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Article(id=1241376204830339569, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241376204247331313, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20230701, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1699977600000, receivedDateStr=2023-11-15, revisedDate=null, revisedDateStr=null, acceptedDate=1708272000000, acceptedDateStr=2024-02-19, onlineDate=1773896750885, onlineDateStr=2026-03-19, pubDate=1714752000000, pubDateStr=2024-05-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773896750885, onlineIssueDateStr=2026-03-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773896750885, creator=13701087609, updateTime=1773896750885, updator=13701087609, issue=Issue{id=1241376204247331313, tenantId=1146029695717560320, journalId=1192105938417971205, year='2024', volume='64', issue='5', pageStart='1331', pageEnd='1682', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1773896750747, creator=13701087609, updateTime=1773897643611, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241379949253284790, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241376204247331313, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241379949253284791, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241376204247331313, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1668, endPage=1682, ext={EN=ArticleExt(id=1241376207002989043, articleId=1241376204830339569, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Development and performance evaluation of a biochemical kit for identification ofEnterobacteriaceae based on numerical identification method, columnId=1194702985843413943, journalTitle=Acta Microbiologica Sinica, columnName=Technology and Method, runingTitle=null, highlight=null, articleAbstract=

[Background] Bacteria ofEnterobacteriaceae are the main pathogens of foodborne and clinical infections, posing a threat to human and animal health. The numerical identification method using API 20E as the "gold standard" is one of the main identification methods forEnterobacteriaceae. However, the existing numerical identification methods have problems such as laborious sample addition, low accuracy, and high prices. [Objective] To develop a semi-automatic, high-precision, and low-cost biochemical kit for identifying the bacteria ofEnterobacteriaceae based on the numerical identification method. [Methods] On the basis of the theoretical model and supporting software of the numerical identification system forEnterobacteriaceae established by our team, we designed and optimized 24 biochemical matrix trace formulations. Furthermore, a semi-automatic freeze-drying identification strip was developed. Using the commercialized numerical identification strip API 20E, MALDI-TOF MS, and 16S rRNA gene sequencing as the controls, we evaluated the performance of the developed biochemical kit that integrated semi-automatic biochemical identification strips and online analysis software. [Results] The biochemical spectra of 458 strains ofEnterobacteriaceae were obtained, with the overall identification accuracy of 98.5% at the genus level and 96.5% at the species level. The kit only needed twice sample addition to obtain the identification results, with the price only 4.46% that of API 20E. The shelf life of the product was 7 days, and the biochemical experiments had repeatability. [Conclusion] We develop a semi-automatic biochemical kit for identification ofEnterobacteriaceae based on the numerical identification method, which has simple operation, a low cost, and high accuracy. This study provides technical support for the identification ofEnterobacteriaceae and the development of biochemical products for numerical identification of other families and genera of bacteria.

, correspAuthors=Qinghua YE, Qingping WU, authorNote=null, correspAuthorsNote=
*E-mail: YE Qinghua,;
E-mail: WU Qingping,
, copyrightStatement=Copyright ©2024 Acta Microbiologica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Lexuan JIN, Qiang WAN, Lulu CAO, Qinghua YE, Xun WANG, Jumei ZHANG, Zhihe CAI, Qingping WU), CN=ArticleExt(id=1241376209532154375, articleId=1241376204830339569, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=基于数值鉴定法的肠杆菌科生化试剂盒的研制及效果评价, columnId=1194702986061517752, journalTitle=微生物学报, columnName=技术与方法, runingTitle=null, highlight=null, articleAbstract=

【背景】肠杆菌科细菌是食源性和临床感染的主要病原菌，对人类和动物健康有重要影响，以API 20E为“金标准”的数值鉴定法是其主要的鉴定方法之一，但现有的数值鉴定方法存在手工单个加样或/和准确率低、价格贵的问题。【目的】研制出半自动化、准确性高及价格低的肠杆菌科细菌数值鉴定生化试剂盒。【方法】在本团队前期建立的肠杆菌科数值鉴定系统理论模型与配套软件的基础上，设计并优化筛选出的24种生化基质微量化配方，在此基础上，研制半自动冻干鉴定条；以现行商品化的数值鉴定条API 20E、MALDI-TOF MS及16S rRNA基因测序为对照，对研制的集半自动生化鉴定条与在线分析软件于一体的生化试剂盒进行效果评价。【结果】共获得458株肠杆菌科细菌的生化谱，在属水平总体鉴定准确率达到98.5%，在种水平总体鉴定准确率达到96.5%；仅需两次加样即可获得鉴定结果，且价格仅为API 20E的4.46%；产品保质期为7 d，生化试验具有稳定可重复性。【结论】本研究研制出的半自动化肠杆菌科细菌数值鉴定生化试剂盒简便经济、准确率高，为肠杆菌科细菌的鉴定提供技术支撑，并为其他科属细菌的数值鉴定生化产品的开发提供技术指引。

, correspAuthors=叶青华, 吴清平, authorNote=null, correspAuthorsNote=null, copyrightStatement=版权所有©《微生物学报》编辑部2024, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=Q1jNpegFzsD/uQz2DT9jAg==, magXml=jV3tKfyYID8ybmwuj4slLg==, pdfUrl=null, pdf=vK0UIKJU1d+whvVy0vo9ng==, pdfFileSize=999096, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=i1KRsuk1ARhS+ehWrGONjQ==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=9P2JPoWlCkW6F+eYHi81CA==, mapNumber=null, authorCompany=null, fund=null, authors=

#These authors contributed equally to this work.

, authorsList=金乐萱, 万强, 曹璐璐, 叶青华, 王迅, 张菊梅, 蔡芷荷, 吴清平)}, authors=[Author(id=1241446113937510483, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1241446114050756698, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, authorId=1241446113937510483, language=EN, stringName=Lexuan JIN, firstName=Lexuan, middleName=null, lastName=JIN, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, address=1 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, Anhui, China
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2 广东省科学院微生物研究所 华南应用微生物国家重点实验室 广东省微生物安全与健康重点实验室国家卫健委微生物食品营养与安全科技创新平台, 广东 广州 510070, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1241446113534857275, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, xref=null, ext=[AuthorCompanyExt(id=1241446113543245883, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, companyId=1241446113534857275, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, Anhui, China), AuthorCompanyExt(id=1241446113551634492, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, companyId=1241446113534857275, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 合肥工业大学食品与生物工程学院, 安徽 合肥 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Beijing: Standards Press of China, 2002 (in Chinese)., articleTitle=null, refAbstract=null), Reference(id=1241446129276080873, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, doi=null, pmid=null, pmcid=null, year=2020, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[21], rfOrder=25, authorNames=null, journalName=中华人民共和国药典: 三部, refType=null, unstructuredReference=国家药典委员会.中华人民共和国药典: 三部[M].11版.北京:中国医药科技出版社,2020., articleTitle=null, refAbstract=null), Reference(id=1241446129452241650, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, doi=null, pmid=null, pmcid=null, year=2020, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[21], rfOrder=26, authorNames=null, journalName=Pharmacopoeia of the People's Republic of China: Part Three, refType=null, unstructuredReference=Chinese Pharmacopoeia Commission.Pharmacopoeia of the People's Republic of China: Part Three[M].11th ed.Beijing:China Medical Science Press,2020 (in Chinese)., articleTitle=null, refAbstract=null), Reference(id=1241446129615819513, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[22], rfOrder=27, authorNames=null, journalName=null, refType=null, unstructuredReference=广东环凯微生物科技有限公司, 广东环凯生物科技有限公司, 上海环凯生物科技有限公司. 一种在疏水性高分子微量多孔载体上实现均匀分液的方法[P]. 中国: CN108341988B. 2020-10-16., articleTitle=null, refAbstract=null), Reference(id=1241446129854894849, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[22], rfOrder=28, authorNames=null, journalName=null, refType=null, unstructuredReference=Guangdong Huankai Microbial Technology Co., Ltd., Guangdong Huankai Biotechnology Co., Ltd., Shanghai Huankai Biotechnology Co., Ltd. . A method for achieving uniform liquid separation on hydrophobic polymer micro porous carriers[P]. China: CN108341988B. 2020-10-16 (in Chinese)., articleTitle=null, refAbstract=null), Reference(id=1241446129980723974, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, doi=10.3390/microorganisms11020287, pmid=null, pmcid=null, year=2023, volume=11, issue=2, pageStart=287, pageEnd=null, url=null, language=null, rfNumber=[23], rfOrder=29, authorNames=null, journalName=Microorganisms, refType=null, unstructuredReference=SURÁNYI BB, ZWIRZITZ B, MOHÁCSI-FARKAS C, ENGELHARDT T, DOMIG KJ.Comparing the efficacy of MALDI-TOF MS and sequencing-based identification techniques (Sanger and NGS) to monitor the microbial community of irrigation water[J].Microorganisms,2023,11(2):287., articleTitle=Comparing the efficacy of MALDI-TOF MS and sequencing-based identification techniques (Sanger and NGS) to monitor the microbial community of irrigation water, refAbstract=null)], funds=[Fund(id=1241446123580215848, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, awardId=2022YFF1100700, language=EN, fundingSource=National Key Research and Development Program of China(2022YFF1100700), fundOrder=null, country=null), Fund(id=1241446123668296236, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, awardId=2022YFF1100700, language=CN, fundingSource=国家重点研发项目(2022YFF1100700), fundOrder=null, country=null), Fund(id=1241446123810902579, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, awardId=2022A1515011740, language=EN, fundingSource=Natural Science Foundation of Guangdong Province(2022A1515011740), fundOrder=null, country=null), Fund(id=1241446123945120315, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, awardId=2022A1515011740, language=CN, fundingSource=广东省自然科学基金(2022A1515011740), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241446113534857275, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, xref=null, ext=[AuthorCompanyExt(id=1241446113543245883, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, companyId=1241446113534857275, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, Anhui, China), AuthorCompanyExt(id=1241446113551634492, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, companyId=1241446113534857275, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 合肥工业大学食品与生物工程学院, 安徽 合肥 230601)]), AuthorCompany(id=1241446113673269316, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, xref=null, ext=[AuthorCompanyExt(id=1241446113681657924, 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tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, xref=null, ext=[AuthorCompanyExt(id=1241446113815875660, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, companyId=1241446113811681355, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3 Guangdong Huankai Biotechnology Co., Ltd., Zhaoqing 526238, Guangdong, China), AuthorCompanyExt(id=1241446113824264269, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, companyId=1241446113811681355, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3 广东环凯生物科技有限公司, 广东 肇庆 526238)])], figs=[ArticleFig(id=1241446119755010449, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Figure 1, caption=Cross section and operation diagram of identification strip[22]., figureFileSmall=v1CBdRwEqAra/vdrT6zuyQ==, figureFileBig=QstkXsUGhkJESWO3FCkCag==, tableContent=null), ArticleFig(id=1241446119859868060, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=图1, caption=鉴定条横截面及操作示意图[22], figureFileSmall=v1CBdRwEqAra/vdrT6zuyQ==, figureFileBig=QstkXsUGhkJESWO3FCkCag==, tableContent=null), ArticleFig(id=1241446120002474406, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Figure 2, caption=Gradient test results of arabinose-reactive sugar fermentation additives.Enterobacter cloacae CMCC(B)45301,Enterobacter sakazakii ATCC51329, andSalmonella paratyphi A CMCC(B)50093 should be positive (with a positive probability of 100 and a color of yellow or chartreuse), whileProteus mirabilis CMCC(B)49005,Proteus vulgaris CMCC(B)49027,Shigella flexneri CMCC(B)51572, andProvidencia rettgeri FSCC(I)205006 should be negative (with a positive probability of 0 and a color of blue or green)., figureFileSmall=kW5mmXQTAH65Rqt5UOEl9g==, figureFileBig=EFLdVjorjDwtZNkP1IDw3Q==, tableContent=null), ArticleFig(id=1241446120128303532, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=图2, caption=阿拉伯糖反应孔糖发酵添加剂梯度试验结果

阴沟肠杆菌CMCC(B)45301、坂崎肠杆菌ATCC51329和甲型副伤寒沙门氏菌CMCC(B)50093应为阳性(阳性率为100，颜色为黄色或黄绿色)，奇异变形杆菌CMCC(B)49005、普通变形杆菌CMCC(B)49027、弗氏志贺氏菌CMCC(B)51572和雷氏普罗维登斯菌FSCC(I)205006应为阴性(阳性率为0，颜色为蓝色或绿色)

, figureFileSmall=kW5mmXQTAH65Rqt5UOEl9g==, figureFileBig=EFLdVjorjDwtZNkP1IDw3Q==, tableContent=null), ArticleFig(id=1241446120266715573, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Figure 3, caption=Identification strip test results ofCronobacter. The reaction sequence of the identification strip is raffinose, sucrose, lactose, maltose, sorbitol, dulcitol, melibiose, trehalose, cellobiose, ONPG, esculin, malonate, citrate, urea, indol, methyl red, phenylalanine, rhamnose, xylose, arabinose, H2S, lysine, ornithine and amino acid control., figureFileSmall=wRkAg38qCtvezS57y2MPGQ==, figureFileBig=38yVYioEeLy2NXjWYUrjEQ==, tableContent=null), ArticleFig(id=1241446120379961788, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=图3, caption=克罗诺菌的鉴定条试验结果, figureFileSmall=wRkAg38qCtvezS57y2MPGQ==, figureFileBig=38yVYioEeLy2NXjWYUrjEQ==, tableContent=null), ArticleFig(id=1241446120539345353, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Figure 4, caption=Comparison test results between identification strips and API 20E of fourEnterobacteriaceae strains with "unacceptable identification results". The reaction sequence of the identification strip is raffinose, sucrose, lactose, maltose, sorbitol, dulcitol, melibiose, trehalose, cellobiose, ONPG, esculin, malonate, citrate, urea, indol, methyl red, phenylalanine, rhamnose, xylose, arabinose, H2S, lysine, ornithine and amino acid control., figureFileSmall=3skhYTAJOtg0p0PeANPHPg==, figureFileBig=4LPOR4vyWbudsoVZAcczjg==, tableContent=null), ArticleFig(id=1241446120669368783, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=图4, caption=四株“不可接受的鉴定结果”的肠杆菌科菌株的鉴定条与API 20E对比试验结果

鉴定条反应依次为棉子糖、蔗糖、乳糖、麦芽糖、山梨醇、卫矛醇、蜜二糖、海藻糖、纤维二糖、邻硝基苯β-D-半乳吡喃糖苷、七叶苷、丙二酸盐、柠檬酸盐、尿酶、靛基质、甲基红、苯丙氨酸、鼠李糖、木糖、阿拉伯糖、硫化氢、赖氨酸、鸟氨酸和氨基酸对照

, figureFileSmall=3skhYTAJOtg0p0PeANPHPg==, figureFileBig=4LPOR4vyWbudsoVZAcczjg==, tableContent=null), ArticleFig(id=1241446120803586519, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Table 1, caption=

Suspicious results of systematic identification ofEnterobacteriaceae standard strains

, figureFileSmall=null, figureFileBig=null, tableContent=
MS resultNumberCodeBiochemical identification resultAppraisal score (%)Similarity (%)Result evaluationAPI 20E result
Shigella dysenteriaeCMCC(B)5110500004110Shigella flexneri, Group B85.2799.99AcceptableShigella spp.
Shigella dysenteriaeCMCC(B)5125206204140Shigella boydii, Group C84.8199.99AcceptableShigella spp.
Shigella dysenteriaeCMCC(B)5105600010100Shigella boydii, Group C41.020.13UnacceptableShigella spp.
Salmonella typhimuriumATCC1402803301177Salmonella enterica subsp.houtenae Ⅳ93.9599.99ExcellentSalmonella spp.
), ArticleFig(id=1241446120967164382, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=表1, caption=

肠杆菌科标准株系统鉴定存疑结果

, figureFileSmall=null, figureFileBig=null, tableContent=
MS resultNumberCodeBiochemical identification resultAppraisal score (%)Similarity (%)Result evaluationAPI 20E result
Shigella dysenteriaeCMCC(B)5110500004110Shigella flexneri, Group B85.2799.99AcceptableShigella spp.
Shigella dysenteriaeCMCC(B)5125206204140Shigella boydii, Group C84.8199.99AcceptableShigella spp.
Shigella dysenteriaeCMCC(B)5105600010100Shigella boydii, Group C41.020.13UnacceptableShigella spp.
Salmonella typhimuriumATCC1402803301177Salmonella enterica subsp.houtenae Ⅳ93.9599.99ExcellentSalmonella spp.
), ArticleFig(id=1241446122510668259, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Table 2, caption=

Suspicious results of systematic identification ofEnterobacteriaceae isolates

, figureFileSmall=null, figureFileBig=null, tableContent=
MS resultNumberCodeBiochemical identification resultAppraisal score (%)Similarity (%)Result evaluationAPI 20E result16S RNA gene identification result
Escherichia coliFSCC(I)1490108643310167Salmonella enterica subsp.diarizonae54.350.00UnacceptableSalmonella arizonaeEscherichia coli
Escherichia coliFSCC(I)1490275143330177Salmonella enterica subsp.diarizonae66.300.00UnacceptableCitrobacter freundiiEscherichia coli
Escherichia coliFSCC(I)1490275243330177Salmonella enterica subsp.diarizonae66.300.00UnacceptableCitrobacter freundiiEscherichia coli
Escherichia coliFSCC(I)14979543310170Escherichia coli, inactive40.224.55UnacceptableEscherichia coli 1Escherichia coli
Escherichia coliFSCC(I)14997755310173Escherichia coli51.210.00UnacceptableSalmonella arizonaeEscherichia coli
Escherichia coliFSCC(I)1490240747310170Escherichia coli,inactive50.83.03UnacceptableEscherichia coli 1Escherichia coli
Escherichia coliFSCC(I)1490272647310172Escherichia coli62.551.10SuspiciousSalmonella arizonaeEscherichia coli
Yersinia frederikseniiFSCC(I)23403622636144Yersinia enterocolitica99.994.76AcceptableYersinia frederiksenii/intermediaYersinia frederiksenii
Yersinia enterocoliticaFSCC(I)23400522214104Yersinia enterocolitica64.670.03SuspiciousYersinia frederiksenii/intermediaYersinia frederiksenii
Yersinia enterocoliticaFSCC(I)23400623236114Yersinia enterocolitica91.280.00AcceptableYersinia frederiksenii/intermediaYersinia frederiksenii
Citrobacter braakiiFSCC(I)13501103253171Citrobacter werkmanii99.93100.00ExcellentCitrobacter youngaeCitrobacter freundii
Citrobacter freundiiFSCC(I)2150118673711147Serratia odorifera biogroup 150.52<0.01UnacceptableCitrobacter freundiiCitrobacter freundii
Citrobacter freundiiFSCC(I)1490171341315171Citrobacter freundii69.840.01UnacceptableCitrobacter freundiiCitrobacter freundii
Citrobacter freundiiFSCC(I)1490316943711171Citrobacter spp.98.8820.06Very good, identify to genus levelCitrobacter braakiiCitrobacter freundii
Citrobacter koseri/amalonaticusFSCC(I)2150111263615174Citrobacter amalonaticus62.940.94SuspiciousCitrobacter koseri/farmeriCitrobacter amalonaticus
Citrobacter koseri/amalonaticusFSCC(I)2150151502615170Citrobacter amalonaticus65.080.01UnacceptableCitrobacter koseri/amalonaticusCitrobacter amalonaticus
Enterobacter sakazakiiFSCC(I)14526971331034Enterobacter sakazakii68.190.00UnacceptableEnterobacter sakazakiiEnterobacter sakazakii
Enterobacter sakazakiiFSCC(I)14565671331054Enterobacter sakazakii57.970.00UnacceptableEnterobacter sakazakiiEnterobacter sakazakii
Enterobacter sakazakiiFSCC(I)14576671731064Enterobacter sakazakii74.790.01UnacceptableEnterobacter sakazakiiEnterobacter sakazakii
Enterobacter cloacaeFSCC(I)21518037771074Enterobacter cloacae61.710.31SuspiciousEnterobacter cloacaeEnterobacter roggenkampii
EnterobacterFSCC(I)21526677771074Enteric group 6996.6033.33GoodEnterobacter cloacaeEnterobacter asburiae
Enterobacter ludwigiiFSCC(I)21510941670570Enteric group 6463.3299.99GoodButtiauxella agrestisPseudocitrobacter faecalis
Enterobacter bugandensisFSCC(I)21511041670570Enteric group 6463.3299.99GoodPantoea spp.Pseudocitrobacter vendiensis
), ArticleFig(id=1241446122644885999, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=表2, caption=

肠杆菌科分离株系统鉴定存疑结果

, figureFileSmall=null, figureFileBig=null, tableContent=
MS resultNumberCodeBiochemical identification resultAppraisal score (%)Similarity (%)Result evaluationAPI 20E result16S RNA gene identification result
Escherichia coliFSCC(I)1490108643310167Salmonella enterica subsp.diarizonae54.350.00UnacceptableSalmonella arizonaeEscherichia coli
Escherichia coliFSCC(I)1490275143330177Salmonella enterica subsp.diarizonae66.300.00UnacceptableCitrobacter freundiiEscherichia coli
Escherichia coliFSCC(I)1490275243330177Salmonella enterica subsp.diarizonae66.300.00UnacceptableCitrobacter freundiiEscherichia coli
Escherichia coliFSCC(I)14979543310170Escherichia coli, inactive40.224.55UnacceptableEscherichia coli 1Escherichia coli
Escherichia coliFSCC(I)14997755310173Escherichia coli51.210.00UnacceptableSalmonella arizonaeEscherichia coli
Escherichia coliFSCC(I)1490240747310170Escherichia coli,inactive50.83.03UnacceptableEscherichia coli 1Escherichia coli
Escherichia coliFSCC(I)1490272647310172Escherichia coli62.551.10SuspiciousSalmonella arizonaeEscherichia coli
Yersinia frederikseniiFSCC(I)23403622636144Yersinia enterocolitica99.994.76AcceptableYersinia frederiksenii/intermediaYersinia frederiksenii
Yersinia enterocoliticaFSCC(I)23400522214104Yersinia enterocolitica64.670.03SuspiciousYersinia frederiksenii/intermediaYersinia frederiksenii
Yersinia enterocoliticaFSCC(I)23400623236114Yersinia enterocolitica91.280.00AcceptableYersinia frederiksenii/intermediaYersinia frederiksenii
Citrobacter braakiiFSCC(I)13501103253171Citrobacter werkmanii99.93100.00ExcellentCitrobacter youngaeCitrobacter freundii
Citrobacter freundiiFSCC(I)2150118673711147Serratia odorifera biogroup 150.52<0.01UnacceptableCitrobacter freundiiCitrobacter freundii
Citrobacter freundiiFSCC(I)1490171341315171Citrobacter freundii69.840.01UnacceptableCitrobacter freundiiCitrobacter freundii
Citrobacter freundiiFSCC(I)1490316943711171Citrobacter spp.98.8820.06Very good, identify to genus levelCitrobacter braakiiCitrobacter freundii
Citrobacter koseri/amalonaticusFSCC(I)2150111263615174Citrobacter amalonaticus62.940.94SuspiciousCitrobacter koseri/farmeriCitrobacter amalonaticus
Citrobacter koseri/amalonaticusFSCC(I)2150151502615170Citrobacter amalonaticus65.080.01UnacceptableCitrobacter koseri/amalonaticusCitrobacter amalonaticus
Enterobacter sakazakiiFSCC(I)14526971331034Enterobacter sakazakii68.190.00UnacceptableEnterobacter sakazakiiEnterobacter sakazakii
Enterobacter sakazakiiFSCC(I)14565671331054Enterobacter sakazakii57.970.00UnacceptableEnterobacter sakazakiiEnterobacter sakazakii
Enterobacter sakazakiiFSCC(I)14576671731064Enterobacter sakazakii74.790.01UnacceptableEnterobacter sakazakiiEnterobacter sakazakii
Enterobacter cloacaeFSCC(I)21518037771074Enterobacter cloacae61.710.31SuspiciousEnterobacter cloacaeEnterobacter roggenkampii
EnterobacterFSCC(I)21526677771074Enteric group 6996.6033.33GoodEnterobacter cloacaeEnterobacter asburiae
Enterobacter ludwigiiFSCC(I)21510941670570Enteric group 6463.3299.99GoodButtiauxella agrestisPseudocitrobacter faecalis
Enterobacter bugandensisFSCC(I)21511041670570Enteric group 6463.3299.99GoodPantoea spp.Pseudocitrobacter vendiensis
), ArticleFig(id=1241446122762326515, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Table 3, caption=

Accuracy of systematic identification ofEnterobacteriaceae

, figureFileSmall=null, figureFileBig=null, tableContent=
GenusTotal bacterial countAccuracy (species level, %)Accuracy (genus level, %)
Enterobacteriaceae standard bacteria3086.7100.0
Escherichia coli9296.796.7
Salmonella78100.0100.0
Yersiniavan6295.2100.0
Serratia41100.0100.0
Citrobactern4993.998.0
Klebsiella27100.0100.0
Cronobacter68100.0100.0
OtherEnterobacteriaceae bacteria1163.672.7
Total45896.598.5
), ArticleFig(id=1241446122904932857, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=表3, caption=

肠杆菌科细菌数值系统鉴定准确率

, figureFileSmall=null, figureFileBig=null, tableContent=
GenusTotal bacterial countAccuracy (species level, %)Accuracy (genus level, %)
Enterobacteriaceae standard bacteria3086.7100.0
Escherichia coli9296.796.7
Salmonella78100.0100.0
Yersiniavan6295.2100.0
Serratia41100.0100.0
Citrobactern4993.998.0
Klebsiella27100.0100.0
Cronobacter68100.0100.0
OtherEnterobacteriaceae bacteria1163.672.7
Total45896.598.5
), ArticleFig(id=1241446123018179069, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Table 4, caption=

Interpretation table for negative and positive qualities of quality control strains

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StrainsATCC8739CMCC(B)49005ATCC13048CMCC(B)50018CMCC(B)51592
Raffinose−−+−−
Sucrose−−+−−
Lactose+−+−−
Maltose+−+++
Sorbitol+−++−
Dulcitol+−−−−
Melibiose+−++−
Trehalose+++−+
Cellobiose−−+−−
ONPG+−+−+
Esculin+−+−−
Malonate−−+−−
Citrate−−+−−
Urea−+−−−
Indol+−−−−
Methyl red++−++
Phenylalanine−+−−−
Adonitol−−+−−
Rhamnose+−+++
Xylose++++−
Arabinose+−+−+
H2S−+−−−
Lysine+−++−
Ornithine−++++
), ArticleFig(id=1241446123152396806, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=表4, caption=

质控菌株阴阳性判读表

, figureFileSmall=null, figureFileBig=null, tableContent=
StrainsATCC8739CMCC(B)49005ATCC13048CMCC(B)50018CMCC(B)51592
Raffinose−−+−−
Sucrose−−+−−
Lactose+−+−−
Maltose+−+++
Sorbitol+−++−
Dulcitol+−−−−
Melibiose+−++−
Trehalose+++−+
Cellobiose−−+−−
ONPG+−+−+
Esculin+−+−−
Malonate−−+−−
Citrate−−+−−
Urea−+−−−
Indol+−−−−
Methyl red++−++
Phenylalanine−+−−−
Adonitol−−+−−
Rhamnose+−+++
Xylose++++−
Arabinose+−+−+
H2S−+−−−
Lysine+−++−
Ornithine−++++
), ArticleFig(id=1241446123278225933, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=EN, label=Table 5, caption=

Stability test results ofEnterobacteriaceae biochemical identification strip

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Strains No.4 ℃, 14 d37 ℃, 9 d42 ℃, 7 d
ATCC8739All correctAll correctAll correct
CMCC(B)49005All correctAll correctSorbitol, cellobiose, ONPG, esculin, malonate (+)
ATCC13048All correctAll correctAll correct
CMCC(B)50018All correctAll correctRaffinose, sucrose, trehalose, ONPG, esculin, malonate (+)
CMCC(B)51592All correctPhenylalanine, H2S (+)Adonitol (+)
), ArticleFig(id=1241446123395666453, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376204830339569, language=CN, label=表5, caption=

肠杆菌科生化鉴定条稳定性试验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains No.4 ℃, 14 d37 ℃, 9 d42 ℃, 7 d
ATCC8739All correctAll correctAll correct
CMCC(B)49005All correctAll correctSorbitol, cellobiose, ONPG, esculin, malonate (+)
ATCC13048All correctAll correctAll correct
CMCC(B)50018All correctAll correctRaffinose, sucrose, trehalose, ONPG, esculin, malonate (+)
CMCC(B)51592All correctPhenylalanine, H2S (+)Adonitol (+)
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基于数值鉴定法的肠杆菌科生化试剂盒的研制及效果评价
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金乐萱 1, 2, # , 万强 3, # , 曹璐璐 1, 2 , 叶青华 2, * , 王迅 3 , 张菊梅 2 , 蔡芷荷 3 , 吴清平 1, 2, *
微生物学报 | 技术与方法 2024,64(5): 1668-1682
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微生物学报 | 技术与方法 2024, 64(5): 1668-1682
基于数值鉴定法的肠杆菌科生化试剂盒的研制及效果评价
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金乐萱1, 2, #, 万强3, #, 曹璐璐1, 2, 叶青华2, * , 王迅3, 张菊梅2, 蔡芷荷3, 吴清平1, 2, *
作者信息
  • 1 合肥工业大学食品与生物工程学院, 安徽 合肥 230601
  • 2 广东省科学院微生物研究所 华南应用微生物国家重点实验室 广东省微生物安全与健康重点实验室国家卫健委微生物食品营养与安全科技创新平台, 广东 广州 510070
  • 3 广东环凯生物科技有限公司, 广东 肇庆 526238
Development and performance evaluation of a biochemical kit for identification ofEnterobacteriaceae based on numerical identification method
Lexuan JIN1, 2, Qiang WAN3, Lulu CAO1, 2, Qinghua YE2, * , Xun WANG3, Jumei ZHANG2, Zhihe CAI3, Qingping WU1, 2, *
Affiliations
  • 1 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, Anhui, China
  • 2 State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Safety and Health, National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, Guangdong, China
  • 3 Guangdong Huankai Biotechnology Co., Ltd., Zhaoqing 526238, Guangdong, China
出版时间: 2024-05-04 doi: 10.13343/j.cnki.wsxb.20230701
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摘要
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【背景】肠杆菌科细菌是食源性和临床感染的主要病原菌，对人类和动物健康有重要影响，以API 20E为“金标准”的数值鉴定法是其主要的鉴定方法之一，但现有的数值鉴定方法存在手工单个加样或/和准确率低、价格贵的问题。【目的】研制出半自动化、准确性高及价格低的肠杆菌科细菌数值鉴定生化试剂盒。【方法】在本团队前期建立的肠杆菌科数值鉴定系统理论模型与配套软件的基础上，设计并优化筛选出的24种生化基质微量化配方，在此基础上，研制半自动冻干鉴定条；以现行商品化的数值鉴定条API 20E、MALDI-TOF MS及16S rRNA基因测序为对照，对研制的集半自动生化鉴定条与在线分析软件于一体的生化试剂盒进行效果评价。【结果】共获得458株肠杆菌科细菌的生化谱，在属水平总体鉴定准确率达到98.5%，在种水平总体鉴定准确率达到96.5%；仅需两次加样即可获得鉴定结果，且价格仅为API 20E的4.46%；产品保质期为7 d，生化试验具有稳定可重复性。【结论】本研究研制出的半自动化肠杆菌科细菌数值鉴定生化试剂盒简便经济、准确率高，为肠杆菌科细菌的鉴定提供技术支撑，并为其他科属细菌的数值鉴定生化产品的开发提供技术指引。

肠杆菌科  /  数值鉴定  /  生化试剂盒  /  效果评价

[Background] Bacteria ofEnterobacteriaceae are the main pathogens of foodborne and clinical infections, posing a threat to human and animal health. The numerical identification method using API 20E as the "gold standard" is one of the main identification methods forEnterobacteriaceae. However, the existing numerical identification methods have problems such as laborious sample addition, low accuracy, and high prices. [Objective] To develop a semi-automatic, high-precision, and low-cost biochemical kit for identifying the bacteria ofEnterobacteriaceae based on the numerical identification method. [Methods] On the basis of the theoretical model and supporting software of the numerical identification system forEnterobacteriaceae established by our team, we designed and optimized 24 biochemical matrix trace formulations. Furthermore, a semi-automatic freeze-drying identification strip was developed. Using the commercialized numerical identification strip API 20E, MALDI-TOF MS, and 16S rRNA gene sequencing as the controls, we evaluated the performance of the developed biochemical kit that integrated semi-automatic biochemical identification strips and online analysis software. [Results] The biochemical spectra of 458 strains ofEnterobacteriaceae were obtained, with the overall identification accuracy of 98.5% at the genus level and 96.5% at the species level. The kit only needed twice sample addition to obtain the identification results, with the price only 4.46% that of API 20E. The shelf life of the product was 7 days, and the biochemical experiments had repeatability. [Conclusion] We develop a semi-automatic biochemical kit for identification ofEnterobacteriaceae based on the numerical identification method, which has simple operation, a low cost, and high accuracy. This study provides technical support for the identification ofEnterobacteriaceae and the development of biochemical products for numerical identification of other families and genera of bacteria.

Enterobacteriaceae  /  numerical identification  /  biochemical kit  /  performance evaluation
金乐萱, 万强, 曹璐璐, 叶青华, 王迅, 张菊梅, 蔡芷荷, 吴清平. 基于数值鉴定法的肠杆菌科生化试剂盒的研制及效果评价. 微生物学报, 2024 , 64 (5) : 1668 -1682 . DOI: 10.13343/j.cnki.wsxb.20230701
Lexuan JIN, Qiang WAN, Lulu CAO, Qinghua YE, Xun WANG, Jumei ZHANG, Zhihe CAI, Qingping WU. Development and performance evaluation of a biochemical kit for identification ofEnterobacteriaceae based on numerical identification method[J]. Acta Microbiologica Sinica, 2024 , 64 (5) : 1668 -1682 . DOI: 10.13343/j.cnki.wsxb.20230701
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肠杆菌科细菌为一大群抗原构造和生物学性状相似的革兰氏阴性杆菌，是氧化酶阴性、发酵型细菌[1]，隶属于变形菌门(Proteobacteria) γ-变形菌纲(Gammaproteobacteria)肠杆菌目(Enterobacteriales)。原核生物标准命名列表(list of prokaryotic names with standing in nomenclature,http://lpsn.dsmz.de/)显示肠杆菌科现有37个有效描述子分类群和46个非有效描述子分类群，其种类繁多且分布广泛，不仅部分致病菌会对生命安全构成不同程度的威胁，大部分作为正常菌群存在于肠道中的菌株在一定条件下也会危害身体健康，肠杆菌科细菌导致的常见疾病有尿路、腹腔、胆道、胃肠道和呼吸道感染以及败血症等[2-5]，造成了卫生医疗系统的沉重负担。食源性致病菌污染已构成全球日益严峻的公共卫生挑战，肠杆菌科细菌作为食品生产的指示菌[6-7]，大多数国家和地区都为此制定了严格的限量标准，同时GB 4789.41—2016《食品安全国家标准食品微生物学检验肠杆菌科检验》明确规定了肠杆菌科中的沙门氏菌、阪崎克罗诺菌、小肠结肠炎耶尔森菌等的检验标准[8]。因此发展简易、快速的肠杆菌科鉴定方法对于临床检验、食品质量控制和监管非常重要。
肠杆菌科与相邻菌科的界限比较清晰，但属间和种间不易鉴别，许多新种的出现也容易混淆鉴定结果。目前肠杆菌科细菌的检测方法主要有生理生化特征检测方法(分离培养基、测试片、数值生化鉴定法)、免疫学检测方法(免疫磁珠、免疫胶体金检测)、分子生物学检测方法(聚合酶链式反应、环介导等温扩增、基因芯片技术、DNA探针)、仪器化检测方法(传感器、仪器联用技术)等[9-11]，其中数值生化鉴定法建立在细菌生化反应特征和数理统计学(包括条件概率理论和贝叶斯定理)的基础上，本质是由传统生化反应检测技术与现代计算机技术结合而成的一种细菌概率鉴定方法[12]。传统鉴定方法操作繁琐且耗时长，无法通过一次性操作达成检验目的，新技术又多需依托大型专业仪器，不易于成为常规方法加以推广。数值生化鉴定法简便高效，无需专业仪器，只需具备基本的计算机素养即可实现在线分析细菌鉴定功能，降低了使用门槛，节省了人力和物力，可满足基层企业和单位的需求，已成为微生物检验的有效工具和商品化细菌鉴定的重要选择。
相较国外成熟的API (Bio Mérieux公司)、Minitek (BD公司)、Micro-ID (REMEL公司)、Enterotube Ⅱ (Roche公司)等系统[13-16]，我国虽然也有自动化细菌鉴定/药敏分析仪的相关报道，但仅见于文献，未见全面推广，如吴诗[17]开发的食源性单增李斯特菌数值鉴定系统；李文明[18]研制的肠杆菌科细菌生化鉴定系统等。我国数值生化鉴定法的商品化进程相对落后，主要表现在4个方面：(1) 能够鉴定的细菌种类较少，覆盖程度与国外产品相比还存在一定差距，应用范围狭窄；(2) 多使用常规传统的西林瓶液体生化试剂，通过对多个反应进行单独判断完成细菌鉴定，反应管分散导致操作繁琐、结果不易判读等缺陷，未能实现标准系统化；(3) 未建立数值鉴定系统核心数据库，因某些生化反应可能受检验方法、地域差异和物种进化等因素影响，导致基础数据即阳性概率存在一定问题，进而降低鉴定结果准确率；(4) 配套的自动化仪器在专业化和智能化程度等方面都与国外产品有一定差距。目前，国内普遍采用国外的数值鉴定系统产品，但进口鉴定板/条作为易耗品也存在价格昂贵、生化试剂反应不稳定等问题。因此，我国亟待开发具有自主知识产权的整套鉴定系统和生化试剂盒产品。
本研究在本团队前期建立的数值鉴定数理模型与配套的在线分析软件的基础上[19]，设计了24种生化基质微量化配方，通过梯度试验对其进行优化，并研制半自动冻干鉴定条，开发了集鉴定条与在线分析软件于一体的肠杆菌科数值鉴定试剂盒，同时结合肠杆菌和其他非苛养革兰氏阴性杆菌鉴定试剂盒(API 20E)、基质辅助激光解析电离飞行时间质谱技术(matrix-assisted laser desorption/ionization-time of flight mass spectrometry, MALDI-TOF MS)及16S rRNA基因测序，对大量肠杆菌科标准菌株和分离菌株进行了效果评价，以期制备出简便、经济、准确率高的肠杆菌科数值鉴定产品，为肠杆菌科细菌的鉴定提供技术支持，并为其他科属细菌的数值鉴定生化产品的开发提供技术指引。
1 材料与方法
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1.1 主要菌株、试剂和仪器
本研究所用458株肠杆菌科细菌菌株，主要包括大肠埃希氏菌(Escherichia coli)、沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、志贺氏菌属(Shigella)、耶尔森氏菌属(Yersiniavan)、柠檬酸杆菌属(Citrobactern)、克雷伯氏菌属(Klebsiella)、克罗诺菌属(Cronobacter)等，所有菌株保存于广东环凯生物科技有限公司。
蛋白胨、酵母粉、牛肉粉，北京索莱宝科技有限公司；色氨酸、半胱氨酸、苯丙氨酸、赖氨酸盐酸盐、鸟氨酸盐酸盐，广州康捷科技有限公司；葡萄糖、棉子糖、蔗糖、乳糖、麦芽糖、山梨醇、卫矛醇、蜜二糖、海藻糖、纤维二糖、侧金盏花醇、鼠李糖、木糖、阿拉伯糖、β-半乳糖苷、无水氯化镁、柠檬酸铁、丙二酸钠、溴麝香草酚蓝、酚红，国药控股股份有限公司；七叶苷、尿素、磷酸二氢铵、硫酸亚铁、磷酸二氢钠、磷酸二氢钾、碳酸钠、氯化钠、柠檬酸钠、硫酸镁，广东广试试剂科技有限公司；无菌液体石蜡、对二甲氨基苯甲醛、对甲基红、10% FeCl3、胰酪大豆胨琼脂培养基、西林瓶液体生化管，广东环凯生物科技有限公司；API 20E，生物梅里埃公司。
电子分析天平、酸度计，赛多利斯公司；高压灭菌锅，株式会社平山制作所；二级生物安全柜，北京东联哈尔仪器制造有限公司；电热恒温培养箱，广东环凯微生物科技有限公司；真空干燥设备，广州金富机械设备有限公司；冷冻离心机、PCR仪，广州市元晞生物科技有限公司；MALDI-TOF MS，布鲁克公司。
1.2 细菌数值鉴定数理模型及在线分析软件的确立
数值鉴定包括数理模型的建立与配套分析软件的编制，其中数理模型的建立包括数值鉴定系统数据库的建立、生化反应的选定、计算方法的确定、结果评价标准的制定，配套分析软件的编制包括生化编码的确定与软件编写。根据本团队前期建立的肠杆菌科细菌数值鉴定数理模型与配套的在线分析软件[19]，为进一步提高对部分菌属鉴定的准确性与制作微量化冻干生化试剂的方便性，进一步优化生化反应的组合。
1.3 微量化冻干生化基质配方设计及优化
基于传统生化管配方和国家标准推荐配方[8,20]，依据肠杆菌科细菌生长特性，设计并优化上述24种生化反应的微量化配方，并探索微量化试剂冻干方法以期达到工艺适用于生产、结果色差明显易判别的目的。
1.4 鉴定条制作及使用、分析方法
1.4.1 鉴定条制作方法
依据生化基质配方优化试验结果，将24种微量化的生化反应试剂制成干燥化的反应底物，并集合在有多个反应小孔的微型容器上制成鉴定条成品(A、B试剂条组成1个测试)，于4 ℃保存备用。
1.4.2 菌悬液制备方法
将肠杆菌科细菌菌株甘油管于无菌环境下接种在TSA培养基上[21]，37 ℃恒温培养18−24 h，挑取纯化后的单菌落接种于新鲜无菌的生理盐水中，稀释成0.5麦氏浊度的均一菌悬液备用。
1.4.3 鉴定条使用方法
鉴定条使用前平衡至室温，从底座中取下鉴定条，并从鉴定条右侧向左掀开贴膜，用微量移液器吸取2 mL菌悬液并小心注入分液槽(应避免菌液提前流入反应孔中)，贴回贴膜，并依次抬起两侧数次，使菌悬液液面达同一高度，然后水平托起分液槽，确保菌液流入各反应孔中，放入底座(图1)。接种菌液后向鼠李糖、木糖和阿拉伯糖反应孔中准确加入30 μL糖发酵添加剂，向尿素、硫化氢、赖氨酸、鸟氨酸、氨基酸对照反应孔中分别滴加3−4滴无菌液体石蜡液封，然后贴紧薄膜，将接种的鉴定条置于(36±1) ℃避光培养18−24 h。培养完毕后，向靛基质(对二甲氨基苯甲醛试剂)、甲基红(对甲基红试剂)、苯丙氨酸(10% FeCl3试剂)反应孔中滴加1−2滴对应试剂。
1.4.4 鉴定条分析方法
滴加完成后，对照比色卡读取A、B试剂条各反应孔阴阳性结果，并将其转化成编码后输入至Numide v1.0[19]数值鉴定软件中进行在线分析，对鉴定结果不理想的菌株参照分析结果提示增加一些补充生化反应进一步确认，以获得较好的鉴定结果。
1.5 肠杆菌科数值鉴定生化试剂盒的效果评价
使用458株肠杆菌科细菌菌株(包括30株标准菌株和428株分离菌株)检验生化试剂盒的准确度。同时采用MALDI-TOF MS鉴定为对照，针对生化数值鉴定条结果与质谱结果不一致的菌株，结合API 20E和16S rRNA基因测序检验结果的正确性。每个试验重复两次，统计已鉴定菌株总数(N0)和在种/属水平上鉴定结果正确菌株总数(N1)，计算准确率(%)=N1/N0。
其中，MALDI-TOF MS技术基于微生物核糖体蛋白和外周蛋白特异性和保守性对菌种进行鉴定，主要原理是通过计算到达检测器的标本电离离子飞行时间而测出质荷比[23]。本研究使用的是MALDI Biotyper微生物快速鉴定系统(bruker daltonics)，借助其食源性致病菌蛋白质指纹图谱数据库与专用软件自动进行分析比对，从而得到鉴定结果。
1.6 肠杆菌科数值生化鉴定条的保质期和稳定性试验
将制备好的鉴定条分别放置于4、37和42 ℃恒温培养箱中，分别在第1、3、5、7、9和14天后进行稳定性和保质期测试，每天的生化试验重复3次，观察质控菌株在鉴定条反应孔中的生长情况。
2 结果与分析
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2.1 微量化冻干生化鉴定条的研制
2.1.1 糖发酵添加剂pH值优化
生化基质原料的配比、微量化的程度与生化反应的灵敏度、准确性、稳定性、时效性等密切相关。以广东环凯生物科技有限公司相应的24种西林瓶液体生化管配方作为微量化冻干的基础配方制备鉴定条，以本团队前期建立的数值鉴定分析软件对肠杆菌科细菌进行测试。
观察试验结果，发现除阿拉伯糖生化试验以外，其余生化反应结果均与预期相符，未发现有问题反应项。部分肠杆菌科菌株的阿拉伯糖反应孔结果颜色介于阳性(黄色)和阴性(蓝色)之间，主观因素影响结果判定。因此，需对其配方进行优化，以避免出现颜色阴阳性辨识不清的情况。
考虑提高营养物质添加量或改善酸碱环境以增强菌株与阿拉伯糖试剂的反应能力和效率，设计两种优化方案：(1) 糖发酵添加剂的pH值从7.00±0.05调至7.40±0.05；(2) 以糖发酵添加剂的添加量为变量，设置I、M、V、C 4个测试组，添加量依次为30、40、50和60 μL梯度。观察结果颜色是否存在差异。
经梯度试验颜色对比发现，适当提高糖发酵添加剂的pH值有助于结果颜色分辨和阴阳性判断，因此选定糖发酵添加剂的pH为7.40±0.05。然而随着糖发酵添加剂添加量的增加，会出现试验结果假阴性判读的情况，如阴沟肠杆菌CMCC(B)45301和坂崎肠杆菌ATCC51329，为避免错误的生化反应结果影响细菌鉴定准确率，选定糖发酵添加剂的添加量为30 μL (图2)。因此，最终确定阿拉伯糖反应孔中糖发酵添加剂的pH为7.40±0.05，添加量为30 μL。
2.1.2 生化反应项目组合优化
按照团队前期选定的生化反应项目组合[19]进行初步测试，结果表明肠杆菌科鉴定条对克罗诺菌(原称阪崎肠杆菌)的鉴定率为82.35% (56/68)，12株克罗诺菌菌株被错误地鉴定为河生肠杆菌生物型Ⅰ，鉴定结果提示不一致试验主要来自丙二酸盐：如图3所示，当丙二酸盐反应为阳性，系统给出的鉴定结果为河生肠杆菌生物型Ⅰ，反之，则为克罗诺菌。克罗诺菌经历了由种到属的变迁，不同的种在丙二酸盐反应上阴阳性不同，这就导致了不同的研究者或者厂家选择的阳性概率数值不同，从而造成鉴定结果的不一致。同时由于克罗诺菌与河生肠杆菌生物型Ⅰ生化特性非常相近，部分菌株仅在丙二酸盐反应孔(12号反应孔)上有出入，难以正确区分两者，可能导致误判。
因此，为提高克罗诺菌的鉴定准确率，增加侧金盏花醇反应，对于该生化反应，坂崎克罗诺菌为阴性(概率为0，颜色为蓝色)，河生肠杆菌为阳性(概率为100，颜色为黄色)。调整后的鉴定系统可正确区分河生肠杆菌和坂崎肠杆菌，由此提升鉴定准确率。
考虑鉴定条制作特性和鉴定结果的准确性，最终确定的24项生化反应分别是[19]：苯丙氨酸脱氨、木糖、棉子糖、吲哚、蔗糖、鸟氨酸脱羧酶、乳糖、邻硝基苯β-D-半乳吡喃糖苷(o-nitrophenyl-β-D-galactopyranoside, ONPG)、尿酶、柠檬酸盐、麦芽糖、赖氨酸脱羧酶、丙二酸盐、山梨醇、卫矛醇、阿拉伯糖、蜜二糖、鼠李糖、七叶苷、甲基红、硫化氢、海藻糖、纤维二糖、侧金盏花醇。以上反应易制备、易判读，适宜应用。鉴定范围覆盖肠杆菌科30属139个种，总计159个分类单元。
2.2 肠杆菌科生化数值鉴定系统的效果评价结果
2.2.1 肠杆菌科标准株的效果评价结果
以优化后生化基质配方为基础制备微量化冻干鉴定条，采用30株肠杆菌科细菌标准菌株为检测对象，以质谱鉴定结果作为对照，比较两者的鉴定结果是否一致，如存在不同的情况，引入API 20E进一步确认结果的正确性，试验结果见表1。其中鉴定百分数表示待测菌的性状与数据库中每一分类单元的符合程度，相似度表示待测菌的性状与之相对应的一个分类单元的最典型生化反应之间的关系，两者数值最高均为100%，与鉴定结果的可靠程度呈正相关关系，它们在不同区间内分别代表不同的结果评价[19]。
结果表明，有3株质谱结果为痢疾志贺氏菌的鉴定结果为弗氏志贺氏菌或鲍氏志贺氏菌，1株质谱结果为鼠伤寒沙门氏菌的鉴定结果为豪顿沙门氏菌。然而在属的水平上，该肠杆菌科鉴定条对所测试的30株标准株的鉴定准确率可达到100%。此外，API 20E肠杆菌科鉴定条对沙门氏菌和志贺氏菌也仅鉴定到属水平(表1)。因此，初步来看本鉴定系统的鉴定结果是可接受的。
2.2.2 肠杆菌科分离株的效果评价结果
使用研制的生化鉴定条和配套的数值系统软件测试428株肠杆菌科细菌分离株，包括92株大肠杆菌分离株、62株耶尔森菌分离株、49株柠檬酸杆菌分离株、27株克雷伯菌分离株、68株坂崎肠杆菌分离株、78株沙门氏菌分离株、41株沙雷氏菌分离株和11株不常见肠杆菌科分离株，其中有23株质谱鉴定结果与数值系统鉴定结果不一致或鉴定结果评价为可疑/不可接受的菌株，结合API 20E对其进行验证，如无法确认菌株具体种属(API 20E、生化鉴定条、质谱鉴定结果均不同)，则进一步开展16S rRNA基因测序。
由表2可知，在所鉴定的23株存疑菌中，共计13株鉴定结果有误或无法鉴定，其中，8株菌的分子检测结果与质谱鉴定结果一致，生化鉴定条在属水平上鉴定错误；13株菌的生化鉴定条在种水平上鉴定错误或无法鉴定；其余菌株生化鉴定条鉴定结果与16S rRNA基因测序比对结果一致，可进一步判定为正确的鉴定结果。
此外，82株大肠埃希氏菌鉴定百分数大于95%，40株沙雷氏菌鉴定百分数大于98%，有57株耶尔森菌鉴定百分数大于97%，73株沙门氏菌鉴定百分数大于90%，38株柠檬酸杆菌鉴定百分数大于98%，26株克雷伯菌鉴定百分数大于97%，51株坂崎肠杆菌鉴定百分数大于98%，总体结果评价较好。另有4株菌的生化鉴定条鉴定结果识别力低，系统给定的结果评价为“不可接受的鉴定结果”，且与16S rRNA基因测序结果不同，分别是大肠埃希氏菌FSCC(I)14901086、FSCC(I)14902751、FSCC(I)14902752和弗氏柠檬酸杆菌FSCC(I)21501186，FSCC(I)14901086的API系统结果属于属的水平，FSCC(I)14902751和FSCC(I)14902752的API系统结果为“不能接受的生化谱”。鉴定条与API 20E无法鉴定以上3株大肠埃希氏菌的原因均来源于吲哚(图4红色箭头)和硫化氢(图4蓝色箭头)，且这2个孔的反应结果在2个产品间是一致的。而FSCC(I)21501186的API 20E鉴定结果与质谱鉴定结果一致，均为弗氏柠檬酸杆菌，鉴定条鉴定结果为气味沙雷氏菌，错误的原因主要来源于赖氨酸(图4蓝色箭头)、鸟氨酸(图4红色箭头)及鼠李糖(图4下划线)。
2.2.3 系统总体鉴定准确率
利用研制的鉴定条对肠杆菌科分离株开展鉴定效果评价，结合质谱法对照、引进API 20E试剂盒对存疑结果进行验证，获得了较为准确的458株菌的生化谱数据，统计结果可知本肠杆菌科细菌鉴定系统在属水平上总体鉴定准确率达到98.5%，在种水平上总体鉴定准确率达到96.5% (表3)，可认为鉴定结果准确性好，且总体鉴定百分数较高，稳定可重复。
2.3 肠杆菌科数值生化鉴定条的保质期和稳定性实验
为保证24个反应均有易于颜色判别明显的阴/阳性结果显示，且质控菌的鉴定百分数应较高，最终选用大肠埃希氏菌ATCC8739、奇异变形杆菌CMCC(B)49005、产气肠杆菌ATCC13048、猪霍乱沙门氏菌CMCC(B)50018及宋内志贺氏菌CMCC(B)51592作为质控菌株(表4)。
如表5所示，42 ℃恒温培养箱中放置7 d后试验，发现棉子糖、蔗糖、山梨醇、海藻糖、纤维二糖、ONPG、七叶苷、丙二酸盐、侧金盏花醇反应均呈现假阳性，鉴定条不可用；37 ℃恒温培养箱中放置9 d后试验，发现苯丙氨酸、硫化氢反应均呈现假阳性，鉴定条不可用。本鉴定条产品需存放于4 ℃冷库，考虑到生化试剂保存和运输的条件，额外设置37 ℃和42 ℃，以时间作为变量进行测定，最早在7 d时反应孔出现假阳性并影响到鉴定结果判定，因而确定本细菌数值鉴定产品的最佳使用期限为7 d。不同批次间试剂3次重复性试验结果均保持一致，说明该产品稳定性较好，具有可重复操作性。
3 讨论与结论
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为有效遏制肠杆菌科细菌感染性疾病的发生，针对目前国产商品化肠杆菌科数值鉴定系统空白的问题和对肠杆菌科细菌快速检测的需求，本研究以大肠埃希氏菌、沙门氏菌、沙雷氏菌、志贺氏菌、耶尔森氏菌、柠檬酸杆菌、克雷伯氏菌等重要条件致病菌为对象，在已有数理模型和配套软件的基础上，参考市售对应生化管配方，开发包含24种生化反应的微量化冻干鉴定条。为进一步验证该鉴定条的适用性，配合前期建立的数值鉴定系统软件，利用肠杆菌科细菌30株标准株和428株分离株联合质谱鉴定、API 20E、16S rRNA基因测序，对本鉴定条进行效果评价。本研究开发的数值鉴定系统范围覆盖了食源性与临床常见的肠杆菌科细菌，在属水平上的总体鉴定准确率达到98.5%，在种水平上的总体鉴定准确率达到96.5%，保质期为7 d，试验稳定可重复，满足了肠杆菌科鉴定的要求。
此外，相较于国内主要的普通生化管和国际上公认的2种商品化肠杆菌科数值鉴定系统API 20E和MID GNA/GNB，鉴定条需手工单个地接种细菌悬浮液，本研究所研制的鉴定条采用独创性的菌液接种方式，成功打破了该局限性，通过手工倾斜法批量接种实现一次性接菌操作，省时省力且降低污染可能性。
综上所述，本研究研制的肠杆菌科细菌数值鉴定系统生化鉴定试剂盒，鉴定结果准确率高，制作使用成本低至API 20E的4.46%，同时独特的半自动接菌方式极大地减轻了工作量。本研究不仅为有效区分肠杆菌科细菌提供了一个高效稳定、质优价廉、操作简便、针对性强的产品，还可促进其他科属细菌数值鉴定系统生化试剂盒的开发。
然而，本研究方法仍有提升空间：(1) 克罗诺菌只能鉴定到属，在种水平上的准确度、可行性较差，后续开发能鉴定7个种的数值鉴定系统；(2) 因部分肠杆菌科菌种不常见，分离的数量缺乏或者很少，生化数据库仍然有待更新，需进一步优化完善。
基金
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  • 国家重点研发项目(2022YFF1100700)
  • 广东省自然科学基金(2022A1515011740)
文献
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参考文献 引证文献
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2024年第64卷第5期
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doi: 10.13343/j.cnki.wsxb.20230701
  • 接收时间：2023-11-15
  • 首发时间：2026-03-19
  • 出版时间：2024-05-04
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  • 收稿日期：2023-11-15
  • 录用日期：2024-02-19
基金
National Key Research and Development Program of China(2022YFF1100700)
国家重点研发项目(2022YFF1100700)
Natural Science Foundation of Guangdong Province(2022A1515011740)
广东省自然科学基金(2022A1515011740)
作者信息
    1 合肥工业大学食品与生物工程学院, 安徽 合肥 230601
    2 广东省科学院微生物研究所 华南应用微生物国家重点实验室 广东省微生物安全与健康重点实验室国家卫健委微生物食品营养与安全科技创新平台, 广东 广州 510070
    3 广东环凯生物科技有限公司, 广东 肇庆 526238

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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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