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Article(id=1217779725867597889, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217779717386715826, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250328003, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1743091200000, receivedDateStr=2025-03-28, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768270911900, onlineDateStr=2026-01-13, pubDate=1750780800000, pubDateStr=2025-06-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768270911900, onlineIssueDateStr=2026-01-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768270911900, creator=13701087609, updateTime=1768270911900, updator=13701087609, issue=Issue{id=1217779717386715826, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='12', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768270909877, creator=13701087609, updateTime=1768299620707, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217900139386163208, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217779717386715826, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217900139386163209, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217779717386715826, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=126, endPage=132, ext={EN=ArticleExt(id=1217779726375108695, articleId=1217779725867597889, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Investigation and analysis of a food poisoning incident caused by bongkrekic acid in Hubei Province, columnId=1217529311867883548, journalTitle=Journal of Food Safety & Quality, columnName=Highlight: Analysis and Monitoring of Toxic and Harmful Substances in Food, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate and analyze a food poisoning incident caused by bongkrekic acid (BA) in Xiaogan City, Hubei Province, isolate and identify the pathogenic bacteria, and detect the toxin. Methods Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure BA levels in 2 exposed food samples (cold rice noodles), 3 food storage environment samples, and 2 patient blood samples. Following GB/T 4789.29—2020 Microbiological examination of food hygiene—Examination of Burkholderia gladioli (Pseudomonas cocovenenans subsp. farinofermentans), microbial identification was performed on 2 exposed food samples and 3 environmental samples, including isolation and culture, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry, 16S rDNA sequencing, real-time quantitative polymerase chain reaction (PCR) analysis, and toxin production experiments. Results The BA concentrations in the 2 patients’ blood samples were 92.2 μg/L and 1059.0 μg/L, respectively. The BA content in the unsold and frozen cold rice noodles was 2.26 mg/kg and 0.39 mg/kg, respectively, while no BA was detected in the environmental samples. Two suspected strains were isolated from the implicated food. VITEK MS MALDI-TOF MS and 16S rDNA sequencing identified both strains as Burkholderia gladioli. Real-time quantitative PCR and toxin production experiments confirmed that both strains could produce BA and tested positive for the bon gene, indicating toxigenic potential. They were comprehensively identified as Burkholderia gladioli pathovar cocovenenans. No Burkholderia gladioli was detected in the environmental samples. Conclusion This food poisoning incident is caused by cold rice noodles contaminated with Burkholderia gladioli pathovar cocovenenans. Relevant authorities need to strengthen food safety supervision to prevent similar poisoning incidents.

, correspAuthors=Xue-Qin LIU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Teng XIE, Huan CHEN, Lin MA, Chi-Hua WANG, Yi ZHENG, Xue-Qin LIU, Fen WANG), CN=ArticleExt(id=1217779727834726585, articleId=1217779725867597889, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=湖北省一起米酵菌酸引起食物中毒事件的调查分析, columnId=1217529312056627244, journalTitle=食品安全质量检测学报, columnName=本期重点：食品中有毒有害物质分析与监测, runingTitle=null, highlight=null, articleAbstract=

目的 调查分析湖北省孝感市一起米酵菌酸(bongkrekic acid, BA)引起的中毒事件, 分离鉴定病原菌, 并检测其毒素。方法 采用超高效液相色谱-串联质谱(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)法测定2份暴露食品(凉皮)、3份食品储存环境样本及2份病例血液样本中BA。参照GB/T 4789.29—2020《食品卫生微生物检验 唐菖蒲伯克霍尔德氏菌(椰毒假单胞菌酵米面亚种)检验》, 对2份暴露食品及3份环境样本中的病原菌进行分离培养、飞行时间质谱、16S rDNA测序、实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)分析、产毒实验等微生物鉴定。结果 两例病例血液样本中BA质量浓度分别为92.2 μg/L、1059.0 μg/L, 待售凉皮与冻存凉皮中BA含量分别为2.26 mg/kg、0.39 mg/kg, 环境样本均未检出BA。从疑似暴露食品中分离出两株可疑菌株, VITEK MS飞行时间质谱仪及16S rDNA测序比对结果表明两株可疑菌株均为唐菖蒲伯克霍尔德氏菌, 实时荧光定量PCR分析及产毒实验结果表明两株可疑菌株均可产生BA且bon基因阳性, 均具备产毒能力, 综合判定为唐菖蒲伯克霍尔德氏菌椰毒致病种。环境样本中未鉴定出唐菖蒲伯克霍尔德氏菌。结论 本次食物中毒事件由受唐菖蒲伯克霍尔德氏菌椰毒致病种污染的凉皮食品引起, 有关部门需加强食品安全监管, 防范类似中毒事件发生。

, correspAuthors=刘雪芹, authorNote=null, correspAuthorsNote=
*刘雪芹(1984—), 女, 硕士, 主管技师, 主要研究方向为食品安全及理化检验。E-mail:
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谢腾(1983—), 男, 主管技师, 主要研究方向为食品安全及微生物检验。E-mail:

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谢腾(1983—), 男, 主管技师, 主要研究方向为食品安全及微生物检验。E-mail:

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Biochemical identification and 16S rDNA sequencing results of isolated strains

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号 VITEK MS结果 16S rDNA测序结果
S1 粘质沙雷氏菌(Serratia marcescens) 粘质沙雷氏菌(Serratia marcescens)
S2 荧光假单胞菌(Pseudomonas fluorescens) 生防假单胞菌(Pseudomonas protegens)
S3 解鸟氨酸拉乌尔菌(Raoultella ornithinolytica) 植生拉乌尔菌(Raoultella planticola)
S4 唐菖蒲伯克霍尔德菌(Burkholderia gladioli) 唐菖蒲伯克霍尔德菌(Burkholderia gladioli)
S5 唐菖蒲伯克霍尔德菌(Burkholderia gladioli) 唐菖蒲伯克霍尔德菌(Burkholderia gladioli)
), ArticleFig(id=1217833925049766895, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725867597889, language=CN, label=表1, caption=

分离菌株的生化鉴定和16S rDNA测序结果

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号 VITEK MS结果 16S rDNA测序结果
S1 粘质沙雷氏菌(Serratia marcescens) 粘质沙雷氏菌(Serratia marcescens)
S2 荧光假单胞菌(Pseudomonas fluorescens) 生防假单胞菌(Pseudomonas protegens)
S3 解鸟氨酸拉乌尔菌(Raoultella ornithinolytica) 植生拉乌尔菌(Raoultella planticola)
S4 唐菖蒲伯克霍尔德菌(Burkholderia gladioli) 唐菖蒲伯克霍尔德菌(Burkholderia gladioli)
S5 唐菖蒲伯克霍尔德菌(Burkholderia gladioli) 唐菖蒲伯克霍尔德菌(Burkholderia gladioli)
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湖北省一起米酵菌酸引起食物中毒事件的调查分析
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谢腾 , 陈焕 , 马琳 , 王赤华 , 郑懿 , 刘雪芹 * , 王芬
食品安全质量检测学报 | 本期重点：食品中有毒有害物质分析与监测 2025,16(12): 126-132
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食品安全质量检测学报 | 本期重点：食品中有毒有害物质分析与监测 2025, 16(12): 126-132
湖北省一起米酵菌酸引起食物中毒事件的调查分析
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谢腾 , 陈焕, 马琳, 王赤华, 郑懿, 刘雪芹* , 王芬
作者信息
  • 黄冈市疾病预防控制中心, 黄冈 438000

    • 谢腾(1983—), 男, 主管技师, 主要研究方向为食品安全及微生物检验。E-mail:

通讯作者:

*刘雪芹(1984—), 女, 硕士, 主管技师, 主要研究方向为食品安全及理化检验。E-mail:
Investigation and analysis of a food poisoning incident caused by bongkrekic acid in Hubei Province
Teng XIE , Huan CHEN, Lin MA, Chi-Hua WANG, Yi ZHENG, Xue-Qin LIU* , Fen WANG
Affiliations
  • Huanggang Center for Disease Control and Prevention, Huanggang 438000, China
出版时间: 2025-06-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250328003
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摘要
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目的 调查分析湖北省孝感市一起米酵菌酸(bongkrekic acid, BA)引起的中毒事件, 分离鉴定病原菌, 并检测其毒素。方法 采用超高效液相色谱-串联质谱(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)法测定2份暴露食品(凉皮)、3份食品储存环境样本及2份病例血液样本中BA。参照GB/T 4789.29—2020《食品卫生微生物检验 唐菖蒲伯克霍尔德氏菌(椰毒假单胞菌酵米面亚种)检验》, 对2份暴露食品及3份环境样本中的病原菌进行分离培养、飞行时间质谱、16S rDNA测序、实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)分析、产毒实验等微生物鉴定。结果 两例病例血液样本中BA质量浓度分别为92.2 μg/L、1059.0 μg/L, 待售凉皮与冻存凉皮中BA含量分别为2.26 mg/kg、0.39 mg/kg, 环境样本均未检出BA。从疑似暴露食品中分离出两株可疑菌株, VITEK MS飞行时间质谱仪及16S rDNA测序比对结果表明两株可疑菌株均为唐菖蒲伯克霍尔德氏菌, 实时荧光定量PCR分析及产毒实验结果表明两株可疑菌株均可产生BA且bon基因阳性, 均具备产毒能力, 综合判定为唐菖蒲伯克霍尔德氏菌椰毒致病种。环境样本中未鉴定出唐菖蒲伯克霍尔德氏菌。结论 本次食物中毒事件由受唐菖蒲伯克霍尔德氏菌椰毒致病种污染的凉皮食品引起, 有关部门需加强食品安全监管, 防范类似中毒事件发生。

唐菖蒲伯克霍尔德氏菌  /  米酵菌酸  /  食物中毒

Objective To investigate and analyze a food poisoning incident caused by bongkrekic acid (BA) in Xiaogan City, Hubei Province, isolate and identify the pathogenic bacteria, and detect the toxin. Methods Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure BA levels in 2 exposed food samples (cold rice noodles), 3 food storage environment samples, and 2 patient blood samples. Following GB/T 4789.29—2020 Microbiological examination of food hygiene—Examination of Burkholderia gladioli (Pseudomonas cocovenenans subsp. farinofermentans), microbial identification was performed on 2 exposed food samples and 3 environmental samples, including isolation and culture, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry, 16S rDNA sequencing, real-time quantitative polymerase chain reaction (PCR) analysis, and toxin production experiments. Results The BA concentrations in the 2 patients’ blood samples were 92.2 μg/L and 1059.0 μg/L, respectively. The BA content in the unsold and frozen cold rice noodles was 2.26 mg/kg and 0.39 mg/kg, respectively, while no BA was detected in the environmental samples. Two suspected strains were isolated from the implicated food. VITEK MS MALDI-TOF MS and 16S rDNA sequencing identified both strains as Burkholderia gladioli. Real-time quantitative PCR and toxin production experiments confirmed that both strains could produce BA and tested positive for the bon gene, indicating toxigenic potential. They were comprehensively identified as Burkholderia gladioli pathovar cocovenenans. No Burkholderia gladioli was detected in the environmental samples. Conclusion This food poisoning incident is caused by cold rice noodles contaminated with Burkholderia gladioli pathovar cocovenenans. Relevant authorities need to strengthen food safety supervision to prevent similar poisoning incidents.

Burkholderia gladioli  /  bongkrekic acid  /  food poisoning
谢腾, 陈焕, 马琳, 王赤华, 郑懿, 刘雪芹, 王芬. 湖北省一起米酵菌酸引起食物中毒事件的调查分析. 食品安全质量检测学报, 2025 , 16 (12) : 126 -132 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250328003
Teng XIE, Huan CHEN, Lin MA, Chi-Hua WANG, Yi ZHENG, Xue-Qin LIU, Fen WANG. Investigation and analysis of a food poisoning incident caused by bongkrekic acid in Hubei Province[J]. Journal of Food Safety & Quality, 2025 , 16 (12) : 126 -132 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250328003
正文
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0 引言
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唐菖蒲伯克霍尔德氏菌(Burkholderia gladioli)椰毒致病种, 也称椰毒假单胞菌酵米面亚种, 是一种严重威胁食品安全的致病菌[1], 该菌广泛存在于自然环境中[2], 尤其容易污染高淀粉含量、高水分的发酵食品[3-4], 如玉米面制品、椰子制品、湿米粉及木耳等。唐菖蒲伯克霍尔德氏菌椰毒致病种的主要代谢产物为米酵菌酸(bongkrekic acid, BA), 其生物活性较强[5]。居民误食受BA污染的食品后, BA在生物体内引起一系列中毒症状, 常见头晕、呕吐、腹痛腹泻、肝肿大等[6]。BA引起的食源性疾病病死率较高, 可达40%, 其流行于我国西北、东南等地区, 严重威胁群众生命安全[7]。近年来, 黑龙江[8]、惠州[9]、广州[10]等地均报道过由唐菖蒲伯克霍尔德氏菌污染食品引发的BA中毒事件。
生物样本中的BA检测方法暂无国家标准, 湿米面等食品样本中BA检测方法前处理复杂、耗时长[11], BA中毒患者发病急, 临床症状与其他疾病无明显区分, 因此建立BA中毒事件的快速溯源方法对及时救助患者有重要的现实意义[12]。超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/ MS)在生物样本的鹅膏肽类等多种毒素检测中显现出快速、准确等优点[13-14], 有研究成功应用UPLC-MS/MS检测血液等生物样本中BA[15-16]。VITEK MS飞行时间质谱仪可快速鉴定可能的病原, 优于传统的生化鉴定试剂盒[17], 结合产毒能力试验可快速确认病菌产毒能力及可能亚种[18]。目前关于BA中毒事件中理化检测结合病原学技术的快速溯源实例报道相对较少, 鉴于此, 本研究采用UPLC-MS/MS快速检测病例血液样本中的BA含量, 确定可能病因后, 利用UPLC-MS/MS筛选BA阳性流行病学相关样本, 对暴露食品中分离出的可疑菌株进行快速生化鉴定后, 直接分析其产毒能力, 确定可能的病菌及其亚种后, 再辅以16S rDNA测序、bon基因簇检测等检测技术进一步确认病菌, 为BA引起的中毒事件中快速确定病因、病原溯源分析和制定科学防控策略提供实践参考。
1 材料与方法
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1.1 样品来源
2024年8月, 孝感市安陆市发生一起疑似米酵菌酸中毒事件, 基于患者特征性临床症状与流行病学调查结果的关联分析, 初步判定患者共同的暴露场所为某凉皮零售摊位, 可疑暴露食品为市售凉皮制品。
环境样本采集: 共采集涉事凉皮摊位的环境样本3份, 包括2份食品加工接触面样本(操作工具、操作台各1份)、1份冷链储存环境样本(冰箱内壁)。参考GB/T 18204.4—2013《公共场所卫生检验方法 第4部分: 公共用品用具微生物》采集涉事凉皮摊位食品加工工具、操作台面及冷链储存环境等样本, 其中, 操作工具(刀具): 使用浸润生理盐水的无菌棉签在刀具表面以切割面为中心各5 cm×3 cm (15 cm2)面积上正反两面各均匀涂抹5次, 将棉拭子放入采样管, 用无菌操作方法去掉手接触部分, 完成采样。操作台面(砧板): 以烧灼灭菌并浸入平皿冷却后的镊子夹取一片浸润生理盐水的检测纸, 于砧板中央处5 cm×5 cm (25 cm2)面积上均匀涂抹5次, 将检测纸放入采样管。储存环境(冰箱): 因凉皮塑料包装完整, 采样处选择为凉皮放置处底部中心5 cm×5 cm (25 cm2)面积, 具体采样过程同操作台面。
食品样本采集: 采集可疑暴露食品样本2份, 其中, 原料型冰冻冻存凉皮包装完整, 整份采集, 共1份, 以下简称“冻存凉皮”, 解冻后即食待售凉皮剩余部分全部采集, 共1份, 以下简称“待售凉皮”。
生物样本采集: 采集2份患者(1例轻度病例、1例重度病例)全血样本。对肘部静脉采血部位进行消毒后, 使用一次性采血针进行穿刺, 用肝素钠抗凝管采集疑似中毒患者静脉血3.0 mL, 置于冷藏盒中避光保存。
所有采集的样本均在4 ℃条件下冷藏保存和运输, 均于采集后24 h内进行实验室检测。
1.2 仪器与试剂
1290UPLC-6475QQQ型超高效液相色谱-质谱/质谱仪[配电喷雾离子源(electrospray ionization, ESI), 美国Agilent公司]; THR-20D型离心机(湖南英泰科技有限公司); 2300TH型数控超声波清洗器(上海安谱实验科技股份有限公司); MFV-24plus型智能氮吹仪(广州得泰仪器科技有限公司); Milli-Q型纯水系(德国Merck公司); VITEK MS全自动快速微生物质谱鉴定系统(法国梅里埃公司); L1-9082电热生化培养箱(上海龙跃仪器设备有限公司); AB2-5S1二级生物安全柜(新加坡ESCO公司); MVS-83立式压力蒸汽灭菌器(大连冰山松洋生物科技有限公司); Archimed X6实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)仪(北京鲲鹏基因科技有限公司); Neofuge 1600R高速冷冻离心机(上海力新仪器有限公司), Zorbax Eclipse Plus C18色谱柱(2.1 mm×50 mm, 1.8 μm)、MAX固相萃取柱(美国Agilent公司)。
甲醇、乙腈[色谱级, 赛默飞世尔科技(中国)有限公司]; 氨水、甲酸(色谱纯, 上海阿拉丁生化科技股份有限公司); 甲醇中米酵菌酸溶液标准物质(质量浓度100 μg/mL, 北方伟业计量集团有限公司); 椰毒假单胞菌酵米面亚种bon基因核酸检测试剂盒(山东美正生物科技有限公司); 含225 mL GVC增菌液的均质袋(即用型)、改良马铃薯葡萄糖琼脂平板、PCFA培养基平板、马铃薯葡萄糖琼脂平板、卵黄琼脂培养基、50%卵黄乳液(青岛海博生物技术有限公司)。均需在有效期内使用。
1.3 实验方法
1.3.1 米酵菌酸的测定
湿米面BA含量的测定方法参考GB 5009.189—2023《食品安全国家标准 食品中米酵菌酸的测定》。
血液样本中BA含量的测定方法参考文献[19-20]。将病例血液样本从冷藏盒中取出, 于室温下振荡摇匀3 min后, 取200 μL全血于1.5 mL EP管中, 加入预先在-20 ℃冷冻的甲醇800 μL, 于室温下振荡摇匀5 min, 在12000 r/min、4 ℃条件下离心5 min, 取上清液0.22 μm滤膜后上机检测。
产毒实验培养液中BA含量的测定方法参考文献[3]。取产毒培养收集的上清液0.5 mL, 以乙腈定容至50 mL, 涡旋, 12000 r/min 离心5 min, 取上清液, 经0.22 μm微孔滤膜过滤后上机检测。
仪器条件为: Zorbax Eclipse Plus C18色谱柱(2.1 mm× 50 mm, 1.8 μm); 柱温30℃; 流量0.40 mL/min; 流动相A为含0.1%甲酸的水溶液, B为乙腈; 进样量10.0 μL。梯度洗脱程序: 0~1 min时, B由20%升至60%; 1.0~3.0 min时, B由60%升至80%; 3.0~5.1 min时, B由80%升至100%; 保持至6.5 min。质谱条件为: 双喷射流电喷雾离子源, 负离子模式; 干燥气温度250 ℃, 干燥气流量10 L/min; 鞘气温度350 ℃, 鞘气流量12 L/min; 雾化器压力40.0 psi; 毛细管喷雾电压4000 V, 喷嘴电压2000 V;多反应监测模式, 母离子(m/z) 485.3; 子离子(m/z) 397.2、441.3, 碎裂电压95 V, 驻留时间100 ms, 碰撞能量分别设置为12~18 V。
根据定性离子对、保留时间定性, 基质匹配外标法定量。BA检出限和定量限分别为1.3 μg/L和4.2 μg/L。
1.3.2 分离纯化与生化鉴定
采用GB/T 4789.29—2020《食品安全国家标准 食品微生物学检验 唐菖蒲伯克霍尔德菌(椰毒假单胞菌酵米面亚种)检验》的方法进行样品处理、增菌、分离, 使用VITEK MS全自动快速微生物质谱鉴定系统进行鉴定。
1.3.3 分子生物学鉴定
将分离纯化后的唐菖蒲伯克霍尔德氏菌平板送往苏州泓迅生物科技股份有限公司进行16S rDNA基因测序。将测序结果在美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)数据库上进行Blast比对分析, 使用Neighbor-Joining method法在分子进化遗传分析(molecular evolutionary genetics analysis, MEGA)上构建系统发育树。使用椰毒假单胞菌酵米面亚种bon基因核酸检测试剂盒(PCR-探针法)进行椰毒假单胞菌酵米面亚种bon基因的检测。
1.3.4 产毒试验
取适量空白湿米面样品放入灭菌锅105 ℃灭菌5 min, 灭菌后样品称取10.0 g放入灭菌的250 mL锥形瓶中, 加入1 mL的108 CFU/mL菌悬液, 再加入50 mL培养液, 于26 ℃连续培养5 d, 利用UPLC-MS/MS测定BA浓度。
1.4 数据处理
使用Excel 2020收集、处理实验数据, 使用Origin 2018绘图。
2 结果与分析
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2.1 米酵菌酸的测定
采用UPLC-MS/MS对2份全血样本(一例轻度病例、一例重度病例)、2份疑似暴露食品样本(待售凉皮样品、冻存凉皮样品各1份)、3份环境样本进行BA毒素检测, 结果表明, 轻度患者、重度患者血液中BA质量浓度分别为92.2 μg/L、1059.0 μg/L。待售凉皮样品、冻存凉皮样品BA含量分别为2.26 mg/kg、0.39 mg/kg, 均高于GB 7096—2014《食品安全国家标准 食用菌及其制品》中BA含量限值0.25 mg/kg, 3份环境样本中均未检出BA。两例病例血液中BA的UPLC-MS/MS色谱图见图1。
2.2 菌落分离与生长特征
将采集的5份样品加入GVC增菌液中, 36 ℃下增菌24 h, 蘸取增菌液分别划线接种于改良马铃薯葡萄糖琼脂平板和PCFA平板上, 36 ℃下培养48 h后观察菌落形态。可疑菌株在改良马铃薯葡萄糖琼脂平板平板上菌落呈紫色, 直径2~4 mm, 圆形轮廓, 表面光滑湿润黏稠, 部分菌落中心凸起。在PCFA平板上菌落呈乳白色, 直径1~2 mm, 表面光滑, 边缘整齐。挑取可疑菌落接种于卵黄琼脂平板上, 36 ℃培养24 h后筛选得到可疑菌株5株(S1、S2、S3、S4和S5), 其中S1~S3来自于环境样本, S4来自于待售凉皮样本, S5来自于冷冻凉皮样本。可疑菌株在卵黄琼脂平板上菌落部分略微凸起, 直径2~4 mm, 周围伴有乳白色混浊环, 表面湿润黏稠, 斜射光下可见菌落带有虹彩环。结果见图2。
2.3 菌株生化鉴定
5株可疑菌株VITEK MS飞行时间质谱图见图3, 生化鉴定结果见表1。S4、S5菌株为唐菖蒲伯克霍尔德菌(Burkholderia gladioli), 其蛋白分布质谱图与标准图谱一致。
2.4 菌株分子生物学鉴定结果
5株可疑菌株16S rDNA基因测序结果见表1, 进一步确定了菌株S4和S5为唐菖蒲伯克霍尔德氏菌。在NCBI基因数据库里选取若干个伯克霍尔德菌属菌株的已知16S序列, 和菌株S4和S5的测序结果进行多序列比对, 构建系统发育树, 结果见图4。菌株S4和S5与唐菖蒲伯克霍尔德氏菌标准菌株聚类到同一分支, 支持率为99%, 亲缘度高。实时荧光定量PCR结果显示S4和S5椰毒假单胞菌bon基因阳性。
2.5 产毒试验
进一步对菌株的BA产毒特性进行研究, 本研究分离出的唐菖蒲伯克霍尔德菌S4、S5均为产毒毒株。S4、S5菌株的BA产量随培养时间增加而增加, 培养4 d内BA浓度快速上升, 并在第5 d趋于稳定。S5菌株产毒能力强于S4毒株, 第5 d时培养液中BA质量浓度分别达到796.7 μg/L、580.2 μg/L, 结果见图5。
3 结论与讨论
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本研究成功运用UPLC-MS/MS在8 min内准确定性患者血液中的毒性物质为BA并定量其浓度, 血液样本操作简单, UPLC-MS/MS分析准确、快速, 实验室生物样本检测总时长低于20 min, 可作为BA中毒事件中生物样本检测的优先考虑方法。既往致病菌病原溯源工作中[21-23]发现, 唐菖蒲伯克霍尔德氏菌属有较多菌种, 部分菌种生化鉴定与16S rDNA测序后仍然无法确认其是否产毒, 本研究发现经生化鉴定后直接进行产毒培养和毒素测定, 明确其产毒能力, 推测其最可能为椰毒假单胞种, 进一步明确患者中毒原因, 以开展相应的救治措施。因此, 以UPLC-MS/MS检测病例血液中BA, 结合生化鉴定后直接产毒培养, 再以16S rDNA测序、bon基因簇检测结果予以佐证[24-25], 可避免溯源时间过长、前处理复杂等问题, 可作为BA中毒事件快速溯源标准流程的实践参考。
研究发现, 湿米面中BA含量较高, 而环境样本均未检出BA, 这可能与操作台及工具清洗频繁, 储存冰箱表面光滑病菌难以附着繁殖有关[26-27]。BA中毒事件的发生大多与食品储存条件不当, 如长时间室温存放或反复冷冻解冻[28-30]以及加工环境不达标[31]等原因有关。本研究也发现, 冷冻储存但时间较长的凉皮仍然存在唐菖蒲伯克霍尔德菌及其代谢产物BA, 且保存不当会使该致病菌产生更多的毒素。
本次事件发生的主要原因是湿米面制品储存条件不当, 商贩食品安全意识薄弱, 因此, 为有效预防BA中毒事件的发生, 建议如下: (1)有关部门应加强BA中毒预防知识的宣传教育工作, 引导消费者、生产厂家及零售摊贩等提高安全意识, 确保湿米面食品保存条件适宜, 同时避免存放时间过久, 减少类似中毒事件的发生; (2)尽快建立理化分析与唐菖蒲伯克霍尔德氏菌病原学鉴定结合的BA中毒事件快速鉴定体系, 及时确定类似事件的病因, 及时对症救助BA中毒患者。
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  • 湖北省公共卫生青年拔尖人才项目(鄂卫通2021-40号)
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2025年第16卷第12期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250328003
  • 接收时间：2025-03-28
  • 首发时间：2026-01-13
  • 出版时间：2025-06-25
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  • 收稿日期：2025-03-28
基金
湖北省公共卫生青年拔尖人才项目(鄂卫通2021-40号)
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    黄冈市疾病预防控制中心, 黄冈 438000

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*刘雪芹(1984—), 女, 硕士, 主管技师, 主要研究方向为食品安全及理化检验。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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