z4,z24:-4040Albany or Duesseldorf(6,8:z4,z24:-)
Enteritidis[1],9,12:g,m:-4310Enteritidis or Gallinarum([1],9,12:-:-)
Infantis6,7,[14]:r:1,54301O抗原基因缺失:预测结果为I -:r:1,5
Poona[1],13,22:z:1,64310Poona or Farmsen(13,23:z:1,6)
Typhimurium[1],4,[5],12:i:1,24301O抗原基因缺失:预测结果为I -:i:1,2
Weltevreden3,10[15]:r:z64301O抗原基因错误:Sokode(9,46:r:z6)
Goldcoast6,8:r:l,w3030Goldcoast or Brikama(8,[20]:r,[i]:l,w)
IV 43:z4,z23:-43:z4,z23:-3201O抗原基因缺失:预测结果为IV -:z4,z23:-
Molade8,[20]:z10:z63030Molade or Wippra(6,8;z10;z6)
Chincol6,8:g,m,[s]:[e,n,x]1010Chincol or Emek(8,20;g,m,s;-)
Choleraesuis6,7:c:1,51010Choleraesuis or Paratyphi C(6,7,[Vi]:c:1,5)or Typhisuis(6,7:c:1,5)
总计4121146
), ArticleFig(id=1324050979867472300, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, language=EN, label=Table 3, caption=

Discordant serotypes predicted by SISTR

, figureFileSmall=null, figureFileBig=null, tableContent=
血清型抗原式菌株数符合菌株数不符合菌株数预测错误情况(菌株数)
Senftenberg1,3,19:g,[s],t:-431Westhampton 3,10[15][15,34]:g,s,t:-(1)
Havana[1],13,23:f,g,[s]:-303Cubana [1],13,23:z29:-(2),Agoueve 13,22:z29:-(1)
I 4,[5],12:i:-I 4,[5],12:i:-303Typhimurium[1],4,[5],12:i:1,2(3)
Fanti13,23:z38:-303Havana [1],13,23:f,g,[s]:-(3)
Orion3,10[15][15,34]:y:1,5202Uganda 3,10[15]:l,z13:1,5(2)
Chicago28:r,[i]:1,5101Nima 28:y:1,5(1)
Goettingen9,12:l,v:e,n,z15101Sandiego 4,[5],12:e,h:e,n,z15(1)
Kingston[1],4,12,[27]:g,s,t:[1,2]101Senftenberg 1,3,19:g,[s],t:-(1)
Kintambo[1],13,23:m,t:-101Larochelle 6,7:e,h:1,2(1)
Lagos[1],4,[5],12:i:1,5101Bovismorbificans 6,8,[20]:r,[i]:1,5(1)
Mississippi[1],13,23:b:1,5101Saintpaul [1],4,[5],12:e,h:1,2(1)
Regent3,10:f,g,[s]:[1,6]101London 3,10[15]:l,v:1,6(1)
总计22319
), ArticleFig(id=1324050979993301421, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1324050969033585002, language=CN, label=表3, caption=

SISTR 预测不符合血清型

, figureFileSmall=null, figureFileBig=null, tableContent=
血清型抗原式菌株数符合菌株数不符合菌株数预测错误情况(菌株数)
Senftenberg1,3,19:g,[s],t:-431Westhampton 3,10[15][15,34]:g,s,t:-(1)
Havana[1],13,23:f,g,[s]:-303Cubana [1],13,23:z29:-(2),Agoueve 13,22:z29:-(1)
I 4,[5],12:i:-I 4,[5],12:i:-303Typhimurium[1],4,[5],12:i:1,2(3)
Fanti13,23:z38:-303Havana [1],13,23:f,g,[s]:-(3)
Orion3,10[15][15,34]:y:1,5202Uganda 3,10[15]:l,z13:1,5(2)
Chicago28:r,[i]:1,5101Nima 28:y:1,5(1)
Goettingen9,12:l,v:e,n,z15101Sandiego 4,[5],12:e,h:e,n,z15(1)
Kingston[1],4,12,[27]:g,s,t:[1,2]101Senftenberg 1,3,19:g,[s],t:-(1)
Kintambo[1],13,23:m,t:-101Larochelle 6,7:e,h:1,2(1)
Lagos[1],4,[5],12:i:1,5101Bovismorbificans 6,8,[20]:r,[i]:1,5(1)
Mississippi[1],13,23:b:1,5101Saintpaul [1],4,[5],12:e,h:1,2(1)
Regent3,10:f,g,[s]:[1,6]101London 3,10[15]:l,v:1,6(1)
总计22319
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多重荧光PCR和基于全基因组测序的沙门菌血清分型方法比较研究
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黄伟峰 , 雷高鹏 , 黄玉兰 , 张林 , 肖桃 , 刘丽 , 吕虹 , 赵雯霞 , 杨小蓉
现代预防医学 | 实验技术及其应用 2026,53(11): 2068-2073
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现代预防医学 |实验技术及其应用 2026 , 53 (11) : 2068 -2073
多重荧光PCR和基于全基因组测序的沙门菌血清分型方法比较研究
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黄伟峰, 雷高鹏, 黄玉兰, 张林, 肖桃, 刘丽, 吕虹, 赵雯霞, 杨小蓉
作者信息
  • 四川省疾病预防控制中心,四川 成都 610041
通讯作者:
杨小蓉,E-mail:
作者简介:

黄伟峰与雷高鹏为共同第一作者

黄伟峰(1987—),男,硕士,副主任技师,研究方向:微生物检验;

雷高鹏(1982—),男,博士,主任技师,研究方向:微生物检验;

Comparative study on Salmonella serotyping based on multiplex qPCR and whole-genome sequencing
Wei-feng HUANG, Gao-peng LEI, Yu-lan HUANG, Lin ZHANG, Tao XIAO, Li LIU, Hong LV, Wen-xia ZHAO, Xiao-rong YANG
Affiliations
  • Sichuan Provincial Center for Disease Control and Prevention, Chengdu, Sichuan 610041, China
出版时间: 2026-06-10 doi: 10.20043/j.cnki.MPM.202511316
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目的

分析比较多重荧光聚合酶链式反应(polymerase chain reaction,PCR)和基于全基因组测序的沙门菌血清分型方法,评估每种方法的分型能力和应用场景。

方法

对四川省覆盖96个血清型的242株沙门菌进行全基因组测序,利用SeqSero2以及SISTR预测血清型,进行一致性比较分析。并运用多重荧光PCR对每种血清型进行鉴定,评估其准确性及适用性。

结果

三种方法检测96种沙门菌血清型,SeqSero2与SISTR的完全符合率均为87.50%(84/96),显著高于多重荧光PCR的52.08%(50/96)。242株沙门菌株中SeqSero2和SISTR鉴定的不符合率分别为2.48%(6/242)、7.85%(19/242)。多重荧光PCR方法仅能准确区分常见血清型;SeqSero2方法不符合主要原因为O抗原基因缺失或错误,部分符合主要原因为H 抗原等位基因相似性干扰,以及不能区分C2血清群中仅O6不同的部分血清型;SISTR方法不符合主要原因是cgMLST聚类误判、O 抗原基因缺失以及不能区分单相变异菌株。

结论

基于基因组测序的血清型预测方法具有较高准确度,在许多应用场景中可替代传统血清凝集试验进行沙门菌血清分型。多重荧光PCR方法虽有局限性,符合率低于基因组测序方法,但其检测时限短,能准确区分常见血清型,可应用于应急快速检测。

沙门菌  /  血清分型  /  全基因组测序  /  多重荧光PCR
Objective

To analyze and compare the Salmonella serotyping methods based on multiplex quantitative polymerase chain reaction (multiplex qPCR) and whole-genome sequencing (WGS), and to evaluate the serotyping capability and application scenarios of each method.

Methods

A total of 242 Salmonella strains isolated in our province, covering 96 serotypes, were subjected to whole-genome sequencing. The serotypes of these strains were predicted using SeqSero2 and SISTR, followed by a concordance analysis of the results. Meanwhile, multiplex qPCR was employed for the identification of each serotype, so as to assess the accuracy and applicability of this method.

Results

Three methods were used to detect 96 Salmonella serovars. The overall concordance rates of SeqSero2 and SISTR were both 87.50% (84/96), which were significantly higher than that of multiplex qPCR (52.08%, 50/96). For the 242 Salmonella strains, the discordance rates of SeqSero2 and SISTR were 2.48% (6/242) and 7.85% (19/242), respectively. The multiplex qPCR method could only accurately distinguish common serotypes. The main reasons for the discordance of the SeqSero2 method included the deletion or error of O antigen genes. The main reasons for partial concordance were the interference from the similarity of H antigen alleles and the inability to distinguish some serovars in the C2 serogroup that differ only in the O6 antigen. The main reasons for the discordance of the SISTR method were cgMLST cluster misclassification, O antigen gene deletion, and the inability to distinguish monophasic variant strains.

Conclusion

The WGS-based serotyping method exhibits high accuracy and can replace the traditional serum agglutination test for Salmonella serotyping in numerous application scenarios. Although the multiplex qPCR method has limitations with a lower concordance rate than WGS-based methods, it features a short detection time and can accurately identify common serotypes, thus being suitable for emergency rapid detection.

Salmonella  /  Serotyping  /  Whole-genome sequencing (WGS)  /  Multiplex qPCR
黄伟峰, 雷高鹏, 黄玉兰, 张林, 肖桃, 刘丽, 吕虹, 赵雯霞, 杨小蓉. 多重荧光PCR和基于全基因组测序的沙门菌血清分型方法比较研究. 现代预防医学, 2026 , 53 (11) : 2068 -2073 . DOI: 10.20043/j.cnki.MPM.202511316
Wei-feng HUANG, Gao-peng LEI, Yu-lan HUANG, Lin ZHANG, Tao XIAO, Li LIU, Hong LV, Wen-xia ZHAO, Xiao-rong YANG. Comparative study on Salmonella serotyping based on multiplex qPCR and whole-genome sequencing[J]. Modern Preventive Medicine, 2026 , 53 (11) : 2068 -2073 . DOI: 10.20043/j.cnki.MPM.202511316
沙门菌是全球重要的食源性病原,血清型是其重要表型之一,与其致病性、耐药性、宿主适应性等均有相关性,血清分型是流行病学调查、疫情溯源和公共卫生监测的基础[1]。依据怀特-考夫曼抗原表(White-Kauffmann-Le Minor scheme, WKL)的传统玻片凝集血清学分型[2],已在全球范围内广泛应用,但该方法存在耗时长、操作繁琐、成本高昂、对实验室检测人员技术经验要求高等问题。近年来,许多实验室利用新技术来鉴别沙门菌血清型,包括微生物质谱[3-4]、聚合酶链式反应(polymerase chain reaction,PCR)及荧光PCR[5-8]、基因芯片[9]和全基因组测序[10](whole-genome sequencing,WGS)。多重荧光PCR血清分型[11]是基于沙门菌不同血清型特有的基因序列差异,如rfb基因簇以确定O抗原,fliC和fljB 确定H1和H2抗原,通过设计特异性引物扩增目标片段进行鉴定。随着WGS技术的发展,已逐渐应用于沙门菌血清型的预测。SeqSero[12]是2015年推出的一款从WGS数据预测沙门菌血清型的软件工具,其核心原理在于通过识别与O和H抗原相关的特异性基因序列,根据WKL方案实现血清型的快速预测。SeqSero2[13]是2019年基于SeqSero 基础上,新增了用于鉴定沙门菌的种、亚种以及某些血清型的序列标记,开发了靶向组装方法,还引入基于k-mer 的算法,不仅加快了血清型预测速度,还提升了对基因组组装的预测准确性。SISTR[14]基于沙门菌基因组拼接草图序列,使用O和H抗原的遗传决定簇数据库以及330位点cgMLST,同时整合了多种基于基因组序列的分子分型方法,实现了对沙门菌血清型的准确预测。本研究分别运用WGS方法和多重荧光PCR方法对沙门菌进行了血清分型,并进行比较分析,评估可靠性,为不同场景下沙门菌血清分型的选择提供借鉴和参考。
四川省食品风险监测和食源性疾病监测等项目分离获得的96种血清型沙门菌,每种血清型随机选取3株(部分血清型4~6株),不足3株的血清型全选,总计242株。所有菌株均进行WGS分析,且通过玻片凝集确定血清型[15]。
血琼脂平板(郑州安图)、营养肉汤(青岛海博)、三糖铁培养基(广州环凯)、沙门菌诊断血清(中创汇科)、细菌DNA提取试剂盒(南京诺唯赞)、沙门菌血清(O、H)基因分型试剂盒(荧光 PCR法)(天津一知);荧光PCR仪(上海宏石)等。
96种血清型随机各选取1个菌株。挑取纯培养菌落至100 μl TE吹打混匀菌体,100 ℃水浴加热10 min,13 000 rpm离心2 min,吸取上清液,即为提取的DNA模板。参照多重荧光 PCR 血清分型检测试剂盒说明书,进行核酸扩增及后续分析。
242株沙门菌离心收集过夜培养物,用DNA提取试剂盒进行基因组提取,送至诺禾致源公司采用华大MGISEQ-2000平台进行测序。核酸浓度及质控标准为:核酸浓度不低于5ng/μl、总量不低于100ng,无严重RNA污染、无明显降解。测序参数为:测序策略为PE150(双端测序,单向读长150bp);原始数据量(碱基数)≥1.5G,Q20高质量数据量≥1G,Q30比例≥85%;基因组10×覆盖度≥95%,基因区覆盖度≥98%,平均覆盖深度≥100×;拼接后数据SCAFFOLD数量<100个,Contig数量<200个。使用Trimmomatic v0.39对下机的原始数据进行测序接头去除及低质量数据过滤,使用SPAdes v3.15.5对过滤后的数据进行拼接后,上传至SeqSero2网站(http://www.denglab.info/SeqSero2)和SISTR网站(https://lfz.corefacility.ca/sistr-app/),在线预测菌株血清型。
以血清凝集试验结果作为金标准,其他分子血清分型结果与之比对。同一血清型所有菌株的分子血清分型结果与凝集试验结果完全一致时,为完全符合。同一血清型的分子血清分型结果中有1株菌不一致时,即为不符合。分子血清分型得出多种可能血清型时,但包含凝集试验血清型时为部分符合,不包含则为不符合。
应用SPSS 30.0软件进行统计学分析。采用Pearson χ2检验对3种方法×3种符合类型(完全符合/部分符合/不符合)进行分析,检验水准α=0.05。
结果显示,三种检测方法的符合率分布存在极显著统计学差异(χ2=47.21,P<0.001)。SeqSero2与SISTR的完全符合率均为87.50%(84/96),显著高于多重荧光PCR的52.08%(50/96);而多重荧光PCR的不符合率为28.13%(27/96),显著高于SeqSero的5.21%(5/96)和SISTR的12.50%(12/96)。SeqSero2与多重荧光PCR、SISTR与多重荧光PCR的符合率分布均存在极显著统计学差异(χ2=29.12、32.05,P均<0.001);而SeqSero2与SISTR的符合率有统计学差异(χ2=9.92,P<0.01),说明虽然SeqSero2检测沙门菌血清型的符合率与SISTR相近,但不符合率显著低于SISTR。见表1。
5个血清型(6个菌株)SeqSero2预测不符合,菌株不符合率分别为2.48%(6/242),原因均为O抗原基因缺失或预测错误。且5个血清型都有预测符合/预测不符合的菌株,分别为:Indiana(4/2)、Infantis(3/1)、Typhimurium(3/1)、Weltevreden(3/1)、IV 43:z4,z23:-(2/1)。
7个血清型(14个菌株)SeqSero2预测部分不符合,主要原因有:C2血清群中仅O6不同的部分血清型不能准确分辨,SeqSero2结果均为两个相近的血清型,如Albany、Goldcoast、Molade、Chincol;血清抗原式相似但生化特征不一样的血清型不能准确分辨,如Choleraesuis;一些血清型的部分菌株缺失特异性基因,也可能得到两个相近的血清型,如Enteritidis缺失肠炎沙门菌的特异性sdf-I基因预测为Gallinarum,Poona的O22相关抗原基因缺失预测为Farmsen。见表2。
表3显示,SISTR 预测不符合菌株分属12个血清型的19株菌,不符合率为7.85%(19/242)。SISTR 预测不符合主要原因为cgMLST聚类误判,可以分为:(1)数据库的集群代表性不足,主要为一些少见血清型,如只有唯一菌株的7个少见血清型Chicago、Goettingen、Kingston、Kintambo、Lagos、Mississippi、Regent;(2)遗传特征相近血清型的 cgMLST 相似度接近,如Havana(3株)、Fanti(3株)、Orion(2株),以及单相变异的血清型,如I 4,[5],12:i:-(3株)。另一个原因为抗原基因缺失或碎片化,4株Senftenberg中有1株被错误预测为Westhampton。
多重荧光 PCR 血清分型结果不符合的27个血清型中,有21个不在试剂盒鉴定范围;其中16个未得出鉴定结果,5个得出错误鉴定结果,即将I 4:b:-鉴定为Limete、将Oranienburg鉴定为Montevideo、将Chincol鉴定为Emek、将Kintambo鉴定为Havana/Agbeni、将Cannstatt鉴定为Senftenberg/ Dessau。有6个在鉴定目录内,但结果不符合;其中5个未得出鉴定结果,分别为:Pomona、Regent、Amoutive、Orion、Liverpool;1个鉴定结果错误,将Kingston鉴定为Derby。Typhimurium、I 4,[5],12:i:-、Enteritidis、London、Derby、Rissen等常见血清型的鉴定结果完全符合。
多重荧光 PCR 血清分型有19株菌结果部分符合。主要表现为O抗原仅O6不同不能区分,如Albany/Duesseldorf、Goldcoast/Brikama、Muenchen/Virginia、Newport/Bardo、Litchfield/Pakistan、Hadar/Istanbul、Molade/Wippra等。A群O2和D1群O9不能区分,如Paratyphi A与Sendai、Miami。H二项抗原H2的e,n,z15和e,n,x不能区分,如Mikawasima/Hartford、Hadar/Glostrup、Sandiego/Chester。H一项抗原H1的含有“l”抗原的不能区分,如l,v和l,w不同的Litchfield/Fayed;l,v和l,z13不同的Uganda/Sinstorf和Goettingen/Napoli。H一项抗原H1的含有“g”抗原的不能区分,如g,m,s和g,s,t不同的Amsterdam/Westhampton;g,m,[s],[t]和f,g,[s]不同的Agbeni/Havana等。
虽然传统玻片凝集方法一直是沙门菌血清分型的金标准,但该方法不但存在耗时长[16]、操作繁琐,而且对于一些特殊的菌株无法进行鉴定:菌株自凝、交叉凝集、抗原不表达等情况,导致无法用免疫学的方法鉴定[17-18]。另外由于实验室检测人员技术和经验、血清质量等原因,以及H抗原二相诱导难度大、单相变异株出现等因素,使准确判定沙门菌血清成为各实验室面临的难题。
随着WGS测序成本不断降低,以及沙门菌血清分型准确率的不断提高,越来越多的实验室使用该方法代替传统血清凝集实验。本研究中,SeqSero2预测242株沙门菌血清型,其中91.74%符合,5.79%为部分符合,与徐秦等人[19]的总体符合率97.81%、张璐等人[20]的符合率97.6%基本一致。SeqSero2预测部分符合的主要原因为C2血清群中存在几对血清型,仅通过有无O6抗原来区分,但O6抗原的遗传决定因子序列差异微小且表达不稳定,导致 k-mer 匹配或靶向组装难以可靠区分,SeqSero2将这类血清型合并处理得到两个血清型结果。日常工作中遇到此类血清型,只需补做玻片凝集实验确认O6是否凝集即可正确判定。另一个原因是某些抗原式相近或一致的血清型,基因层面无法区分,只能通过生化实验来区分,如Paratyphi C、Choleraesuis和Typhisuis。SeqSero2 预测不符合的主要原因为O抗原基因缺失或部分缺失,如一株Weltevreden(3,10[15]:r:z6)预测错误为Sokode(9,46:r:z6),通过与其他Weltevreden菌株进行比对,发现此株缺失O-3,10_wzx基因,而其他菌株同时携带O-3,10_wzx基因和O-9,46_wzy基因,导致误判为Sokode,最终结合玻片凝集才正确鉴定其抗原表型。而导致O抗原基因缺失的原因,Laura Uelze等人[21]认为建库试剂存在序列偏好性,导致O抗原的rfb 基因簇片段化程度较高,使相关基因的小片段从基因组文库中被去除,或在组装过程中被拆分到两个contig中,从而影响识别。因此进行WGS时,可采取以下措施避免rfb 基因簇片段化:提取DNA 时避免降解,确保基因组片段完整;选择低 GC偏好的文库制备试剂盒;优化测序参数;使用长读长测序平台。
本研究中SISTR预测不符合率为7.85%(19/242)高于SeqSero2的2.48%(6/242),与陈丹妮等人[22]的准确率96.67%、Laura Uelze等人[21]的准确率94% 评估结果略有不同,可能是因为本研究中选用了更多的血清型(96种),包含了Havana、Cubana等易混淆血清型以及罕见血清型沙门菌,由于这些血清型cgMLST集群分散,聚类分析时聚到其他相近血清型中。而SISTR采用 85%的 cgMLST基因相似度作为聚类阈值血清型预测机制,当不同血清型的 cgMLST 相似度接近 85% 时,会被误判为同一集群。其次SISTR进行预测是基于组装的测序数据,这些数据因为前文提及的原因,产生低质量组装(N50<50 000 bp)会导致rfb簇碎片化,SISTR无法提取完整wzx/wzy序列,无法确定O抗原;同时组装质量差也会导致cgMLST核心基因断裂或缺失,使计算的相似度偏离真实值。因此,严格组装质量控制,尤其是采用长读长测序的完整基因组,可以提高SISTR 预测准确性。此外,对于一些单相变异的血清型,如I 4,[5],12:i:-,SISTR基于cgMLST聚类方法,没有SeqSero2 基于抗原基因方法的预测更可靠,这也是本研究中SISTR预测不符合率较高的重要原因。
本研究采用12个四重荧光PCR反应体系,理论上可检测200多种血清型。但对96种血清型检测的完全符合率为52.08%,低于董晓根等人[23]报道的85.26%(81/95),可能原因是本研究选用了更多血清型,且包含了更多的相近或者少见血清型,而导致不能区分或错误鉴定。其最主要原因为这些相近血清型的抗原编码基因非常相似,PCR存在交叉反应。虽然多重荧光PCR血清分型方法与WGS血清预测方法原理类似,都是基于菌株抗原基因进行判定,但由于多重荧光PCR方法的局限性,检测的抗原基因数量有限,且无法区分相似度高的基因靶点,导致其检测的血清型种类和符合率都低于基因组测序方法。因此多重荧光PCR方法适用于应急快速检测和特定血清型的常规监测。
SeqSero2和SISTR血清型预测方法准确度较高,两者结合可将准确率提升至98%左右,在许多应用场景中可替代玻片凝集试验。但目前测序设备价格昂贵、测序流程难度大,以及细菌基因组生物信息学分析技术门槛高,制约了WGS血清分型在基层的应用和普及。多重荧光PCR血清分型技术及设备要求低,并且检测时间短,方便各级检验机构应用;但该方法具有局限性,只能准确鉴定常见血清型,仍不能完全替代传统方法。随着检测的血清型越来越丰富,不同实验室可根据自身需求建立适用于本地区的血清型检测方案。通过统计我省历年来沙门菌血清型分布,单相鼠伤寒、鼠伤寒、肠炎、伦敦、里森,5种血清型占比达到80%以上,因此可以针对本地区前3~5种血清型设计多重荧光PCR引物用于快速检测,对PCR不能区分的,可以结合玻片凝集试验和WGS综合判定,从而能高效、快速、准确、低成本地对沙门菌进行血清分型。
  • 海南省科技厅卫生健康科技创新联合青年项目计划(WSJK2024QN083)
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2026年第53卷第11期
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doi: 10.20043/j.cnki.MPM.202511316
  • 接收时间:2025-11-22
  • 首发时间:2026-11-02
  • 出版时间:2026-06-10
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  • 收稿日期:2025-11-22
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海南省科技厅卫生健康科技创新联合青年项目计划(WSJK2024QN083)
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    四川省疾病预防控制中心,四川 成都 610041

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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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