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Article(id=1241675633621790843, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241675628051755031, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202403536, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1711641600000, receivedDateStr=2024-03-29, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773968140273, onlineDateStr=2026-03-20, pubDate=1732464000000, pubDateStr=2024-11-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773968140273, onlineIssueDateStr=2026-03-20, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773968140273, creator=13701087609, updateTime=1773968140273, updator=13701087609, issue=Issue{id=1241675628051755031, tenantId=1146029695717560320, journalId=1227665162245664772, year='2024', volume='51', issue='22', pageStart='4033', pageEnd='4224', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773968138945, creator=13701087609, updateTime=1773968595676, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241677543783322543, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241675628051755031, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241677543783322544, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241675628051755031, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=4184, endPage=4190, ext={EN=ArticleExt(id=1241675634892664991, articleId=1241675633621790843, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Study on the application of blood plate-paper bridge method in Salmonella serotyping, columnId=1228016572065837304, journalTitle=Modern Preventive Medicine, columnName=Experimental Technology and Applications, runingTitle=null, highlight=null, articleAbstract=
Objective

To comprehensively analyze the influencing factors of the blood plate-paper bridge method for H antigen induction and identification, and to investigate its mechanism in order to promote the application of this method in Salmonella serotyping.

Methods

The experimental conditions of the blood plate-paper bridge method for Salmonella serotyping were optimized by comparing the effects of different paper bridge materials on the induction and identification of H antigen, scanning the surface of different paper bridge materials by scanning electron microscope to explore the mechanism of the effects, and combining the factors of environmental humidity and saline dosage. The optimized method was compared with the simple plate method of GB 4789.4 standard for serotyping 40 strains of Salmonella.

Results

The matte surface of CN110 and CN95 nitrocellulose membranes had similar bridging effect with ordinary filter paper, and the difference was not statistically significant (P>0.05), while the effect of filter membrane was significantly lower than that of ordinary filter paper (P<0.05). Salmonella spread better at higher humidity, and there was a significant difference between the results at 80% humidity compared with those of the 50% and 20% groups (P<0.016 7). The spreading effect was significantly improved with increasing amount of saline (P<0.001), and the optimal amount was 15 μL. Forty foodborne Salmonella strains were identified using the two methods, and the method in this study was able to accurately identify Salmonella serotypes in one go, whereas the simple plate method required one to three inductions.

Conclusion

The blood plate-paper bridge method optimized in this study is easy to operate and has a significant induction effect, which is suitable for the identification of Salmonella serotypes. This method can be used as an effective complement to Salmonella serotyping methods to improve the efficiency and accuracy of foodborne disease traceability.

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目的

综合分析血平板-纸桥法进行H抗原诱导和鉴定的影响因素，探究其机理，以促进该方法在沙门氏菌血清分型中的应用。

方法

通过比较不同纸桥材质对H抗原诱导和鉴定的影响，采用扫描电镜对不同纸桥材质表面进行扫描，探究其影响机理，并结合环境湿度和生理盐水用量等因素，优化沙门氏菌血清分型的血平板-纸桥法实验条件。采用优化后的方法与GB 4789.4标准的简易平板法，对40株沙门氏菌进行血清型鉴定进行比较。

结果

CN110和CN95硝酸纤维素膜的哑光面与普通滤纸的过桥效果相近，差异无统计学意义（P>0.05），而过滤滤膜效果显著低于普通滤纸（P<0.05）。沙门氏菌在更高湿度下蔓延效果更好，80%湿度下与50%、20%组的结果相比存在显著差异（P<0.016 7）。随着生理盐水量的增加，蔓延效果显著提升（P<0.001），最优量是15 μL。使用两种方法对40株食源性沙门氏菌进行鉴定，本研究方法能一次性准确鉴定沙门氏菌血清型，而简易平板法需1至3次诱导。

结论

本研究所优化的血平板-纸桥法操作简便且诱导效果显著，适合于沙门氏菌血清型的鉴定。此方法可作为沙门氏菌血清分型方法的有效补充，提高食源性疾病的溯源效率和准确性。

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吕敬章，E-mail: ；
林燕奎，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=h1v7cOU4O0NPuyvazvKE5w==, magXml=Xj9T6GKuWhMLGemWELQQTQ==, pdfUrl=null, pdf=wKYzQOy2FJuSrfyJhLqxBA==, pdfFileSize=991969, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=wLg6+zC4mgzLSJyrNIjVsw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=YNzfX0LlBAFiDTHE0xnBVA==, mapNumber=null, authorCompany=null, fund=null, authors=

涂晓波（1988—），男，本科，工程师，研究方向：食品安全检验

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涂晓波（1988—），男，本科，工程师，研究方向：食品安全检验

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涂晓波（1988—），男，本科，工程师，研究方向：食品安全检验

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Nature, 2019, 575(7784): 664-668., articleTitle=An evolutionarily stable strategy to colonize spatially extended habitats, refAbstract=null)], funds=[Fund(id=1241826549700297031, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, awardId=spaq-2019-50, language=CN, fundingSource=食品安全国家标准立项计划(spaq-2019-50), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241826540204393434, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, xref=1., ext=[AuthorCompanyExt(id=1241826540208587739, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, companyId=1241826540204393434, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Food Inspection and Quarantine Technology Center of Shenzhen Customs District, Shenzhen, Guangdong 518000, China), AuthorCompanyExt(id=1241826540216976348, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, companyId=1241826540204393434, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.深圳海关食品检验检疫技术中心，广东　深圳　518000)]), AuthorCompany(id=1241826540313445348, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, xref=2., ext=[AuthorCompanyExt(id=1241826540321833958, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, companyId=1241826540313445348, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.深圳市检验检疫科学研究院)])], figs=[ArticleFig(id=1241826548005798149, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=EN, label=Fig.1, caption=Blood plate-paper bridge method for Salmonella phase inversion, figureFileSmall=4C4KV4zXpWcGWscd7OS4mg==, figureFileBig=bV41aIDBG8xaODeZogG79w==, tableContent=null), ArticleFig(id=1241826548119044361, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=CN, label=图1, caption=用于沙门氏菌相位反转的血平板-纸桥法, figureFileSmall=4C4KV4zXpWcGWscd7OS4mg==, figureFileBig=bV41aIDBG8xaODeZogG79w==, tableContent=null), ArticleFig(id=1241826548257456402, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=EN, label=Fig.2, caption=Images of different paper bridge materials under SEM electron microscopy（2 000×）, figureFileSmall=4xgZIY9T/CRWCSxuDZDXkw==, figureFileBig=2fBErfpAqcCKJkEQnWIofw==, tableContent=null), ArticleFig(id=1241826548366508310, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=CN, label=图2, caption=不同纸桥材料的SEM电镜下扫描图（2 000×）, figureFileSmall=4xgZIY9T/CRWCSxuDZDXkw==, figureFileBig=2fBErfpAqcCKJkEQnWIofw==, tableContent=null), ArticleFig(id=1241826548496531740, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=EN, label=Table 1, caption=

Effects of different paper bridge materials on Salmonella paper bridge method

, figureFileSmall=null, figureFileBig=null, tableContent=
材料血平板接触面沙门氏菌菌种
马流产沙门氏菌
ATCC 9842		鼠伤寒沙门氏菌
ATCC 14028		婴儿沙门氏菌
ATCC 51741		鸭沙门氏菌
ATCC 9270		肠炎沙门氏菌
ATCC13076
阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数
CN110光面0505050505
哑光面5050505050
CN95光面0505050505
哑光面5050505050
过滤滤膜光面0523052341
哑光面1450235050
普通滤纸正面5050414150
反面5050504150
), ArticleFig(id=1241826548626555166, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=CN, label=表1, caption=

不同纸桥材料对沙门氏菌纸桥法的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
材料血平板接触面沙门氏菌菌种
马流产沙门氏菌
ATCC 9842		鼠伤寒沙门氏菌
ATCC 14028		婴儿沙门氏菌
ATCC 51741		鸭沙门氏菌
ATCC 9270		肠炎沙门氏菌
ATCC13076
阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数
CN110光面0505050505
哑光面5050505050
CN95光面0505050505
哑光面5050505050
过滤滤膜光面0523052341
哑光面1450235050
普通滤纸正面5050414150
反面5050504150
), ArticleFig(id=1241826548756578597, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=EN, label=Table 2, caption=

Statistical analysis of the effects of different paper bridge materials on the paper bridge method for Salmonella（n=40）

, figureFileSmall=null, figureFileBig=null, tableContent=
材料阳性率（%）χ2值aP值a
普通滤纸94//
CN110哑光面1001.5630.546
CN95哑光面1001.5630.546
过滤滤膜哑光面726.9810.013
), ArticleFig(id=1241826548857241898, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=CN, label=表2, caption=

不同纸桥材料对沙门氏菌纸桥法效果的统计分析（n=40）

, figureFileSmall=null, figureFileBig=null, tableContent=
材料阳性率（%）χ2值aP值a
普通滤纸94//
CN110哑光面1001.5630.546
CN95哑光面1001.5630.546
过滤滤膜哑光面726.9810.013
), ArticleFig(id=1241826548924350766, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=EN, label=Table 3, caption=

Effects of different environmental humidity on Salmonella paper bridge method

, figureFileSmall=null, figureFileBig=null, tableContent=
材料相对湿度
（%）		沙门氏菌菌种
鼠伤寒沙门氏菌
ATCC 14028		阿巴特图巴沙门菌
ATCC 35640		肠炎沙门氏菌
ATCC 13076		山夫登堡沙门氏菌
（2020国本级盲样）		合计
阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数
滤纸2005233205515
5041414123146
8050505050200
χ2值////25.055
P值0.006a0.231a0.725a0.009a<0.001
CN1102041413214119
5023414141146
8050505050200
χ2值////11.200
P值0.251a1.000a0.725a0.051a0.004
), ArticleFig(id=1241826549037596979, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=CN, label=表3, caption=

不同环境湿度对沙门氏菌纸桥法的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
材料相对湿度
（%）		沙门氏菌菌种
鼠伤寒沙门氏菌
ATCC 14028		阿巴特图巴沙门菌
ATCC 35640		肠炎沙门氏菌
ATCC 13076		山夫登堡沙门氏菌
（2020国本级盲样）		合计
阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数
滤纸2005233205515
5041414123146
8050505050200
χ2值////25.055
P值0.006a0.231a0.725a0.009a<0.001
CN1102041413214119
5023414141146
8050505050200
χ2值////11.200
P值0.251a1.000a0.725a0.051a0.004
), ArticleFig(id=1241826549159231799, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=EN, label=Table 4, caption=

Effects of different amounts of physiological saline on paper bridge method

, figureFileSmall=null, figureFileBig=null, tableContent=
生理盐水量（μl）沙门氏菌菌种
鼠伤寒沙门氏菌
ATCC 14028		阿巴特图巴沙门菌
ATCC 35640		肠炎沙门氏菌
ATCC 13076		山夫登堡沙门氏菌
（2020国本级盲样）		合计
阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数
0（对照）05050505020
523322314812
1041414123146
1550505050200
χ2值////17.143
P值0.231a0.725a0.231a0.068a<0.001
), ArticleFig(id=1241826549259895098, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=CN, label=表4, caption=

不同生理盐水量对沙门氏菌纸桥法的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
生理盐水量（μl）沙门氏菌菌种
鼠伤寒沙门氏菌
ATCC 14028		阿巴特图巴沙门菌
ATCC 35640		肠炎沙门氏菌
ATCC 13076		山夫登堡沙门氏菌
（2020国本级盲样）		合计
阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数阳性数阴性数
0（对照）05050505020
523322314812
1041414123146
1550505050200
χ2值////17.143
P值0.231a0.725a0.231a0.068a<0.001
), ArticleFig(id=1241826549364752701, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=EN, label=Table 5, caption=

Identification results of serotypes of Salmonella detected in food

, figureFileSmall=null, figureFileBig=null, tableContent=
沙门氏菌血清型拉丁名称抗原结构群组诱导次数
血平板-纸桥法简易平板法
甲型副伤寒S.Paratyphi A1,2,12 :a :1,5A12
马流产S.Abortusequi4,12 : - : e,n,xB11
鼠伤寒S.Typhimurium1,4,12 :i :1,2B11
乙型副伤寒S.Paratyphi B 1,4,12 :b :1,2B11
德尔卑S.Derby1,4,12 : f,g : 1,2B12
阿贡纳S.Agona1,4,12 : f,g,s : 1,2B11
蒙得维的亚S.Montevideo6,7, 14 : g,m,s : 1,2,7C111
纽波特S.Newport6,8,20 : e,h : 1,2C211
肯塔基S.Kentucky8,20 : i : z6C211
肠炎S.Enteritidis1,9,12 : g,m : 1,7D11
巴拿马S.Panama1,9,12 : l,v : 1,5D11
鸭S.Anatum3, {15,34} : e,h : 1,6E111
阿巴特图巴S.Abaetetuba11: k : 1,5F11
伦敦S.London3,{10}{15} : l,v : 1,6E112
旺兹沃思S.Wandsworth39: b : 1,2Q11
海德尔堡S.Heidelberg1,4,12 :r :1,2B12
婴儿S.Infantis6,7, 14 : r : 1,5C113
圣保罗S.Saintpaul1,4,12 :e,h :1,2B11
维尔肖S.Virchow6,7,14 : r : 1,2C111
病牛S.Bovismorbificans6,8,20 : r :1,5C211
斯坦利S.Stanly1,4,12, :d :1,2B11
乌干达S.Uganda3,{15} : l,z13 : 1,5E111
阿顿道夫S.Altendorf4,12:c:1,7B11
普利茅斯S.Plymouth9,46:d:z6D211
韦太夫雷登S.Weltevreden 3,{15} : r : z6E111
科瓦利斯S.Corvallis8,20:z4,z23:z6C312
火鸡S.Meleagridis3, {15,34} : e,h : l,wE111
巴雷利S.Bareilly6,7,14:y:1,5C111
黄金海岸S.Goldcoast6,8:r:l,wC211
布伦登卢普S.Braenderup6,7, 14 : e,h : e,n,z15C111
姆班达卡S.Mbandaka 6,7, 14 : z10 : e,n,z15C111
利奇菲尔德S.Litchfield6,8 : l,v : 1,2C211
波茨坦S.Potsdam 6,7,14 : l,v : e,n,z15C111
印地安纳S.Indiana 1,4,12 :z :1,7B11
明斯特S.Muenster 3,{15,34} : e,h :1,5E111
塞罗S.Cerro6,14,18:z4,z23:1,5K11
奥古斯坦堡S.Augustenborg6,7,14:i:1,2C111
利文斯通S.Livingstone6,7,14:d:l,wC112
列克星敦S.Lexington 3,{15,34}:z10:1,5E111
巴尔多S.Bardo8: e,h : 1,2C311
), ArticleFig(id=1241826549469610303, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675633621790843, language=CN, label=表5, caption=

食品中检出沙门菌的血清型鉴定结果

, figureFileSmall=null, figureFileBig=null, tableContent=
沙门氏菌血清型拉丁名称抗原结构群组诱导次数
血平板-纸桥法简易平板法
甲型副伤寒S.Paratyphi A1,2,12 :a :1,5A12
马流产S.Abortusequi4,12 : - : e,n,xB11
鼠伤寒S.Typhimurium1,4,12 :i :1,2B11
乙型副伤寒S.Paratyphi B 1,4,12 :b :1,2B11
德尔卑S.Derby1,4,12 : f,g : 1,2B12
阿贡纳S.Agona1,4,12 : f,g,s : 1,2B11
蒙得维的亚S.Montevideo6,7, 14 : g,m,s : 1,2,7C111
纽波特S.Newport6,8,20 : e,h : 1,2C211
肯塔基S.Kentucky8,20 : i : z6C211
肠炎S.Enteritidis1,9,12 : g,m : 1,7D11
巴拿马S.Panama1,9,12 : l,v : 1,5D11
鸭S.Anatum3, {15,34} : e,h : 1,6E111
阿巴特图巴S.Abaetetuba11: k : 1,5F11
伦敦S.London3,{10}{15} : l,v : 1,6E112
旺兹沃思S.Wandsworth39: b : 1,2Q11
海德尔堡S.Heidelberg1,4,12 :r :1,2B12
婴儿S.Infantis6,7, 14 : r : 1,5C113
圣保罗S.Saintpaul1,4,12 :e,h :1,2B11
维尔肖S.Virchow6,7,14 : r : 1,2C111
病牛S.Bovismorbificans6,8,20 : r :1,5C211
斯坦利S.Stanly1,4,12, :d :1,2B11
乌干达S.Uganda3,{15} : l,z13 : 1,5E111
阿顿道夫S.Altendorf4,12:c:1,7B11
普利茅斯S.Plymouth9,46:d:z6D211
韦太夫雷登S.Weltevreden 3,{15} : r : z6E111
科瓦利斯S.Corvallis8,20:z4,z23:z6C312
火鸡S.Meleagridis3, {15,34} : e,h : l,wE111
巴雷利S.Bareilly6,7,14:y:1,5C111
黄金海岸S.Goldcoast6,8:r:l,wC211
布伦登卢普S.Braenderup6,7, 14 : e,h : e,n,z15C111
姆班达卡S.Mbandaka 6,7, 14 : z10 : e,n,z15C111
利奇菲尔德S.Litchfield6,8 : l,v : 1,2C211
波茨坦S.Potsdam 6,7,14 : l,v : e,n,z15C111
印地安纳S.Indiana 1,4,12 :z :1,7B11
明斯特S.Muenster 3,{15,34} : e,h :1,5E111
塞罗S.Cerro6,14,18:z4,z23:1,5K11
奥古斯坦堡S.Augustenborg6,7,14:i:1,2C111
利文斯通S.Livingstone6,7,14:d:l,wC112
列克星敦S.Lexington 3,{15,34}:z10:1,5E111
巴尔多S.Bardo8: e,h : 1,2C311
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血平板-纸桥法在沙门氏菌血清分型中的应用探究
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涂晓波 1, 2 , 马淑棉 1, 2 , 金晓蕾 1, 2 , 匡燕云 1, 2 , 刘慧玲 1, 2 , 牛娜 1, 2 , 赵芳 1, 2 , 吕敬章 1, 2 , 林燕奎 1, 2
现代预防医学 | 实验技术及其应用 2024,51(22): 4184-4190
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现代预防医学 | 实验技术及其应用 2024, 51(22): 4184-4190
血平板-纸桥法在沙门氏菌血清分型中的应用探究
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涂晓波1, 2, 马淑棉1, 2, 金晓蕾1, 2, 匡燕云1, 2, 刘慧玲1, 2, 牛娜1, 2, 赵芳1, 2, 吕敬章1, 2 , 林燕奎1, 2
作者信息
  • 1.深圳海关食品检验检疫技术中心，广东　深圳　518000
  • 2.深圳市检验检疫科学研究院

    • 涂晓波（1988—），男，本科，工程师，研究方向：食品安全检验

通讯作者:

吕敬章，E-mail: ；
林燕奎，E-mail:
Study on the application of blood plate-paper bridge method in Salmonella serotyping
Xiao-bo TU1, 2, Shu-mian MA1, 2, Xiao-lei JIN1, 2, Yan-yun KUANG1, 2, Hui-ling LIU1, 2, Na NIU1, 2, Fang ZHAO1, 2, Jing-zhang LV1, 2 , Yan-kui LIN1, 2
Affiliations
  • Food Inspection and Quarantine Technology Center of Shenzhen Customs District, Shenzhen, Guangdong 518000, China
出版时间: 2024-11-25 doi: 10.20043/j.cnki.MPM.202403536
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摘要
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目的

综合分析血平板-纸桥法进行H抗原诱导和鉴定的影响因素，探究其机理，以促进该方法在沙门氏菌血清分型中的应用。

方法

通过比较不同纸桥材质对H抗原诱导和鉴定的影响，采用扫描电镜对不同纸桥材质表面进行扫描，探究其影响机理，并结合环境湿度和生理盐水用量等因素，优化沙门氏菌血清分型的血平板-纸桥法实验条件。采用优化后的方法与GB 4789.4标准的简易平板法，对40株沙门氏菌进行血清型鉴定进行比较。

结果

CN110和CN95硝酸纤维素膜的哑光面与普通滤纸的过桥效果相近，差异无统计学意义（P>0.05），而过滤滤膜效果显著低于普通滤纸（P<0.05）。沙门氏菌在更高湿度下蔓延效果更好，80%湿度下与50%、20%组的结果相比存在显著差异（P<0.016 7）。随着生理盐水量的增加，蔓延效果显著提升（P<0.001），最优量是15 μL。使用两种方法对40株食源性沙门氏菌进行鉴定，本研究方法能一次性准确鉴定沙门氏菌血清型，而简易平板法需1至3次诱导。

结论

本研究所优化的血平板-纸桥法操作简便且诱导效果显著，适合于沙门氏菌血清型的鉴定。此方法可作为沙门氏菌血清分型方法的有效补充，提高食源性疾病的溯源效率和准确性。

血平板-纸桥法  /  沙门氏菌  /  血清分型
Objective

To comprehensively analyze the influencing factors of the blood plate-paper bridge method for H antigen induction and identification, and to investigate its mechanism in order to promote the application of this method in Salmonella serotyping.

Methods

The experimental conditions of the blood plate-paper bridge method for Salmonella serotyping were optimized by comparing the effects of different paper bridge materials on the induction and identification of H antigen, scanning the surface of different paper bridge materials by scanning electron microscope to explore the mechanism of the effects, and combining the factors of environmental humidity and saline dosage. The optimized method was compared with the simple plate method of GB 4789.4 standard for serotyping 40 strains of Salmonella.

Results

The matte surface of CN110 and CN95 nitrocellulose membranes had similar bridging effect with ordinary filter paper, and the difference was not statistically significant (P>0.05), while the effect of filter membrane was significantly lower than that of ordinary filter paper (P<0.05). Salmonella spread better at higher humidity, and there was a significant difference between the results at 80% humidity compared with those of the 50% and 20% groups (P<0.016 7). The spreading effect was significantly improved with increasing amount of saline (P<0.001), and the optimal amount was 15 μL. Forty foodborne Salmonella strains were identified using the two methods, and the method in this study was able to accurately identify Salmonella serotypes in one go, whereas the simple plate method required one to three inductions.

Conclusion

The blood plate-paper bridge method optimized in this study is easy to operate and has a significant induction effect, which is suitable for the identification of Salmonella serotypes. This method can be used as an effective complement to Salmonella serotyping methods to improve the efficiency and accuracy of foodborne disease traceability.

Blood plate-paper bridge method  /  Salmonella  /  Serotyping
涂晓波, 马淑棉, 金晓蕾, 匡燕云, 刘慧玲, 牛娜, 赵芳, 吕敬章, 林燕奎. 血平板-纸桥法在沙门氏菌血清分型中的应用探究. 现代预防医学, 2024 , 51 (22) : 4184 -4190 . DOI: 10.20043/j.cnki.MPM.202403536
Xiao-bo TU, Shu-mian MA, Xiao-lei JIN, Yan-yun KUANG, Hui-ling LIU, Na NIU, Fang ZHAO, Jing-zhang LV, Yan-kui LIN. Study on the application of blood plate-paper bridge method in Salmonella serotyping[J]. Modern Preventive Medicine, 2024 , 51 (22) : 4184 -4190 . DOI: 10.20043/j.cnki.MPM.202403536
正文
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沙门氏菌被认为是全球最重要的病原体之一[1]，主要通过摄入受污染食物或接触受感染动物而传播[2]，能引发食物中毒、胃肠炎、伤寒等多种疾病。在中国，沙门氏菌是导致食物中毒的主要病原体之一，涉及约40%的细菌性食物中毒案例[3-4]。
沙门氏菌属属于肠杆菌科中的一个复杂菌属，其血清分型主要基于White–Kauffman–Le Minor（WKLM）表[5]。到目前为止，WKLM表已列出超过2 000种沙门氏菌血清型。血清分型是鉴定沙门氏菌的主要方法，通过与特异性抗体的凝集反应来识别沙门氏菌的O抗原和H抗原。O抗原为脂多糖中的糖聚合物[6]，而H抗原是构成鞭毛的蛋白质亚单位[7]。其中，H抗原的确定耗时较长。沙门氏菌通常具有双相型，编码H1和H2两种H抗原基因[8]。它们受协调性调控，任一时刻仅表达一种H抗原[9]，需通过位相诱导分别测定H1和H2抗原[10-11]。单相分离株的血清分型需多次相位转换确认H2抗原不表达[12]。目前，H抗原的常用鉴定方法为GB 4789.4中的简易平板法[13]，但其有诱导效果差，生长控制困难和凝集反应受半固体琼脂干扰等缺点[14]。血平板-纸桥法是一种新兴的分型方法，通过在血平板上制作凹槽，放置纸桥，在纸桥上滴加生理盐水或抗血清，诱导沙门氏菌的相位转换，完成分型鉴定。具有操作简便、快速高效和经济实用的优势，可应用于临床诊断和食品污染溯源等领域，已被ISO采纳[15]。但是该方法受材质、湿度等因素影响，本研究首次对其影响因素进行综合分析，对其影响机制进行探究，提高分型效率和准确性，应对食源性疾病日益严峻的挑战。
1　材料与方法
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1.1　菌株来源
本研究所用的40株沙门氏菌菌株，包括5株采购于美国模式培养物集存库（ATCC）的标准菌株（马流产沙门氏菌ATCC 9842、鼠伤寒沙门氏菌ATCC 14028、婴儿沙门氏菌ATCC 51741、鸭沙门氏菌ATCC 9270及肠炎沙门氏菌ATCC 13076）；其余35株为近年来深圳海关食检中心从进出口食品中分离出的沙门氏菌菌株。
1.2　材料和仪器
氯化钠（广州牌），Swarm琼脂培养基（北京陆桥），血琼脂平板（北京陆桥），沙门氏菌属诊断血清（丹麦SSI）；无菌刀片，无菌镊子，普通滤纸（杭州双圈），CN95及CN110硝酸纤维素膜（深圳百穗康），0.22 μm过滤滤膜（德国Merck）；恒温恒湿培养箱（德国宾得/KBWF240），电子天平（梅特勒/ML4002T），高压灭菌锅（日本HIRAYAMA/HV-85），温湿度计（德图仪表/608-H1），热场发射扫描电镜（捷克FEI/Quanta 400F）。
1.3　实验方法
1.3.1　简易平板法
本方法依据GB 4789.4对沙门氏菌H抗原的鉴定方法进行。将菌株接种于Swarm琼脂中央，于36 ℃下培养24 h。之后，从其边缘部分取菌进行H1相血清型鉴定。然后在Swarm琼脂平板表面滴加H1相转相血清，待血清渗透入琼脂，在血清区域中心点接种待检菌株。再次于36 ℃下培养24 h，最后从蔓延生长的菌苔边缘取菌落进行H2相血清型鉴定。
1.3.2　血平板-纸桥法
使用无菌手术刀沿血平板中心线切去0.6 cm宽的琼脂条，使培养基被分割成两个无接触的区域A和B。然后用无菌小镊子夹取2至3根“纸桥”，搭设在A和B两个区域之间，从而形成2~3条纸桥连接这两个区域。当待检菌株的H1相抗原未检出时，可以在纸桥上加入生理盐水，以促进抗原良好生长，随后进行鉴定。使用接种环取少量菌苔，轻轻涂抹在A区域纸桥边缘的血平板上，然后在36 ℃下倒置培养24 h。之后从B区域新生长的菌苔边缘取菌，以进行H1相的鉴定。在纸桥上滴加待检菌株H1相转相血清，再用接种环取少量菌苔轻轻涂抹于A区域纸桥边缘的血平板上，并在36 ℃下倒置培养24 h。之后在B区域的新生长菌苔边缘取菌，进行H2相的鉴定。
如图1所示，血琼脂平板中间被移除了0.6 cm宽的条带，形成槽。细菌接种在纸桥左侧的A区，标示①所指示。孵育后，相位诱导表达出替代的鞭毛抗原，并向右端迁移。经过24 h孵育，可以在纸桥右侧的B区观察到细菌生长，用②指示。本研究中，以沙门氏菌从A区蔓延生长至B区判定为“阳性”，以沙门氏菌未从A区蔓延生长至B区判定为“阴性”。
1.3.3　不同纸桥材料对比
本试验选用普通滤纸、过滤滤膜、CN110及CN95硝酸纤维素膜作为纸桥材料。实验菌株为马流产沙门菌ATCC 9842等6株标准菌株。不同材料的接触面各设置5次平行实验。依照1.3.2操作，对不同材料的试验结果进行比较。对于效果较好的纸桥材料，通过扫描电镜进行观察分析。
1.3.4　不同环境湿度对比
采用试验结果较好的纸桥材料，利用恒温恒湿培养箱设置20%、50%、80%相对湿度的环境，并设立相应的对照组进行比较试验。同时，使用湿度计确保各环境湿度准确无误。不同环境湿度各设置5次平行实验。依照1.3.2操作，将血平板置于不同相对湿度环境中，并在36 ℃恒温条件下倒置培养24 h。
1.3.5　不同生理盐水量对比
依照1.3.2操作，在A和B区域搭建好纸桥后，在纸桥上分别滴加5 μL、10 μL、15 μL的生理盐水。不同生理盐水量各设置5次平行实验。同时设立未加生理盐水的空白对照，以观察不同盐水量对菌种过桥的影响。
1.3.6　血清型鉴定对比
对优化后的血平板-纸桥法和简易平板法进行比较，使用5株标准菌株、35株分离株共计40株沙门氏菌（分属WKLM表中的11个群）进行血清分型鉴定，并对比两种方法的诱导次数和鉴定结果。
1.3.7　统计学分析
采用SPSS 27.0软件对数据资料进行分析，使用χ2检验或Fisher精确检验对计数资料进行统计分析，检验水准α =0.05。
2　结果与分析
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2.1　不同纸桥材料对沙门氏菌的影响
不同纸桥材料对沙门氏菌影响的具体结果可参见表1。
从表1结果来看，当CN110和CN95硝酸纤维素膜的哑光面接触血平板时，所有试验菌株均能蔓延生长过桥。马流产沙门菌和婴儿沙门菌在过滤滤膜上只有部分蔓延过桥。多数试验菌株能够通过普通滤纸的正反面蔓延过桥。
选取效果较好的材料，和普通滤纸进行比较，根据样本数据，采用Fisher精确检验进行统计分析。表2结果显示，普通滤纸的阳性率为94%，整体表现可靠。CN110哑光面和CN95哑光面的阳性率为100%，效果与普通滤纸相近，差异无统计学意义（P>0.05）。过滤滤膜哑光面的阳性率为72%，低于普通滤纸，且差异有统计学意义（P<0.05）。
2.2　硝酸纤维膜CN110、CN95和普通滤纸的扫描电镜观察
使用热场发射扫描电镜(捷克FEI/Quanta 400F)在10.5 mm工作距离、2 000倍放大、20.0 kV电压和高真空模式等相同实验条件下对不同纸桥材料进行扫描的图像。图2显示，CN110和CN95的哑光面材料表面是粗糙且多孔的，而光面则较为平滑；滤纸的正面和背面呈现纤维状结构。
结合表1的结果可知，沙门氏菌经由CN110和CN95膜的哑光面及滤纸过桥的效果较好，但不通过其光面过桥。而CN110和CN95膜的哑光面均较为粗糙，CN95的孔径比CN110膜更大。通过纸桥材料表面微观结构可以发现，材料的粗糙度、孔隙结构和纤维状层是关键因素。粗糙且多孔的表面更适合沙门氏菌的生长和蔓延，而平滑表面由于接触面积减少，不利于细菌的过桥。
2.3　不同环境湿度的结果
依据2.1的试验结果，本研究选用滤纸和CN110硝酸纤维素膜作为纸桥材料，依据1.3.4操作，结果见表3。实验显示，滤纸在20%、50%、80%湿度下阳性率分别为25%、70%、100%，CN110膜在20%、50%、80%湿度下阳性率分别为55%、70%、100%；显示滤纸在不同湿度下的结果差异有统计学意义（χ2=25.055，P<0.001），CN110膜在不同湿度下的结果差异有统计学意义（χ2=11.200，P=0.004<0.016 7）；采用Bonferroni法进行两两比较，滤纸在不同湿度下结果均存在显著差异，CN110膜在80%湿度下与50%、20%组的结果相比存在显著差异。综上，无论是滤纸还是CN110膜，方法的阳性率随相对湿度增加而显著增加。目前确定的最佳环境相对湿度为80%。
2.4　不同生理盐水量的试验结果
按照1.3.5的操作步骤进行实验，具体结果见表4。在无生理盐水条件下，四种沙门氏菌均未蔓延过桥。滴加5 μL生理盐水时，总阳性率为40%。滴加10 μL时，总阳性率为70%。滴加15 μL时，所有菌株均过桥。结果显示，随着生理盐水量的增加，沙门氏菌纸桥法的阳性率显著增加（χ2=17.143，P<0.001），采用Bonferroni法进行两两比较，15 μL组与10 μL组、5 μL组的阳性率相比存在显著差异。考虑到如果生理盐水量过大，游离水可能形成一个替代路径，将细菌输送至抗血清屏障，从而导致相位转换失败[16]。因此，目前确定的最佳生理盐水量为15 μL。
2.5　血清型鉴定比对试验
使用改进的血平板-纸桥法和简易平板法对40株沙门氏菌进行H抗原诱导和血清型鉴定。血平板-纸桥法一次诱导即可准确鉴定所有沙门氏菌的血清型。简易平板法需2次诱导才能鉴定出甲型副伤寒沙门菌等六种菌株，而婴儿沙门菌则需3次诱导。结果表明，血平板-纸桥法比简易平板法传代次数少、耗时短、结果更准确。
3　结论与讨论
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沙门氏菌的感染是一个复杂的过程，首先通过特定的粘附素与肠上皮细胞表面受体结合，侵入细胞内部。在细胞内，通过形成包含体在宿主细胞内存活并增殖[17]。沙门氏菌的鞭毛不仅为其提供运动性，还参与其趋化性和毒力的表达[18]。鞭毛上的H抗原交替表达，即鞭毛H相变异，是沙门氏菌逃避宿主免疫识别的一种机制[19]。因此，精确地诱导和鉴定H抗原对于沙门氏菌的血清分型至关重要。本研究中，血平板-纸桥法通过血平板提供了必要的营养成分，支持沙门氏菌的生长和鞭毛抗原的表达。通过在纸桥上滴加抗血清，来创建抗血清屏障，进一步诱导H抗原的表达。
本研究拓展了纸桥材料的选择范围，结果显示硝酸纤维素膜哑光面的效果与普通滤纸相近，硝酸纤维素膜光面和过滤滤膜的效果差于普通滤纸。通过观察表面微观结构，粗糙且多孔的表面更适合沙门氏菌的生长和蔓延。这与细菌的迁移行为受表面粗糙度、孔隙结构和化学性质（如疏水性或亲水性）等多种因素的影响有关。表面粗糙度增加会降低微生物与介质之间的排斥能垒，从而增加微生物的迁移能力。孔隙度和孔隙大小分布影响细菌的迁移路径和速度[20]。
本研究中，最优环境相对湿度为80%，最优生理盐水量为15 μL。这与沙门氏菌的迁移能力受鞭毛游动的影响有关。在干燥环境中，细菌的迁移能力会受到限制，因为缺乏足够的水分会影响鞭毛的正常功能。而在适量的水分条件下，有助于细菌的游动和扩散[21]。
与简易平板法相比，血平板-纸桥法更为高效。简易平板法通常需要传代1至3次后才能进行血清分型。血平板-纸桥法可直接在血平板上进行诱导和血清分型，仅需少量抗血清便能有效诱导沙门氏菌的鞭毛抗原，一个血平板能检测3至5株分离株。考虑到血平板-纸桥法的特性和优点，基层实验室可以在GB4789.4血清学鉴定方法的基础上，利用血平板-纸桥法进一步诱导和鉴定H抗原不明确的沙门氏菌株。这为准确鉴定沙门氏菌提供了一种可靠且快速的补充方法，对于食源性疾病快速诊断和溯源具有重要意义。
基金
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  • 食品安全国家标准立项计划(spaq-2019-50)
文献
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参考文献 引证文献
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2024年第51卷第22期
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doi: 10.20043/j.cnki.MPM.202403536
  • 接收时间：2024-03-29
  • 首发时间：2026-03-20
  • 出版时间：2024-11-25
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  • 收稿日期：2024-03-29
基金
食品安全国家标准立项计划(spaq-2019-50)
作者信息
    1.深圳海关食品检验检疫技术中心，广东　深圳　518000
    2.深圳市检验检疫科学研究院

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吕敬章，E-mail: ；
林燕奎，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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