微生物的就业前景在哪方面应用缺乏，有比较有前景
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时间：2011-10-09 03:49
微生物在医学的应用有哪些?
1.微生物能够致病,能够造成食品、布匹、皮革等发霉腐烂,但微生物也有有益的一面.最早是弗莱明从青霉菌抑制其它细菌的生长中发现了青霉素,这对医药界来讲是一个划时代的发现.抗生素的使用在第二次世界大战中挽救了无数人的生命.2.通过基因组研究揭示微生物的遗传机制,发现重要的功能基因并在此基础上发展疫苗,开发新型抗病毒、抗细菌、真菌药物,将对有效地控制新老传染病的流行,促进医疗健康事业的迅速发展和壮大!&3.一直沿用至今天的巴斯德消毒法（60～65℃作短时间加热处理,杀死有害微生物的一种消毒法）和家蚕软化病问题的解决也是巴斯德的重要贡献,它不仅在实践上解决了当时法国酒变质和家蚕软化病的实际问题,而且也推动了微生物病原学说的发展,并深刻影响医学的发展.
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扫描下载二维码微生物在哪方面应用缺乏,有比较有前景
微生物学主要研究与医学有关的病原微生物的生物学性状、传染致病的机理、免疫学的基本理论、诊断技术和特异性防治措施等,以达到控制和消灭传染性疾病和与微生物有关的免疫性疾病,保障人类健康的目的,微生物学是一门...
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科研单位、医院中心实验室、大学教研室、CDC、微生物检验执法部门、微生物发酵工厂、甚至食品厂、各大分子生物学公司、诊断试剂公司等。我在网络购买试剂的时候，经常看见公司招聘专业人员解决他们的诊断试剂的克隆表达题目，国内的大的诊断试剂公司、生物公司非常多。这些都是微生物方面较好的发展方向，但是你要有足够的能力哦，最好有硕士以上的学历，希望采纳！...
医药，农业，食品（比如酿酒业）
医药比较好 貌似生物能源前景很好 要是能造出廉价生物燃料你就发了
微生物在环保领域，特别是优势的环保功能菌应用方面研究及应用前景好
扫描下载二维码导读：微生物在食品工业的应用，摘要：叙述了微生物与食品工业的关系，微生物在食品的应用，微生物在食品应用工业的发展前景，关键词：微生物食品工业发酵应用前景，微生物是所有形体微小、单细胞或者个体结构简单的多细胞以至没有细胞结构的低等生物的，微生物总类繁多、分布广、代谢类型多、代谢能力强、生长繁殖快、易培养、易变异、适应，使微生物与人类的关系非常密切，微生物不仅在自然界物资循环中起着非常重要的作用，而且在
微生物在食品工业的应用
摘要：叙述了微生物与食品工业的关系，微生物在食品的应用，微生物在食品应用工业的发展前景。
关键词：微生物
应用前景。
微生物是所有形体微小、单细胞或者个体结构简单的多细胞以至没有细胞结构的低等生物的总称。微生物总类繁多、分布广、代谢类型多、代谢能力强、生长繁殖快、易培养、易变异、适应能力强，正是上述特性，使微生物与人类的关系非常密切，微生物不仅在自然界物资循环中起着非常重要的作用，而且在食品工业的应用中也非常广泛。本文叙述了微生物在食品工业中的应用，讨论了微生物的广阔发展前景。
微生物与食品工业的关系
随着人们对微生物认识的不断深入，微生物已被广泛应用于食品生产。今天基因工程、固定化酶、固定化细胞等先进技术的应用，进一步发掘了微生物在食品工业中的巨大发展潜能。微生物在食品工业生产中有非常大的好处，例如可以制作面包，酒；霉菌可制作豆酱、酱油；乳酸菌可制作泡菜、酸奶等；当然也有危害，我们要充分利用微生物有利的方面为食品工业服务，消除器有害影响，为人类造福。 二
微生物在食品生产中的应用
食醋是我国劳动人民在长期的生产实践中制造出来的一种酸性调味品。它能增进食欲，帮助消化，在人们饮食生活中不可缺少。在我国的中医药学中醋也有一定的用途。全国各地生产的食醋品种较多。著名的山西陈醋、镇江香醋、四川麸醋、东北白醋、江浙玫瑰米醋、福建红曲醋等是食醋的代表品种。食醋按加工方法可分为合成醋、酿造醋、再制醋三大类。其中产量最大且与我们关系最为密切的是酿造醋，它是用粮食等淀粉质为原料，经微生物制曲、糖化、酒精发酵、醋酸发酵等阶段酿制而成。其主要成分除醋酸(3%～5%)外，还含有各种氨基酸、有机酸、糖类、维生素、醇和酯等营养成分及风味成分，具有独特的色、香、味。它不仅是调味佳品，长期食用对身体健康也十分有益。
面包是产小麦国家的主食，几乎世界各国都有生产。它是以面粉为主要原料，以酵母菌、糖、油脂和鸡蛋为辅料生产的发酵食品，其营养丰富，组织蓬松，易于消化吸收，食用方便，深受消费者喜爱。酵母是生产面包必不可少的生物松软剂。面包酵母是一种单细胞生物，属真菌类，学名为啤酒酵母。面包酵母有圆形、椭圆形等多种形态。以椭圆形的用于生产较好。酵母为兼性厌氧性微生物，在有氧及无氧条件下都可以进行发酵。
我国是一个酒类生产大国，也是一个酒文化文明古国，在应用酵母菌酿酒的领域里，有着举足轻重的地位。许多独特的酿酒工艺在世界上独领风骚，深受世界各国赞誉，同时也为我国经济繁荣作出了重要贡献。
酿酒具有悠久的历史，产品种类繁多如：黄酒、白酒、啤酒、果酒等品种。而且形成了各种类型的名酒，如绍兴黄酒、贵州茅台酒、青岛啤酒等。酒的品种不同，酿酒所用的酵母以及酿造工艺也不同，而且同一类型的酒各地也有自己独特的工艺。
啤酒是以优质大麦芽为主要原料，大米、酒花等为辅料，经过制麦、糖化、啤酒酵母发酵等工序酿制而成的一种含有C02、低酒精浓度和多种营养成分的饮料酒。它是世界上产量最大的酒种之一。
生产用霉菌菌种：淀粉的糖化、蛋白质的水解均是通过霉菌产生的淀粉酶和蛋白质水解酶进行的。通常情况是先进行霉菌培养制曲。淀粉、蛋白质原料经过蒸煮糊化加入种曲，在一定温度下培养，曲中由霉菌产生的各种酶起作用，将淀粉、蛋白质分解成糖、氨基酸等水解产物。
酱类包括大豆酱、蚕豆酱、面酱、豆瓣酱、豆豉及其加工制品，都是由一些粮食和油料作物为主要原料，利用以米曲霉为主的微生物经发酵酿制的。酱类发酵制品营养丰富，易于消化吸收，即可作小菜，又是调味品，具有特有的色、香、味，价格便宜，是一种受欢迎的大众化调味品。
用于酱类生产的霉菌主要是米曲霉（Asp.oryzae），生产上常用的有沪酿3.042，黄曲霉Cr-1菌株（不产生毒素），黑曲霉（Asp. Nigerf-27）等。所用的曲霉具有较强的蛋白酶、淀粉酶及纤维素酶的活力，它们把原料中的蛋白质分解为氨基酸，淀粉变为糖类，在其他微生物的共同作用下生成醇、酸、酯等，形成酱类特有的风味。
4.1.酱油：
酱油生产中常用的霉菌有米曲霉、黄曲霉和黑曲霉等，应用于酱油生产的曲霉菌株应符合如下条件：不产黄曲霉毒素；蛋白酶、淀粉酶活力高，有谷氨酰胺酶活力；生长快速、培养条件粗放、抗杂菌能力强；不产生异味，制曲酿造的酱制品风味好。
L-谷氨酸钠俗称味精。L-谷氨酸发酵生产菌种主要有棒状杆菌、短杆菌属、小杆菌属的细菌。L-谷氨酸钠发酵工艺主要有4个阶段：①摇瓶种子培养。即采用适合的液体培养基培养菌种。②二级种子培养。③L-谷氨酸发酵生产。④L-谷氨酸钠的提取。在L-谷氨酸发酵生产时期的谷氨酸合成阶段，菌体浓度基本不变，糖与尿素分解后产生的α-酮戊二酸和氨主要用于合成谷氨酸。此阶段应为菌种提供生长最适宜的条件，如及时添加尿素，调节最适PH7.2~7.4，提高温度到谷氨酸合成的最适温度并大量通气，保证菌体的有氧呼吸，使它大量产生代谢产物提高产量。
发酵生产柠檬酸。柠檬酸发酵主要采用的菌种是黑曲霉。黑曲霉能利用淀粉分解出代谢产物柠檬酸。柠檬酸发酵工艺流程为：种子的扩大培养→柠檬酸的发酵。
微生物在食品工业的前景
1.固定化酶在食品工业中的应用
1.1.应用于食品酶制剂的生产
利用基因工程技术，不但可以成倍地提高酶的活力，而且还可将生物酶基因克隆到微生物中，构建基因工程菌来生产酶。转基因微生物生产酶有许多优点，如产量高、品质均一、稳定性好、价格低等。第一个应用于食品的基因工程酶为凝乳酶，它是制造干酪过程中起凝乳作用的关键酶，传统来源是从小牛皱胃液中提取，造成全球性小牛短缺，酶成本不断提高。人们曾经尝试用微生物代用品或其他动物来源的凝乳酶，但效果均不理想；基因工程解决了这一难题。近20 年来用基因工程菌发酵生产的食品酶制剂主要有：凝乳酶、α－淀粉酶、葡萄糖氧化酶、葡萄糖异构酶、转化酶、普鲁多糖酶( 茁霉多糖酶) 、脂肪酶、α－半乳糖苷酶、β－半乳糖苷酶、α－乙酰乳酸脱羧酶、溶菌酶、碱性蛋白酶等。
1.2.固定化葡萄糖异构酶在高果糖浆生产中的应用
固定化葡萄糖异构酶是世界上生产规模最大的一种固定化酶，1973年就已应用在工业化生产， 它可以用来催化玉米糖浆和淀粉生产高甜度的高果糖糖浆。
2.酶制剂在食品保鲜方面的应用
随着人们对食品的要求不断提高和科学技术的不断进步，一种崭新的食品保鲜技术―酶法保鲜技术正在崛起。酶法保鲜技术是利用生物酶的高效的催化作用，防止或消除外界因素对食品的不良影响，从而保持食品原有的优良品质和特性的技术。由于酶具有专一性强、催化效率高、作用条件温和等特点，可广泛地应用于各种食品的保鲜，有效地防止外界因素，特别是氧化和微生物对食品所造成的不良影响。
葡萄糖氧化酶是一种氧化还原酶，它可催化葡萄糖和氧反应，生成葡萄糖酸和双氧水。将葡萄糖氧化酶与食品一起置于密封容器中，在有葡萄糖存在的条件下，该酶可有效地降低或消除密封容器中的氧气，从而有效地防止食品成分的氧化作用，起到食品保鲜作用。
3.作为畜禽饲料添加剂
中国是蛋白原料缺乏的国家，随着饲料工业的迅速发展和生产的高度集约化，对优质饲料蛋白原料的需求日趋增大，目前饲料优质蛋白原料的主要来源是鱼粉，而作为一种亚稀缺资源，鱼粉已经在各主要产地如秘鲁等国受到严格限产保护。需求的膨胀和来源的快速减少，正是目前饲料优质蛋白原料面临的尴尬处境。一些西方发达国家先行一步，将解决优质饲料蛋白来源的目光投向了生物技术产品―单细胞蛋白。
4.生产SCP的微生物
在工业生产中，作为蛋白质资源的微生物菌体，特别的酵母菌和细菌，它们都能利用糖类原料生产菌体蛋白，究竟采用酵母菌和细菌哪种更好呢？这在很大程度上取决于生产SCP的原料。在20世纪60年代末和70年代初期，开发了多种由烷烃类物质产生的SCP工艺，能够利用烷烃的微生物主要有细菌和放线菌，如产碱杆菌、假单孢菌、节杆菌、短杆菌等，其次为酵母菌属。
结束语：随着生物技术的飞速发展,利用基因工程对微生物进行菌种改造从根本上解决发酵食品生产工艺中的问题,已在食品工业中开展试用。中科院微生 物所与青岛啤酒集团正在联合研制构建具有熟化期短、絮凝性强等优良特性,又保持原有口味的青岛啤酒酵母工程菌。将生物技术应用到食品工业必将对中国食品行业的发展产生重要的影响。功能性食品及食品添加剂等各方面的生产都将与微生物紧密相连，微生物将为食品工业的发展开辟更广泛的前景。微生物在食品工业中的应用也远远不仅如此，今后将会发挥越来越重要的作用。
总而言之，我们应该展望未来，食品工业将成为现代生物技术应用最广阔、最活跃、最富有挑战性的领域。要充分利用世界生物技术迅猛发展的契机，重视
发酵工程技术的研究，促进我国食品工业的改革，实现我国食品工业健康有序的发展。
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[2] 郑华学．试论生物技术在我国食品工业中的应用[J]．2003，(5)：99―102．
[3] 姬德衡．发酵工程在功能食品开发中的应用[J]．食品技术，2002，(7)：9-13.
[4] 何国庆．食品发酵与酿造工艺学[M]．北京:中国农业出版社，．
[5] 金滨锋．现代生物技术在食品工业中的应用[J]．生物技术世界，2007，(8)：36-39．
[6] 史先振．现代发酵工程技术在食品领域的应用研究进展[J].中国酿造，2005，(2)：1 ―4．
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相关内容搜索质谱技术在临床微生物实验室中的应用前景
罗燕萍. 质谱技术在临床微生物实验室中的应用前景.检验医学,): 97-100LUO Yanping. The prospects of mass spectrometry application on microbiology. Labratory Medicine,): 97-100&&
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质谱技术在临床微生物实验室中的应用前景
解放军总医院微生物科,北京 100853
作者简介:罗燕萍,女,1960年生,硕士,主任技师,主要从事临床微生物耐药机制研究。
目前应用于临床微生物实验室的基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS) 是一种直接从完整细菌中获得蛋白指纹图谱,从而对细菌进行快速鉴定的方法,由于可以对常见细菌,诺卡菌、分枝杆菌、厌氧菌、酵母菌及丝状真菌进行快速和准确的鉴定,近年来备受推崇。该文对这次质谱专题刊登的1篇综述和4篇论著进行总结评价。相信随着相关研究的不断深入,这项技术在临床微生物实验室将有更广阔的应用空间。
基质辅助激光解析电离飞行时间质谱;
临床微生物实验室
中图分类号:R446.5
文献标志码:A
文章编号:15)02-0097-04
The prospects of mass spectrometry application on microbiology
LUO Yanping
Department of Microbiology, Chinese PLA General Hospital , Beijing 100853, China
The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) technique can be used to generate protein fingerprint signatures from whole bacterial cells. By comparing these fingerprints to a database of reference spectra by the various algorithms, bacteria can be rapidly identified. MALDI-TOF MS can be used for accurate and rapid identification of various microorganisms, such as common bacteria, Nocardia, Mycobacterium, anaerobe, yeast and filamentous fungi. MALDI-TOF MS have recently been widely introduced. The characteristics of one review and four papers published in the special subject about MALDI-TOF MS were reviewed. The development of studies in MALDI-TOF MS will play an important role in the application of mass spectrometry in clinical microbiological laboratories.
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry;
Bacteria identification;
Clinical microbiology laboratory
引言自20世纪80年代起, 质谱技术就已经成为科学研究中用于蛋白分析的强大工具。随着技术的不断成熟和广泛使用, 其在微生物检验常规诊断中的作用越来越受到关注, 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)已经进入临床微生物实验室用于病原菌鉴定, 与传统的表型鉴定及分子生物学技术相比, MALDI-TOF MS快速、准确、成本低廉。本刊本期集中刊登“ 质谱技术在临床微生物检验中的应用” 专题, 旨在使临床和实验室工作者对其有一个全面的认识和了解。一、MALDI-TOF MS目前的应用现状传统的细菌鉴定流程通常是:根据细菌在不同培养基上的生长情况和菌落形态, 应用一些简便、快速的试验如革兰染色、触酶、氧化酶等进行初步分类, 然后通过手工生化试验或自动鉴定系统等来完成鉴定[, ]。虽然有些试验几分钟即可完成, 但大部分情况下, 完成常规细菌鉴定至少需要8~18 h或更长的时间(如苛养菌)。分子生物学如聚合酶链反应(polymerase chain reactuin, PCR)、DNA 芯片、微阵列技术等方法虽然敏感性高, 但费用高昂, 且对操作人员、设备和环境有较严格的要求, 不适合常规应用。MALDI-TOF MS是将完整的病原菌细胞(intact-cell, IC)直接进行检测, 无需蛋白提纯, 样本准备很简便, 也有人称之为 IC MALDI-TOF MS。许多研究表明, IC MALDI-TOF MS敏感性很高, 可以区分表型相似甚至相同的菌株, 提供属、种、型水平的鉴定。目前报道使用质谱进行微生物鉴定方面的问题主要集中在质谱数据库容量和有些特定种属无法明确区分上。二、质谱专题收录论文的主要内容此次专题共刊登1篇综述和4篇论著。综述文章“ MALDI-TOF MS在临床微生物检验中的应用” , 从MADLI-TOF MS的基本原理、在微生物鉴定中的应用、在科研中的应用等三方面比较全面地介绍了目前MALDI-TOF MS在临床微生物实验室应用的现状及前景。质谱技术对以葡萄球菌和链球菌为代表的革兰阳性球菌鉴定到种水平的准确率> 99%, 对肠杆菌科细菌鉴定到种水平的准确率为96.60%, 非发酵菌为98.75%, 结核分枝杆菌为94.90%, 非结核分枝杆菌为94.60%, 对以拟杆菌属为代表的厌氧菌可以达到97.50%, 以酵母菌为主的真菌为97.30%。对临床常见分离菌鉴定到种水平的准确率很高, 使质谱技术在临床微生物实验室中得到广泛应用。另外, 从阳性血培养和尿液样本中直接鉴定病原菌作为一个研究方向也在大量的研究尝试之中, 尚未完全应用于常规鉴定。利用特异峰值的分析对耐药基因、血清分型、毒力等的研究也在不断进行中。本专题中“ MALDI-TOF MS在中段尿样本细菌直接检测中的应用” 一文, 应用MALDI-TOF MS快速、直接检测中段尿样本中的细菌, 尝试建立中段尿快速细菌检测和鉴定方法。其结论与国外相关研究相似:涂靶板之前必须将尿液样本进行离心处理; 较高的细菌计数可以提高鉴定到种的正确率; 如果是2种以上细菌混合感染, 鉴定结果是否正确取决于2种细菌的比例。该方法可比常规尿培养鉴定方法提早40 h左右报告鉴定结果。血流感染是一种严重的感染性疾病, 当血培养瓶报告阳性时, 微生物实验室只能给临床医师暂报涂片结果, 较快明确病原菌是微生物实验室亟待解决的课题。目前国内、外很多学者尝试分子生物学方法如实时荧光定量PCR以及其它一些快速方法如微阵列技术、杂交探针、流式细胞术等直接从血培养阳性瓶中鉴定细菌。近几年还有一些研究使用MALDI-TOF MS直接从阳性瓶中鉴定细菌。该方法首先最重要的一步是将细菌从细胞组分中分离出来, FERRONI等[]在样本中环节做了很大的改进, 血培养瓶报阳性后, 应用温和去污剂裂解细胞膜, 这一步只需要几分钟, 从而使整个鉴定过程缩短到不足30 min。本专题中“ 分离胶促凝管联合 MALDI-TOF MS直接检测血培养阳性细菌” 一文, 采用分离胶促凝管预处理方法, 其优点是成本低廉, 速度快(平均15 min/样本), 安全性好, 没有过多的转移、洗涤等工序, 不容易引起气溶胶等生物安全风险。缺点是纯度不佳, 由于没有额外的洗涤步骤, 最终得到的菌体富集物始终还是含有部分细胞碎片, 其对革兰阳性菌和革兰阴性菌的检出率分别为80.10%和86.90%; 联合法对念珠菌的检出率较低, 仅为22.20%, 还有进一步提升和改进的空间, 期待通过方法学的改进使质谱技术直接实时鉴定血培养阳性瓶中细菌成为可能。有学者将MALDI-TOF MS 与其它方法做比对进行细菌鉴定, 得出的结论是质谱技术对常规分离细菌的鉴定正确率相对于其它方法而言较高。“ MicroflexTM MALDI-TOF MS和Vitek 2 Compact全自动微生物分析系统对肠杆菌科细菌鉴定能力的比较” 一文, 将关注点放在肠杆菌科细菌上, 这2种仪器对细菌种的鉴定符合率分别为97.10%和83.30%, 对于阴沟肠杆菌复合群内的阴沟肠杆菌、霍氏肠杆菌和路德维希肠杆菌, 普通变性杆菌复合群中的普通变形杆菌和潘氏变形杆菌, 液化沙雷菌群中的液化沙雷菌, 小肠结肠耶尔森菌群中的小肠结肠耶尔森菌, 质谱技术通常可以鉴定到种水平, 而后者只能鉴定到群, 印证了质谱技术对于常规分离细菌鉴定到种的正确率较高的结论。“ 两种MALDI-TOF MS系统快速鉴定血培养中白念珠菌以外酵母样真菌” 一文, 比较了2种不同MALDI-TOF MS系统对白念珠菌以外酵母样真菌的鉴定率, 目前市场上有不同厂家的质谱仪尽管原理相似, 但各有自己的特点, 该文的结论指出Autoflex MALDI-TOF MS 对念珠菌尤其是近平滑念珠菌复合体鉴定的分辨率较高, 但本研究对比的菌株数略少(59株, 包括11种真菌), 结论需要更大、更多样化样本的支持。如在LACROIX等[]的两种质谱技术在念珠菌鉴定的研究中, 样本量达到1 383株, 最终2种方法的鉴定正确率均为98.30%, 相差并不显著。本次质谱专题收录的文章主要涉及到鉴定方法的比对和从血培养阳性及尿液样本中直接检测鉴定细菌, 这也是近几年国内外相关研究的热点, 我们也期待有更多质谱应用方面的研究, 推动质谱技术在临床微生物领域的应用范围进一步扩大。三、MALDI-TOF MS未来的发展方向近年来, 越来越多的临床微生物实验室开始引入MALDI-TOF MS进行常规样本的细菌和真菌鉴定, 随着研究的不断深入, MALDI-TOF MS的应用领域也将变得越来越广泛, 主要有3个方面:在流行病学中的应用、从患者样本中直接检测病原菌和对耐药机制的检测。(一)流行病学微生物实验室、感染控制人员和临床医师面临的挑战之一是在某个临床菌株暴发流行时可以得到菌株代表分类的特异数据, 如沙门菌、链球菌等, 为了准确得到血清型、亚型或其它分类, 实验室必须耗费更多的时间做进一步检测, 而且许多菌株分型需要特殊的试验方法、仪器、耗材等。因此, 条件有限的实验室就需要外送做检测, 可能导致检测时间延长、丢失重要的流行病学数据、增加检测费用、结果不准确等。但如果在微生物实验室内快速、准确完成重要数据的结果将会帮助临床医师、护士、感控人员和卫生机构处理、追踪感染病原菌并掌握其流行状况。目前, 通过大量菌株的群、属、种分析, 以及许多菌株分型研究的深入, MALDI-TOF MS所具备的鉴定这些高度相关菌株的能力将使临床微生物实验室的影响力提升。将来可能出现更加精密的MALDI-TOF MS仪器, 不但可以鉴定病原菌, 同时可以快速、准确地提供菌株的流行病学数据, 这将使临床微生物实验室在医院感染控制、病原菌暴发、耐药菌国家监测及生物防御等领域扮演重要角色。(二)MALDI-TOF MS直接从患者样本鉴定细菌因为不需要对目标物质进行扩增以及其鉴定病原菌的高敏感性, MALDI-TOF MS直接从样本中检测细菌的能力可能取代PCR。通过对这些患者样本处理方法的改良, 主要包括去除蛋白、核酸、细胞碎片等可以影响分析的组分, MALDI-TOF MS直接从样本中进行细菌鉴定的能力会进一步提高。以下是直接从不同样本类型中鉴定的报道。1.尿液样本目前的研究表明, 对于计数> 105的尿液样本, 通过低速离心去除细胞碎片和白细胞, 高速离心得到细菌, 再用甲酸和乙腈处理得到蛋白用于质谱分析, 鉴定准确率> 90%[, ]。存在的问题是MALDI-TOF MS尚不能对混合感染的尿液进行准确鉴定; 没有标准化的尿液前期处理流程。而上述的处理流程是目前被证实较简便、快速且保证质谱鉴定准确的方法[, ]。总之, MALDI-TOF MS在单一细菌的尿液样本中直接检测具有很强大的、准确的鉴定能力。2.脑脊液细菌性脑膜炎是临床最严重的感染之一, 快速、准确的检测非常重要, 目前通过MALDI-TOF MS直接检测脑脊液细菌的研究较少, 有一份报道肺炎链球菌引起的脑膜炎, 前期处理流程与尿液样本相似[]。3.血培养阳性瓶中直接鉴定目前有许多从阳性血培养瓶中准确鉴定病原菌的方法, 但由于使用的软件不同, 血培养瓶(培养系统)不同, 因此, 无法客观地比较这些方法的优劣。有的研究认为提取细胞蛋白优于完整细胞分析, 无碳颗粒的血培养瓶会得到准确的鉴定率, 而含有碳颗粒的血培养瓶则结果不好[, , , ]。总之, 从血培养瓶中直接检测细菌和真菌可以显著地缩短鉴定时间[, ], 随着研究的深入, 这些检测方法也会进一步改进, 以获得更方便可行的操作和更高的敏感性, 提高革兰阳性菌及念珠菌等病原菌的检出能力。(三)耐药性检测微生物实验室不但要提供精确的病原菌鉴定结果, 同时还要检测其敏感性, 为临床医生提供治疗依据。常规的药物敏感性试验方法比较费时, 一些酶联免疫、凝集等方法只能局限于少数细菌。MALDI-TOF MS可为耐药基因检测提供一个很好的平台, 可以分析几乎所有的耐药机制, 目前报道的方法主要基于下列几种原理:分析抗菌药物及被修饰后的产物; 分析细胞组分; 分析核糖体DNA甲基化; 检测突变等。1.通过抗菌药物的变化检测酶活性如直接检测β -内酰胺酶活性, 检测方法基本相似:收集新鲜培养的待测细菌, 与β -内酰胺分子反应后, 用MALDI-TOF MS检测β -内酰胺分子及其降解产物的特异峰[, ]。有文献报道通过检测美罗培南及其降解产物, 证实了30株产IPM-7、VIM-2的铜绿假单胞菌, 产VIM-1、KPC-2、NDM-1的肠杆菌产生碳青霉烯酶, 其敏感性和特异性均超过95%。检测β -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[, , , , ]。2.MALDI-TOF MS直接检测耐药决定基团对多耐药细菌蛋白组学的研究可以建立耐药机制相关的主要蛋白指纹图谱, 最近一种检测耐万古霉素肠球菌的方法已经被证实有效。这种方法可以在微生物实验室用于vanB阳性屎肠球菌的快速检测[]。用MALDI-TOF MS区分耐甲氧西林金黄色葡萄球菌和甲氧西林敏感金黄色葡萄球菌时有一定挑战, 尽管有相关研究, 但方法需要进一步优化和验证[]。到目前为止, 还没有一种方法可以检测其它的如β -内酰胺酶耐药决定基团的特异性峰值[]。3.分析细胞壁组分几乎一半以上的抗菌药物的作用靶位位于细胞壁上, 因此, 细菌细胞壁在抗菌药物耐药中的重要性不言而喻。其它抗菌药物则需要越过细胞壁屏障到达它们的作用靶点。对革兰阴性杆菌外膜蛋白的蛋白组学和基因组学研究表明, 膜孔蛋白、外排泵、脂多糖等细胞组分系统主要是通过对不同抗菌药物泵入和泵出的调节而表现耐药的[]。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及2D电泳结合MALDI-TOF MS指纹图谱可以用来鉴定耐药和敏感菌株表达水平不同的外膜及周浆蛋白, 建立一个综合的数据库来研究各种细胞组分, 在耐药检测中发挥不同的作用[, ]。4. 检测耐药机制MALDI-TOF MS通过分析DNA来检测耐药机制, 如可以通过检测单核苷酸多态性来鉴定SHV-型、TEM-型超广谱β -内酰胺酶等[, ]。5.抗菌药物检测MALDI-TOF MS检测的相对分子质量范围较大, 而抗菌药物的相对分子质量通常< 1 000因此, 基质和较高的背景干扰使得这项技术在检测抗菌药物时变得复杂。LIN等通过方法学的改进, 目前已成功检测出水杨酰胺等6种抗菌药物, 未来这种方法有可能用于耐药机制及检测血样本中的微生物毒素和药物等[]。随着质谱数据库及分析软件的不断更新, 所有病原菌鉴定的时间都会缩短。尽管一些方法目前还不能常规应用于微生物实验室, 但随着这些检测方法的不断优化和标准化, 一定会有广阔的应用前景。实验技术不断发展, 使得临床微生物实验室从曾经最慢速服务的实验室逐步转变为一个可以快速、准确为患者提供结果的充满活力的新实验室。我们相信MALDI-TOF MS在未来一定会在临床微生物实验室扮演更重要的角色。
The authors have declared that no competing interests exist.
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... 一、MALDI-TOF MS目前的应用现状传统的细菌鉴定流程通常是:根据细菌在不同培养基上的生长情况和菌落形态,应用一些简便、快速的试验如革兰染色、触酶、氧化酶等进行初步分类,然后通过手工生化试验或自动鉴定系统等来完成鉴定[1,2] ...
... 一、MALDI-TOF MS目前的应用现状传统的细菌鉴定流程通常是:根据细菌在不同培养基上的生长情况和菌落形态,应用一些简便、快速的试验如革兰染色、触酶、氧化酶等进行初步分类,然后通过手工生化试验或自动鉴定系统等来完成鉴定[1,2] ...
... 该方法首先最重要的一步是将细菌从细胞组分中分离出来,FERRONI等[3]在样本中环节做了很大的改进,血培养瓶报阳性后,应用温和去污剂裂解细胞膜,这一步只需要几分钟,从而使整个鉴定过程缩短到不足30 min ...
. ):153-158
1 Service de Mycologie-Parasitologie, H&pital Saint-Louis AP-HP, Universit& Paris Diderot Paris 7, Paris, France 2 Laboratoire de Mycologie-Parasitologie CHU, Nantes, France 3 Laboratoire de Mycologie-Parasitologie CHRU, Inserm U995, Lille, France 4 Service de Microbiologie, H&pital Necker-Enfants Malades AP-HP, Universit& Paris-Descartes, Paris, France 5 Laboratoire de Mycologie-Parasitologie CHU, Bordeaux, France 6 Laboratoire de Mycologie-Parasitologie CHU, Tours, France 7 Laboratoire de Mycologie-Parasitologie CHU, Poitiers, France 8 Laboratoire de Mycologie-Parasitologie H&pital Saint-Antoine AP-HP, Paris, France
Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas& and Bruker Biotyper&, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level ( for Andromas&;
for Bruker Biotyper&) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.
... 如在LACROIX等[4]的两种质谱技术在念珠菌鉴定的研究中,样本量达到1 383株,最终2种方法的鉴定正确率均为98 ...
... 90%[5,6] ...
... 90%[5,6] ...
... 而上述的处理流程是目前被证实较简便、快速且保证质谱鉴定准确的方法[6,7] ...
. , :231-235
... 而上述的处理流程是目前被证实较简便、快速且保证质谱鉴定准确的方法[6,7] ...
... 细菌性脑膜炎是临床最严重的感染之一,快速、准确的检测非常重要,目前通过MALDI-TOF MS直接检测脑脊液细菌的研究较少,有一份报道肺炎链球菌引起的脑膜炎,前期处理流程与尿液样本相似[8] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 总之,从血培养瓶中直接检测细菌和真菌可以显著地缩短鉴定时间[13,14],随着研究的深入,这些检测方法也会进一步改进,以获得更方便可行的操作和更高的敏感性,提高革兰阳性菌及念珠菌等病原菌的检出能力 ...
Background With long delays observed between sampling and availability of results, the usefulness of blood cultures in the context of emergency infectious diseases has recently been questioned. Among methods that allow quicker bacterial identification from growing colonies, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was demonstrated to accurately identify bacteria routinely isolated in a clinical biology laboratory. In order to speed up the identification process, in the present work we attempted bacterial identification directly from blood culture bottles detected positive by the automate. Methodology/Principal Findings We prospectively analysed routine MALDI-TOF identification of bacteria detected in blood culture by two different protocols involving successive centrifugations and then lysis by trifluoroacetic acid or formic acid. Of the 562 blood culture broths detected as positive by the automate and containing one bacterial species, 370 (66%) were correctly identified. Changing the protocol from trifluoroacetic acid to formic acid improved identification of Staphylococci , and overall correct identification increased from 59% to 76%. Lack of identification was observed mostly with viridans streptococci, and only one false positive was observed. In the 22 positive blood culture broths that contained two or more different species, only one of the species was identified in 18 samples, no species were identified in two samples and false species identifications were obtained in two cases. The positive predictive value of bacterial identification using this procedure was 99.2%. Conclusions/Significance MALDI-TOF MS is an efficient method for direct routine identification of bacterial isolates in blood culture, with the exception of polymicrobial samples and viridans streptococci. It may replace routine identification performed on colonies, provided improvement for the specificity of blood culture broths growing viridans streptococci is obtained in the near future.
... 总之,从血培养瓶中直接检测细菌和真菌可以显著地缩短鉴定时间[13,14],随着研究的深入,这些检测方法也会进一步改进,以获得更方便可行的操作和更高的敏感性,提高革兰阳性菌及念珠菌等病原菌的检出能力 ...
... -内酰胺分子及其降解产物的特异峰[15,16] ...
... -内酰胺分子及其降解产物的特异峰[15,16] ...
. ):193-204
1.Chinese Academy of Sciences National Chromatographic R & A Center, Dalian Institute of Chemical Physics Dalian 116023 People’s Republic of China Dalian 116023 People’s Republic of China
Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) is widely used in a variety of fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range for obtaining molecular weights and for structural characterization of macromolecules. In this article we summarize recent developments in matrix additives, new matrices, and sample-pretreatment methods using off-probe or on-probe techniques or nanomaterials for MALDI-TOF-MS analysis of biological samples.
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
. ):e31676
Rapid detection of carbapenem-resistant Acinetobacter baumannii strains is critical and will benefit patient care by optimizing antibiotic therapies and preventing outbreaks. Herein we describe the development and successful application of a mass spectrometry profile generated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) that utilized the imipenem antibiotic for the detection of carbapenem resistance in a large series of A. baumannii clinical isolates from France and Algeria. A total of 106 A. baumannii strains including 63 well-characterized carbapenemase-producing and 43 non-carbapenemase-producing strains, as well as 43 control strains (7 carbapenem-resistant and 36 carbapenem-sensitive strains) were studied. After an incubation of bacteria with imipenem for up to 4 h, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. The presence and absence of peaks representing imipenem and its natural metabolite was analyzed. The result was interpreted as positive for carbapenemase production if the specific peak for imipenem at 300.0 m/z disappeared during the incubation time and if the peak of the natural metabolite at 254.0 m/z increased as measured by the area under the curves leading to a ratio between the peak for imipenem and its metabolite being A. baumannii clinical isolates, showed a sensitivity of 100.0% and a specificity of 100.0%. Our study is the first to demonstrate that this quick and simple assay can be used as a routine tool as a point-of-care method for the identification of A. baumannii carbapenemase-producers in an effort to prevent outbreaks and the spread of uncontrollable superbugs.
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
J. Proteome Res.. ):79&84
Gero P. Hooff
Jeroen J. A. van Kampen
Roland J. W. Meesters
Alex van Belkum
Wil H. F. Goessens
Theo M. Luider
+ Department of Neurology, Laboratory of Neuro-Oncology and Clinical and Cancer Proteomics, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands
? Department of Microbiology and Infectious Diseases, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands *G. P. Hooff, Laboratory of Neuro-Oncology and Clinical and Cancer Proteomics, Department of Neurology, University Medical Center Rotterdam (Erasmus MC), Dr. Molewaterplein 50, Room Ae-307, 3015 GE Rotterdam, The Netherlands. Phone: + . Fax: + . E-mail: g.hooff@erasmusmc.nl .
Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in Gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry.
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
... 这种方法可以在微生物实验室用于vanB阳性屎肠球菌的快速检测[22] ...
. , :81-86
... 用MALDI-TOF MS区分耐甲氧西林金黄色葡萄球菌和甲氧西林敏感金黄色葡萄球菌时有一定挑战,尽管有相关研究,但方法需要进一步优化和验证[23] ...
. 2012, :-
1. Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Worts Causeway, Cambridge, CB1 8RN, UK
2. Cancer Genomics Laboratory, Centre Hospitalier Universitaire de Québec, 2705 Laurier Boulevard, T3-57, Quebec City, QC, Canada
3. Genetics and Population Health Division, Queensland Institute of Medical Research, 300 Herston Rd, Herston, Brisbane, QLD 4006, Australia
143. INSERM U1052, CNRS UMR5286, Université Lyon 1, Cancer Research Center of Lyon, 28 rue La?nnec, Lyon, 69373, France
4. Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Hospitalier Universitaire de Lyon/Centre Léon Bérard, 28 rue La?nnec, Lyon, 69373, France
5. Section of Genetic Oncology, Dept. of Laboratory Medicine, University and University Hospital of Pisa, Via Roma 57, 56125, Pisa, Italy
6. Department of Oncology, Lund University Hospital, Lund, Sweden
7. Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden
8. Department of Oncology, Karolinska University Hospital, Stockholm, Sweden
9. Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden
10. Department of Oncology, Sahlgrenska University Hospital, Gothenburg, Sweden
11. Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
12. Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland
13. Department of Genetics and Pathology, Pomeranian Medical University, Szczecin and Postgraduate School of Molecular Medicine, Warsaw Medical University, Warsaw, Poland
14. Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre, Spanish Network on Rare Diseases (CIBERER), Madrid, Spain
15. Institute of Biology and Molecular Genetics, Universidad de Valladolid (IBGM-UVA), Valladolid, Spain
16. Oncology unit. Hospital clinico Universitario “Lozano Blesa”, Zaragoza, Spain
17. Human Genetics Group and Genotyping Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Centre, Spanish Network on Rare Diseases (CIBERER), Madrid, Spain
18. Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
19. Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The Netherlands
20. Department of Clinical Genetics, Academic Meical Center, Amsterdam, The Netherlands
21. Department of Clinical Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands
22. Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands
23. Department of Clinical Genetics and GROM, School for Oncology and Developmental Biology, MUMC, Maastricht, The Netherlands
24. Department of Clinical Genetics and GROM, School for Oncology and Developmental Biology, MUMC, Maastricht, The Netherlands
25. Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
26. Department of Clinical Genetics, Family Cancer Clinic, Erasmus University Medical Center, Rotterdam, The Netherlands
27. Department of Clinical Genetics, Family Cancer Clinic, Erasmus University Medical Center, Rotterdam, The Netherlands
28. Department of Medical Genetics, University Medical Center Utrecht, PO Box 8 AB, Utrecht, The Netherlands
29. Department of Genetics, University Medical Center, Groningen University, Groningen, The Netherlands
31. Genetic Medicine, Manchester Academic Health Sciences Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK
32. Clinical Genetics, Guy’s and St. Thomas’ NHS Foundation Trust, London, UK
33. Oncogenetics Team, The Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Kragujevac, UK
34. Yorkshire Regional Genetics Service, Leeds, UK
35. Ferguson-Smith Centre for Clinical Genetics, Yorkhill Hospitals, Glasgow, UK
36. West Midlands Regional Genetics Service, Birmingham Women’s Hospital Healthcare NHS Trust, Edgbaston, Birmingham, UK
37. Sheffield Clinical Genetics Service, Sheffield Children’s Hospital, Sheffield, UK
38. Department of Clinical Genetics, East Anglian Regional Genetics Service, Addenbrookes Hospital, Cambridge, UK
39. Institute of Genetic Medicine, Centre for Life, Newcastle Upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, UK
40. Department of Clinical Genetics, Royal Devon & Exeter Hospital, Exeter, UK
41. Medical Genetics Unit, St George’s, University of London, Kragujevac, UK
42. Northern Ireland Regional Genetics Centre, Belfast Health and Social Care Trust, and Department of Medical Genetics, Queens University Belfast, Belfast, UK
43. Oxford Regional Genetics Service, Churchill Hospital, Oxford, UK
44. All Wales Medical Genetics Services, University Hospital of Wales, Cardiff, UK
45. Clinical Genetics Department, St Michael’s Hospital, Bristol, UK
46. North West Thames Regional Genetics Service, Kennedy-Galton Centre, Harrow, UK
47. Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA
48. Clinical Molecular Genetics Laboratory, Fox Chase Cancer Center, Philadelphia, PA, USA
144. Unité INSERM U830, Institut Curie, Paris, France
145. Université Paris Descartes, Faculté de Médecine, Paris, France
49. Service de Génétique Oncologique, Institut Curie, Paris, France
146. Université Paris Descartes, Faculté de Pharmacie, Paris, France
50. Service de Génétique Oncologique, Institut Curie, Paris, France
51. Service de Génétique Oncologique, Institut Curie, 26 rue d’Ulm, Paris, France
52. Service de Génétique Oncologique, Institut Curie, Paris, France
53. INSERM U1052, CNRS UMR5286, Université Lyon 1, Centre de Recherche en Cancérologie de Lyon, Lyon, France
54. Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Hospices Civils de Lyon/Centre Léon Bérard, Lyon, France
55. Service de Génétique, Institut de Cancérologie Gustave Roussy, Villejuif, France and INSERM U946, Fondation Jean Dausset, Paris, France
56. Consultation de Génétique, Département de Médecine, Institut de Cancérologie Gustave Roussy, Villejuif, France
57. Département Oncologie génétique, Prévention et Dépistage, INSERM CIC-P9502, Institut Paoli-Calmettes/Université d’Aix-Marseille II, Marseille, France
58. Centre Antoine Lacassagne, Nice, France
59. Service de Génétique Clinique Chromosomique et Moléculaire, Centre Hospitalier Universitaire de St Etienne, St Etienne, France
60. Laboratoire de Génétique Chromosomique, H?tel Dieu Centre Hospitalier, BP 1125, Chambéry, France
61. Service de Génétique, Centre Hospitalier Universitaire Bretonneau, Tours, France
63. Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, UT, 84112, USA
64. Division of Population Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA
65. Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Surgery, Harvard Medical School, 27 Drydock Avenue, Boston, MA, 02210, USA
66. Department of Epidemiology, Columbia University, New York, NY, USA
67. Centre for Molecular, Environmental, Genetic and Analytic (MEGA) Epidemiology, Melbourne School of Population Health, Level 1, The University of Melbourne, 723 Swanston Street, VIC 3010, Kragujevac, Australia
68. Department of Epidemiology, Cancer Prevention Institute of California, 2201 Walnut Avenue, Suite 300, Fremont, CA, 94538, USA
69. Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Kragujevac, Australia
70. Department of Dermatology, University of Utah School of Medicine, 30 North 1900 East, SOM 4B454, Salt Lake City, UT, 84132, USA
71. Dept of OB/GYN and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria
72. Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
73. Department of Pathology, Landspitali, University Hospital, Reykjavik Iceland and Faculty of Medicine, University of Iceland, Reykjavik, Iceland
74. Epidemiology Research Program, American Cancer Society, Atlanta, GA, USA
75. Department of Environmental Medicine, NYU Cancer Institute, New York University School of Medicine, New York, NY, USA
76. Clinical Cancer Genetics Laboratory, Memorial Sloane Kettering Cancer Center, New York, NY, USA
77. Statistical and Data Center, Roswell Park Cancer Institute, Buffalo, NY, USA
78. Australia New Zealand (ANZGOG), Westmead Hospital, Sydney, Australia
79. Ohio State University, Columbus Cancer Council, Columbus, OH, USA
80. Evanston CCOP - NorthShore University Health System, University of Chicago, Chicago, IL, USA
81. Southern Pines Women’s Health Center, P.C., University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
82. Sarasota Memorial Healthcare, Tufts Medical Center, Sarasota, Florida, USA
83. Department of Molecular Virology, Immunology and Medical Genetics and Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
84. Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV - IRCCS, Padua, Italy
85. U.O.C. di Oncologia, ULSS5 Ovest Vicentino, Kragujevac, Italy
86. Laboratory of Molecular Oncology, N.N. Petrov Institute of Oncology, St.-Petersburg, Russia
87. Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA
88. Latvian Biomedical Research and Study Centre, Kragujevac, Latvia
89. Genetic Counselling Unit, Hereditary Cancer Program, IDIBELL-Catalan Institute of Oncology, Barcelona, Spain
90. Molecular Diagnostic Unit, Hereditary Cancer Program, IDIBELL-Catalan Institute of Oncology, Barcelona, Spain
91. Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
92. Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA
93. Women’s Cancer Program at the Samuel Oschin Comprehensive Cancer Institute at Cedars-Sinai Medical Center, Los Angeles, CA, USA
94. Department of Molecular Genetics, National Institute of Oncology, Budapest, Hungary
147. University Malaya Cancer Research Institute, University Malaya Medical Centre, Kuala Lumpur, Malaysia
95. Cancer Research Initiatives Foundation, Sime Darby Medical Centre, Kragujevac, Malaysia
96. Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, CA, USA
97. UCSF Cancer Risk Program, University of California, San Francisco, CA, USA
98. Cancer Genetics Laboratory, Department of Genetics, University of Pretoria, Kragujevac, South Africa
99. Oncogenetics Laboratory. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital, Barcelona, Spain
100. The Hong Kong Hereditary Breast Cancer Family Registry, The Universtiy of Hong K Cancer Genetics Center, Hong Kong Sanatorium and Hospital, Kragujevac, Hong Kong
101. Centre of Familial Breast and Ovarian Cancer, Department of Gynaecology and Obstetrics and Centre for Integrated Oncology (CIO), University hospital of Cologne, Cologne, Germany
102. Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany
103. Department of Gynaecology and Obstetrics, Division of Tumour Genetics, Klinikum rechts der Isar, Technical University Munich, Munich, Germany
104. Department of Gynaecology and Obstetrics, Ludwig-Maximilian University Munich, Munich, Germany
105. Department of Gynaecology and Obstetrics, University Hospital of Schleswig-Holstein, Campus Kiel, Christian-Albrechts University Kiel, Kiel, Germany
106. Institute of Human Genetics, University Hospital of Schleswig-Holstein, Campus Kiel, Christian-Albrechts University Kiel, Kiel, Germany
107. Department of Gynaecology and Obstetrics, University Hospital Düsseldorf, Heinrich-Heine University, Düsseldorf, Germany
108. Institute of Human Genetics, University of Münster, Münster, Germany
109. Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany
110. Institute of Human Genetics, Campus Virchov Klinikum, Charite Berlin, Germany
111. Department of Gynaecology and Obstetrics, University Hospital Ulm, Ulm, Germany
112. Centre of Familial Breast and Ovarian Cancer, Department of Medical Genetics, Institute of Human Genetics, University Würzburg, Würzburg, Germany
113. Institute of Human Genetics, Department of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany
114. Department of Gynaecology and Obstetrics, University Hospital Carl Gustav Carus, Technical University, Dresden, Germany
115. Institute of Human Genetics, University Regensburg, Regensbirg, Germany
116. Institute of Human Genetics, University Hospital Frankfurt a.M., Germany Molecular Oncology Laboratory, Hospital Clinico San Carlos, Madrid, Spain
117. Molecular Oncology Laboratory, Hospital Clinico San Carlos, Martin Lagos s/n, Madrid, Spain
118. Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Central Hospital, Biomedicum Helsinki, P.O. BOX 700, 00029 HUS, Helsinki, Finland
119. Faculty of Medicine - Medicine and Medical Specialties, Université de Montréal Hemato-oncology service, H?pital du Sacré-Coeur de Montréal, 5400 Gouin Blvd West Montreal, QC, Kragujevac, Canada
121. Department of Population Sciences, Beckman Research Institute of City of Hope, Duarte, CA, USA
122. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
123. Department of Medical Genetics, Mayo Clinic, Rochester, MN, USA
124. Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA
125. Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Preventive and Predicted Medicine, Fondazione IRCCS Istituto Nazionale Tumouri (INT), Milan, Italy
149. IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy
126. Unit of Medical Genetics, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale Tumouri (INT), Milan, Italy
127. Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia (IEO), Milan, Italy
128. Department of Experimental Oncology, Istituto Europeo di Oncologia, Milan, Italy
150. Consortium for Genomics Technology (Cogentech), Milan, Italy
129. Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico, IRCCS, Aviano (PN), Italy
130. Medical Genetics Unit, Department of Clinical Physiopathology, University of Florence, Firenze, Italy
131. Department of Molecular Medicine, “Sapienza” University of Rome, Rome, Italy
132. Clinical Genetics Branch, DCEG, NCI, Room EPS 7032, Rockville, MD, 20852, USA
133. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
151. Cancer Care Ontario, Departments of Molecular Genetics and Laboratory Medicine and Pathobiology, University of Toronto, ON, Kragujevac, Canada
134. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
152. Department of Laboratory Medicine and Pathobiology, University of Toronto, ON, Kragujevac, Canada
135. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
153. Department of Laboratory Medicine, and the Keenan Research Centre of the Li Ka Shing Knowledge Institute, St Michael’s Hospital, Toronto, ON, Canada
136. Ontario Cancer Genetics Network, Cancer Care Ontario, Toronto, ON, Canada
137. Department of Clinical Genetics, Odense University Hospital, Kragujevac, Denmark
138. Department of Clincial Genetics, Rigshospital and Copenhagen University, Kragujevac, Denmark
139. Department of Clinical Genetics, Skejby Hospital, Aarhus, Denmark
140. Department of Clinical Genetics, Vejle Hospital, Kragujevac, Denmark
141. Department of Laboratory Medicine and Pathology, Health Sciences Research, Mayo Clinic, Rochester, MN, USA
142. Cancer Genomics Laboratory, Centre Hospitalier Universitaire de Québec, Canada Research Chair in Oncogenetics, Department of Molecular Medicine, Faculty of Medicine, Laval University, 2705 Laurier Boulevard, T3-57, Quebec City, QC, Canada
... -内酰胺酶耐药决定基团的特异性峰值[24] ...
... 对革兰阴性杆菌外膜蛋白的蛋白组学和基因组学研究表明,膜孔蛋白、外排泵、脂多糖等细胞组分系统主要是通过对不同抗菌药物泵入和泵出的调节而表现耐药的[25] ...
... 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及2D电泳结合MALDI-TOF MS指纹图谱可以用来鉴定耐药和敏感菌株表达水平不同的外膜及周浆蛋白,建立一个综合的数据库来研究各种细胞组分,在耐药检测中发挥不同的作用[26,27] ...
... 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及2D电泳结合MALDI-TOF MS指纹图谱可以用来鉴定耐药和敏感菌株表达水平不同的外膜及周浆蛋白,建立一个综合的数据库来研究各种细胞组分,在耐药检测中发挥不同的作用[26,27] ...
... -内酰胺酶等[28,29] ...
. , :385-391
... -内酰胺酶等[28,29] ...
Anal. Chem.. ):
Po-Chiao Lin
Mei-Chun Tseng
Yu-Ju Chen
Chun-Cheng Lin
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan and Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan, and Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Functionalized magnetic nanoparticles (MNPs) were synthesized to serve as laser desorption/ionization elements as well as solid-phase extraction probes for simultaneous enrichment and detection of small molecules in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Two laser-absorbing matrices were each conjugated onto MNP to give MNP@matrix which provided high ionization efficiency and background-free detection in MS leading to unambiguous identification of target small molecules in a complex mixture. MNP@matrix was demonstrated to serve as a general matrix-free additive in MALDI-TOF MS analysis of structurally distinct small molecules. Also, MNP@matrix provides a simple, rapid, and reliable quantitative assay for small molecules by mass without either the use of an internal standard or an isotopic labeling tag. Furthermore, the affinity extraction of small molecules from complex biofluid was achieved by probe protein-conjugated MNP@matrix without laborious purification. We demonstrated that a nanoprobe-based assay is a cost-effective, rapid, and accurate platform for robotic screening of small molecules.
... LIN等通过方法学的改进,目前已成功检测出水杨酰胺等6种抗菌药物,未来这种方法有可能用于耐药机制及检测血样本中的微生物毒素和药物等[30] ...
质谱技术在临床微生物实验室中的应用前景}
1.微生物能够致病,能够造成食品、布匹、皮革等发霉腐烂,但微生物也有有益的一面.最早是弗莱明从青霉菌抑制其它细菌的生长中发现了青霉素,这对医药界来讲是一个划时代的发现.抗生素的使用在第二次世界大战中挽救了无数人的生命.2.通过基因组研究揭示微生物的遗传机制,发现重要的功能基因并在此基础上发展疫苗,开发新型抗病毒、抗细菌、真菌药物,将对有效地控制新老传染病的流行,促进医疗健康事业的迅速发展和壮大!&3.一直沿用至今天的巴斯德消毒法（60～65℃作短时间加热处理,杀死有害微生物的一种消毒法）和家蚕软化病问题的解决也是巴斯德的重要贡献,它不仅在实践上解决了当时法国酒变质和家蚕软化病的实际问题,而且也推动了微生物病原学说的发展,并深刻影响医学的发展.
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扫描下载二维码微生物在哪方面应用缺乏,有比较有前景
微生物学主要研究与医学有关的病原微生物的生物学性状、传染致病的机理、免疫学的基本理论、诊断技术和特异性防治措施等,以达到控制和消灭传染性疾病和与微生物有关的免疫性疾病,保障人类健康的目的,微生物学是一门...
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科研单位、医院中心实验室、大学教研室、CDC、微生物检验执法部门、微生物发酵工厂、甚至食品厂、各大分子生物学公司、诊断试剂公司等。我在网络购买试剂的时候，经常看见公司招聘专业人员解决他们的诊断试剂的克隆表达题目，国内的大的诊断试剂公司、生物公司非常多。这些都是微生物方面较好的发展方向，但是你要有足够的能力哦，最好有硕士以上的学历，希望采纳！...
医药，农业，食品（比如酿酒业）
医药比较好 貌似生物能源前景很好 要是能造出廉价生物燃料你就发了
微生物在环保领域，特别是优势的环保功能菌应用方面研究及应用前景好
扫描下载二维码导读：微生物在食品工业的应用，摘要：叙述了微生物与食品工业的关系，微生物在食品的应用，微生物在食品应用工业的发展前景，关键词：微生物食品工业发酵应用前景，微生物是所有形体微小、单细胞或者个体结构简单的多细胞以至没有细胞结构的低等生物的，微生物总类繁多、分布广、代谢类型多、代谢能力强、生长繁殖快、易培养、易变异、适应，使微生物与人类的关系非常密切，微生物不仅在自然界物资循环中起着非常重要的作用，而且在
微生物在食品工业的应用
摘要：叙述了微生物与食品工业的关系，微生物在食品的应用，微生物在食品应用工业的发展前景。
关键词：微生物
应用前景。
微生物是所有形体微小、单细胞或者个体结构简单的多细胞以至没有细胞结构的低等生物的总称。微生物总类繁多、分布广、代谢类型多、代谢能力强、生长繁殖快、易培养、易变异、适应能力强，正是上述特性，使微生物与人类的关系非常密切，微生物不仅在自然界物资循环中起着非常重要的作用，而且在食品工业的应用中也非常广泛。本文叙述了微生物在食品工业中的应用，讨论了微生物的广阔发展前景。
微生物与食品工业的关系
随着人们对微生物认识的不断深入，微生物已被广泛应用于食品生产。今天基因工程、固定化酶、固定化细胞等先进技术的应用，进一步发掘了微生物在食品工业中的巨大发展潜能。微生物在食品工业生产中有非常大的好处，例如可以制作面包，酒；霉菌可制作豆酱、酱油；乳酸菌可制作泡菜、酸奶等；当然也有危害，我们要充分利用微生物有利的方面为食品工业服务，消除器有害影响，为人类造福。 二
微生物在食品生产中的应用
食醋是我国劳动人民在长期的生产实践中制造出来的一种酸性调味品。它能增进食欲，帮助消化，在人们饮食生活中不可缺少。在我国的中医药学中醋也有一定的用途。全国各地生产的食醋品种较多。著名的山西陈醋、镇江香醋、四川麸醋、东北白醋、江浙玫瑰米醋、福建红曲醋等是食醋的代表品种。食醋按加工方法可分为合成醋、酿造醋、再制醋三大类。其中产量最大且与我们关系最为密切的是酿造醋，它是用粮食等淀粉质为原料，经微生物制曲、糖化、酒精发酵、醋酸发酵等阶段酿制而成。其主要成分除醋酸(3%～5%)外，还含有各种氨基酸、有机酸、糖类、维生素、醇和酯等营养成分及风味成分，具有独特的色、香、味。它不仅是调味佳品，长期食用对身体健康也十分有益。
面包是产小麦国家的主食，几乎世界各国都有生产。它是以面粉为主要原料，以酵母菌、糖、油脂和鸡蛋为辅料生产的发酵食品，其营养丰富，组织蓬松，易于消化吸收，食用方便，深受消费者喜爱。酵母是生产面包必不可少的生物松软剂。面包酵母是一种单细胞生物，属真菌类，学名为啤酒酵母。面包酵母有圆形、椭圆形等多种形态。以椭圆形的用于生产较好。酵母为兼性厌氧性微生物，在有氧及无氧条件下都可以进行发酵。
我国是一个酒类生产大国，也是一个酒文化文明古国，在应用酵母菌酿酒的领域里，有着举足轻重的地位。许多独特的酿酒工艺在世界上独领风骚，深受世界各国赞誉，同时也为我国经济繁荣作出了重要贡献。
酿酒具有悠久的历史，产品种类繁多如：黄酒、白酒、啤酒、果酒等品种。而且形成了各种类型的名酒，如绍兴黄酒、贵州茅台酒、青岛啤酒等。酒的品种不同，酿酒所用的酵母以及酿造工艺也不同，而且同一类型的酒各地也有自己独特的工艺。
啤酒是以优质大麦芽为主要原料，大米、酒花等为辅料，经过制麦、糖化、啤酒酵母发酵等工序酿制而成的一种含有C02、低酒精浓度和多种营养成分的饮料酒。它是世界上产量最大的酒种之一。
生产用霉菌菌种：淀粉的糖化、蛋白质的水解均是通过霉菌产生的淀粉酶和蛋白质水解酶进行的。通常情况是先进行霉菌培养制曲。淀粉、蛋白质原料经过蒸煮糊化加入种曲，在一定温度下培养，曲中由霉菌产生的各种酶起作用，将淀粉、蛋白质分解成糖、氨基酸等水解产物。
酱类包括大豆酱、蚕豆酱、面酱、豆瓣酱、豆豉及其加工制品，都是由一些粮食和油料作物为主要原料，利用以米曲霉为主的微生物经发酵酿制的。酱类发酵制品营养丰富，易于消化吸收，即可作小菜，又是调味品，具有特有的色、香、味，价格便宜，是一种受欢迎的大众化调味品。
用于酱类生产的霉菌主要是米曲霉（Asp.oryzae），生产上常用的有沪酿3.042，黄曲霉Cr-1菌株（不产生毒素），黑曲霉（Asp. Nigerf-27）等。所用的曲霉具有较强的蛋白酶、淀粉酶及纤维素酶的活力，它们把原料中的蛋白质分解为氨基酸，淀粉变为糖类，在其他微生物的共同作用下生成醇、酸、酯等，形成酱类特有的风味。
4.1.酱油：
酱油生产中常用的霉菌有米曲霉、黄曲霉和黑曲霉等，应用于酱油生产的曲霉菌株应符合如下条件：不产黄曲霉毒素；蛋白酶、淀粉酶活力高，有谷氨酰胺酶活力；生长快速、培养条件粗放、抗杂菌能力强；不产生异味，制曲酿造的酱制品风味好。
L-谷氨酸钠俗称味精。L-谷氨酸发酵生产菌种主要有棒状杆菌、短杆菌属、小杆菌属的细菌。L-谷氨酸钠发酵工艺主要有4个阶段：①摇瓶种子培养。即采用适合的液体培养基培养菌种。②二级种子培养。③L-谷氨酸发酵生产。④L-谷氨酸钠的提取。在L-谷氨酸发酵生产时期的谷氨酸合成阶段，菌体浓度基本不变，糖与尿素分解后产生的α-酮戊二酸和氨主要用于合成谷氨酸。此阶段应为菌种提供生长最适宜的条件，如及时添加尿素，调节最适PH7.2~7.4，提高温度到谷氨酸合成的最适温度并大量通气，保证菌体的有氧呼吸，使它大量产生代谢产物提高产量。
发酵生产柠檬酸。柠檬酸发酵主要采用的菌种是黑曲霉。黑曲霉能利用淀粉分解出代谢产物柠檬酸。柠檬酸发酵工艺流程为：种子的扩大培养→柠檬酸的发酵。
微生物在食品工业的前景
1.固定化酶在食品工业中的应用
1.1.应用于食品酶制剂的生产
利用基因工程技术，不但可以成倍地提高酶的活力，而且还可将生物酶基因克隆到微生物中，构建基因工程菌来生产酶。转基因微生物生产酶有许多优点，如产量高、品质均一、稳定性好、价格低等。第一个应用于食品的基因工程酶为凝乳酶，它是制造干酪过程中起凝乳作用的关键酶，传统来源是从小牛皱胃液中提取，造成全球性小牛短缺，酶成本不断提高。人们曾经尝试用微生物代用品或其他动物来源的凝乳酶，但效果均不理想；基因工程解决了这一难题。近20 年来用基因工程菌发酵生产的食品酶制剂主要有：凝乳酶、α－淀粉酶、葡萄糖氧化酶、葡萄糖异构酶、转化酶、普鲁多糖酶( 茁霉多糖酶) 、脂肪酶、α－半乳糖苷酶、β－半乳糖苷酶、α－乙酰乳酸脱羧酶、溶菌酶、碱性蛋白酶等。
1.2.固定化葡萄糖异构酶在高果糖浆生产中的应用
固定化葡萄糖异构酶是世界上生产规模最大的一种固定化酶，1973年就已应用在工业化生产， 它可以用来催化玉米糖浆和淀粉生产高甜度的高果糖糖浆。
2.酶制剂在食品保鲜方面的应用
随着人们对食品的要求不断提高和科学技术的不断进步，一种崭新的食品保鲜技术―酶法保鲜技术正在崛起。酶法保鲜技术是利用生物酶的高效的催化作用，防止或消除外界因素对食品的不良影响，从而保持食品原有的优良品质和特性的技术。由于酶具有专一性强、催化效率高、作用条件温和等特点，可广泛地应用于各种食品的保鲜，有效地防止外界因素，特别是氧化和微生物对食品所造成的不良影响。
葡萄糖氧化酶是一种氧化还原酶，它可催化葡萄糖和氧反应，生成葡萄糖酸和双氧水。将葡萄糖氧化酶与食品一起置于密封容器中，在有葡萄糖存在的条件下，该酶可有效地降低或消除密封容器中的氧气，从而有效地防止食品成分的氧化作用，起到食品保鲜作用。
3.作为畜禽饲料添加剂
中国是蛋白原料缺乏的国家，随着饲料工业的迅速发展和生产的高度集约化，对优质饲料蛋白原料的需求日趋增大，目前饲料优质蛋白原料的主要来源是鱼粉，而作为一种亚稀缺资源，鱼粉已经在各主要产地如秘鲁等国受到严格限产保护。需求的膨胀和来源的快速减少，正是目前饲料优质蛋白原料面临的尴尬处境。一些西方发达国家先行一步，将解决优质饲料蛋白来源的目光投向了生物技术产品―单细胞蛋白。
4.生产SCP的微生物
在工业生产中，作为蛋白质资源的微生物菌体，特别的酵母菌和细菌，它们都能利用糖类原料生产菌体蛋白，究竟采用酵母菌和细菌哪种更好呢？这在很大程度上取决于生产SCP的原料。在20世纪60年代末和70年代初期，开发了多种由烷烃类物质产生的SCP工艺，能够利用烷烃的微生物主要有细菌和放线菌，如产碱杆菌、假单孢菌、节杆菌、短杆菌等，其次为酵母菌属。
结束语：随着生物技术的飞速发展,利用基因工程对微生物进行菌种改造从根本上解决发酵食品生产工艺中的问题,已在食品工业中开展试用。中科院微生 物所与青岛啤酒集团正在联合研制构建具有熟化期短、絮凝性强等优良特性,又保持原有口味的青岛啤酒酵母工程菌。将生物技术应用到食品工业必将对中国食品行业的发展产生重要的影响。功能性食品及食品添加剂等各方面的生产都将与微生物紧密相连，微生物将为食品工业的发展开辟更广泛的前景。微生物在食品工业中的应用也远远不仅如此，今后将会发挥越来越重要的作用。
总而言之，我们应该展望未来，食品工业将成为现代生物技术应用最广阔、最活跃、最富有挑战性的领域。要充分利用世界生物技术迅猛发展的契机，重视
发酵工程技术的研究，促进我国食品工业的改革，实现我国食品工业健康有序的发展。
[1] 邓紫筠. 微生物在食品工业中的应用[J]. 农业与技术， ，（3）：15-17.
[2] 郑华学．试论生物技术在我国食品工业中的应用[J]．2003，(5)：99―102．
[3] 姬德衡．发酵工程在功能食品开发中的应用[J]．食品技术，2002，(7)：9-13.
[4] 何国庆．食品发酵与酿造工艺学[M]．北京:中国农业出版社，．
[5] 金滨锋．现代生物技术在食品工业中的应用[J]．生物技术世界，2007，(8)：36-39．
[6] 史先振．现代发酵工程技术在食品领域的应用研究进展[J].中国酿造，2005，(2)：1 ―4．
包含总结汇报、IT计算机、考试资料、旅游景点、党团工作、教学教材、计划方案、出国留学、外语学习、文档下载以及微生物在食品方面的应用等内容。
相关内容搜索质谱技术在临床微生物实验室中的应用前景
罗燕萍. 质谱技术在临床微生物实验室中的应用前景.检验医学,): 97-100LUO Yanping. The prospects of mass spectrometry application on microbiology. Labratory Medicine,): 97-100&&
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质谱技术在临床微生物实验室中的应用前景
解放军总医院微生物科,北京 100853
作者简介:罗燕萍,女,1960年生,硕士,主任技师,主要从事临床微生物耐药机制研究。
目前应用于临床微生物实验室的基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS) 是一种直接从完整细菌中获得蛋白指纹图谱,从而对细菌进行快速鉴定的方法,由于可以对常见细菌,诺卡菌、分枝杆菌、厌氧菌、酵母菌及丝状真菌进行快速和准确的鉴定,近年来备受推崇。该文对这次质谱专题刊登的1篇综述和4篇论著进行总结评价。相信随着相关研究的不断深入,这项技术在临床微生物实验室将有更广阔的应用空间。
基质辅助激光解析电离飞行时间质谱;
临床微生物实验室
中图分类号:R446.5
文献标志码:A
文章编号:15)02-0097-04
The prospects of mass spectrometry application on microbiology
LUO Yanping
Department of Microbiology, Chinese PLA General Hospital , Beijing 100853, China
The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) technique can be used to generate protein fingerprint signatures from whole bacterial cells. By comparing these fingerprints to a database of reference spectra by the various algorithms, bacteria can be rapidly identified. MALDI-TOF MS can be used for accurate and rapid identification of various microorganisms, such as common bacteria, Nocardia, Mycobacterium, anaerobe, yeast and filamentous fungi. MALDI-TOF MS have recently been widely introduced. The characteristics of one review and four papers published in the special subject about MALDI-TOF MS were reviewed. The development of studies in MALDI-TOF MS will play an important role in the application of mass spectrometry in clinical microbiological laboratories.
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry;
Bacteria identification;
Clinical microbiology laboratory
引言自20世纪80年代起, 质谱技术就已经成为科学研究中用于蛋白分析的强大工具。随着技术的不断成熟和广泛使用, 其在微生物检验常规诊断中的作用越来越受到关注, 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)已经进入临床微生物实验室用于病原菌鉴定, 与传统的表型鉴定及分子生物学技术相比, MALDI-TOF MS快速、准确、成本低廉。本刊本期集中刊登“ 质谱技术在临床微生物检验中的应用” 专题, 旨在使临床和实验室工作者对其有一个全面的认识和了解。一、MALDI-TOF MS目前的应用现状传统的细菌鉴定流程通常是:根据细菌在不同培养基上的生长情况和菌落形态, 应用一些简便、快速的试验如革兰染色、触酶、氧化酶等进行初步分类, 然后通过手工生化试验或自动鉴定系统等来完成鉴定[, ]。虽然有些试验几分钟即可完成, 但大部分情况下, 完成常规细菌鉴定至少需要8~18 h或更长的时间(如苛养菌)。分子生物学如聚合酶链反应(polymerase chain reactuin, PCR)、DNA 芯片、微阵列技术等方法虽然敏感性高, 但费用高昂, 且对操作人员、设备和环境有较严格的要求, 不适合常规应用。MALDI-TOF MS是将完整的病原菌细胞(intact-cell, IC)直接进行检测, 无需蛋白提纯, 样本准备很简便, 也有人称之为 IC MALDI-TOF MS。许多研究表明, IC MALDI-TOF MS敏感性很高, 可以区分表型相似甚至相同的菌株, 提供属、种、型水平的鉴定。目前报道使用质谱进行微生物鉴定方面的问题主要集中在质谱数据库容量和有些特定种属无法明确区分上。二、质谱专题收录论文的主要内容此次专题共刊登1篇综述和4篇论著。综述文章“ MALDI-TOF MS在临床微生物检验中的应用” , 从MADLI-TOF MS的基本原理、在微生物鉴定中的应用、在科研中的应用等三方面比较全面地介绍了目前MALDI-TOF MS在临床微生物实验室应用的现状及前景。质谱技术对以葡萄球菌和链球菌为代表的革兰阳性球菌鉴定到种水平的准确率> 99%, 对肠杆菌科细菌鉴定到种水平的准确率为96.60%, 非发酵菌为98.75%, 结核分枝杆菌为94.90%, 非结核分枝杆菌为94.60%, 对以拟杆菌属为代表的厌氧菌可以达到97.50%, 以酵母菌为主的真菌为97.30%。对临床常见分离菌鉴定到种水平的准确率很高, 使质谱技术在临床微生物实验室中得到广泛应用。另外, 从阳性血培养和尿液样本中直接鉴定病原菌作为一个研究方向也在大量的研究尝试之中, 尚未完全应用于常规鉴定。利用特异峰值的分析对耐药基因、血清分型、毒力等的研究也在不断进行中。本专题中“ MALDI-TOF MS在中段尿样本细菌直接检测中的应用” 一文, 应用MALDI-TOF MS快速、直接检测中段尿样本中的细菌, 尝试建立中段尿快速细菌检测和鉴定方法。其结论与国外相关研究相似:涂靶板之前必须将尿液样本进行离心处理; 较高的细菌计数可以提高鉴定到种的正确率; 如果是2种以上细菌混合感染, 鉴定结果是否正确取决于2种细菌的比例。该方法可比常规尿培养鉴定方法提早40 h左右报告鉴定结果。血流感染是一种严重的感染性疾病, 当血培养瓶报告阳性时, 微生物实验室只能给临床医师暂报涂片结果, 较快明确病原菌是微生物实验室亟待解决的课题。目前国内、外很多学者尝试分子生物学方法如实时荧光定量PCR以及其它一些快速方法如微阵列技术、杂交探针、流式细胞术等直接从血培养阳性瓶中鉴定细菌。近几年还有一些研究使用MALDI-TOF MS直接从阳性瓶中鉴定细菌。该方法首先最重要的一步是将细菌从细胞组分中分离出来, FERRONI等[]在样本中环节做了很大的改进, 血培养瓶报阳性后, 应用温和去污剂裂解细胞膜, 这一步只需要几分钟, 从而使整个鉴定过程缩短到不足30 min。本专题中“ 分离胶促凝管联合 MALDI-TOF MS直接检测血培养阳性细菌” 一文, 采用分离胶促凝管预处理方法, 其优点是成本低廉, 速度快(平均15 min/样本), 安全性好, 没有过多的转移、洗涤等工序, 不容易引起气溶胶等生物安全风险。缺点是纯度不佳, 由于没有额外的洗涤步骤, 最终得到的菌体富集物始终还是含有部分细胞碎片, 其对革兰阳性菌和革兰阴性菌的检出率分别为80.10%和86.90%; 联合法对念珠菌的检出率较低, 仅为22.20%, 还有进一步提升和改进的空间, 期待通过方法学的改进使质谱技术直接实时鉴定血培养阳性瓶中细菌成为可能。有学者将MALDI-TOF MS 与其它方法做比对进行细菌鉴定, 得出的结论是质谱技术对常规分离细菌的鉴定正确率相对于其它方法而言较高。“ MicroflexTM MALDI-TOF MS和Vitek 2 Compact全自动微生物分析系统对肠杆菌科细菌鉴定能力的比较” 一文, 将关注点放在肠杆菌科细菌上, 这2种仪器对细菌种的鉴定符合率分别为97.10%和83.30%, 对于阴沟肠杆菌复合群内的阴沟肠杆菌、霍氏肠杆菌和路德维希肠杆菌, 普通变性杆菌复合群中的普通变形杆菌和潘氏变形杆菌, 液化沙雷菌群中的液化沙雷菌, 小肠结肠耶尔森菌群中的小肠结肠耶尔森菌, 质谱技术通常可以鉴定到种水平, 而后者只能鉴定到群, 印证了质谱技术对于常规分离细菌鉴定到种的正确率较高的结论。“ 两种MALDI-TOF MS系统快速鉴定血培养中白念珠菌以外酵母样真菌” 一文, 比较了2种不同MALDI-TOF MS系统对白念珠菌以外酵母样真菌的鉴定率, 目前市场上有不同厂家的质谱仪尽管原理相似, 但各有自己的特点, 该文的结论指出Autoflex MALDI-TOF MS 对念珠菌尤其是近平滑念珠菌复合体鉴定的分辨率较高, 但本研究对比的菌株数略少(59株, 包括11种真菌), 结论需要更大、更多样化样本的支持。如在LACROIX等[]的两种质谱技术在念珠菌鉴定的研究中, 样本量达到1 383株, 最终2种方法的鉴定正确率均为98.30%, 相差并不显著。本次质谱专题收录的文章主要涉及到鉴定方法的比对和从血培养阳性及尿液样本中直接检测鉴定细菌, 这也是近几年国内外相关研究的热点, 我们也期待有更多质谱应用方面的研究, 推动质谱技术在临床微生物领域的应用范围进一步扩大。三、MALDI-TOF MS未来的发展方向近年来, 越来越多的临床微生物实验室开始引入MALDI-TOF MS进行常规样本的细菌和真菌鉴定, 随着研究的不断深入, MALDI-TOF MS的应用领域也将变得越来越广泛, 主要有3个方面:在流行病学中的应用、从患者样本中直接检测病原菌和对耐药机制的检测。(一)流行病学微生物实验室、感染控制人员和临床医师面临的挑战之一是在某个临床菌株暴发流行时可以得到菌株代表分类的特异数据, 如沙门菌、链球菌等, 为了准确得到血清型、亚型或其它分类, 实验室必须耗费更多的时间做进一步检测, 而且许多菌株分型需要特殊的试验方法、仪器、耗材等。因此, 条件有限的实验室就需要外送做检测, 可能导致检测时间延长、丢失重要的流行病学数据、增加检测费用、结果不准确等。但如果在微生物实验室内快速、准确完成重要数据的结果将会帮助临床医师、护士、感控人员和卫生机构处理、追踪感染病原菌并掌握其流行状况。目前, 通过大量菌株的群、属、种分析, 以及许多菌株分型研究的深入, MALDI-TOF MS所具备的鉴定这些高度相关菌株的能力将使临床微生物实验室的影响力提升。将来可能出现更加精密的MALDI-TOF MS仪器, 不但可以鉴定病原菌, 同时可以快速、准确地提供菌株的流行病学数据, 这将使临床微生物实验室在医院感染控制、病原菌暴发、耐药菌国家监测及生物防御等领域扮演重要角色。(二)MALDI-TOF MS直接从患者样本鉴定细菌因为不需要对目标物质进行扩增以及其鉴定病原菌的高敏感性, MALDI-TOF MS直接从样本中检测细菌的能力可能取代PCR。通过对这些患者样本处理方法的改良, 主要包括去除蛋白、核酸、细胞碎片等可以影响分析的组分, MALDI-TOF MS直接从样本中进行细菌鉴定的能力会进一步提高。以下是直接从不同样本类型中鉴定的报道。1.尿液样本目前的研究表明, 对于计数> 105的尿液样本, 通过低速离心去除细胞碎片和白细胞, 高速离心得到细菌, 再用甲酸和乙腈处理得到蛋白用于质谱分析, 鉴定准确率> 90%[, ]。存在的问题是MALDI-TOF MS尚不能对混合感染的尿液进行准确鉴定; 没有标准化的尿液前期处理流程。而上述的处理流程是目前被证实较简便、快速且保证质谱鉴定准确的方法[, ]。总之, MALDI-TOF MS在单一细菌的尿液样本中直接检测具有很强大的、准确的鉴定能力。2.脑脊液细菌性脑膜炎是临床最严重的感染之一, 快速、准确的检测非常重要, 目前通过MALDI-TOF MS直接检测脑脊液细菌的研究较少, 有一份报道肺炎链球菌引起的脑膜炎, 前期处理流程与尿液样本相似[]。3.血培养阳性瓶中直接鉴定目前有许多从阳性血培养瓶中准确鉴定病原菌的方法, 但由于使用的软件不同, 血培养瓶(培养系统)不同, 因此, 无法客观地比较这些方法的优劣。有的研究认为提取细胞蛋白优于完整细胞分析, 无碳颗粒的血培养瓶会得到准确的鉴定率, 而含有碳颗粒的血培养瓶则结果不好[, , , ]。总之, 从血培养瓶中直接检测细菌和真菌可以显著地缩短鉴定时间[, ], 随着研究的深入, 这些检测方法也会进一步改进, 以获得更方便可行的操作和更高的敏感性, 提高革兰阳性菌及念珠菌等病原菌的检出能力。(三)耐药性检测微生物实验室不但要提供精确的病原菌鉴定结果, 同时还要检测其敏感性, 为临床医生提供治疗依据。常规的药物敏感性试验方法比较费时, 一些酶联免疫、凝集等方法只能局限于少数细菌。MALDI-TOF MS可为耐药基因检测提供一个很好的平台, 可以分析几乎所有的耐药机制, 目前报道的方法主要基于下列几种原理:分析抗菌药物及被修饰后的产物; 分析细胞组分; 分析核糖体DNA甲基化; 检测突变等。1.通过抗菌药物的变化检测酶活性如直接检测β -内酰胺酶活性, 检测方法基本相似:收集新鲜培养的待测细菌, 与β -内酰胺分子反应后, 用MALDI-TOF MS检测β -内酰胺分子及其降解产物的特异峰[, ]。有文献报道通过检测美罗培南及其降解产物, 证实了30株产IPM-7、VIM-2的铜绿假单胞菌, 产VIM-1、KPC-2、NDM-1的肠杆菌产生碳青霉烯酶, 其敏感性和特异性均超过95%。检测β -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[, , , , ]。2.MALDI-TOF MS直接检测耐药决定基团对多耐药细菌蛋白组学的研究可以建立耐药机制相关的主要蛋白指纹图谱, 最近一种检测耐万古霉素肠球菌的方法已经被证实有效。这种方法可以在微生物实验室用于vanB阳性屎肠球菌的快速检测[]。用MALDI-TOF MS区分耐甲氧西林金黄色葡萄球菌和甲氧西林敏感金黄色葡萄球菌时有一定挑战, 尽管有相关研究, 但方法需要进一步优化和验证[]。到目前为止, 还没有一种方法可以检测其它的如β -内酰胺酶耐药决定基团的特异性峰值[]。3.分析细胞壁组分几乎一半以上的抗菌药物的作用靶位位于细胞壁上, 因此, 细菌细胞壁在抗菌药物耐药中的重要性不言而喻。其它抗菌药物则需要越过细胞壁屏障到达它们的作用靶点。对革兰阴性杆菌外膜蛋白的蛋白组学和基因组学研究表明, 膜孔蛋白、外排泵、脂多糖等细胞组分系统主要是通过对不同抗菌药物泵入和泵出的调节而表现耐药的[]。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及2D电泳结合MALDI-TOF MS指纹图谱可以用来鉴定耐药和敏感菌株表达水平不同的外膜及周浆蛋白, 建立一个综合的数据库来研究各种细胞组分, 在耐药检测中发挥不同的作用[, ]。4. 检测耐药机制MALDI-TOF MS通过分析DNA来检测耐药机制, 如可以通过检测单核苷酸多态性来鉴定SHV-型、TEM-型超广谱β -内酰胺酶等[, ]。5.抗菌药物检测MALDI-TOF MS检测的相对分子质量范围较大, 而抗菌药物的相对分子质量通常< 1 000因此, 基质和较高的背景干扰使得这项技术在检测抗菌药物时变得复杂。LIN等通过方法学的改进, 目前已成功检测出水杨酰胺等6种抗菌药物, 未来这种方法有可能用于耐药机制及检测血样本中的微生物毒素和药物等[]。随着质谱数据库及分析软件的不断更新, 所有病原菌鉴定的时间都会缩短。尽管一些方法目前还不能常规应用于微生物实验室, 但随着这些检测方法的不断优化和标准化, 一定会有广阔的应用前景。实验技术不断发展, 使得临床微生物实验室从曾经最慢速服务的实验室逐步转变为一个可以快速、准确为患者提供结果的充满活力的新实验室。我们相信MALDI-TOF MS在未来一定会在临床微生物实验室扮演更重要的角色。
The authors have declared that no competing interests exist.
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... 一、MALDI-TOF MS目前的应用现状传统的细菌鉴定流程通常是:根据细菌在不同培养基上的生长情况和菌落形态,应用一些简便、快速的试验如革兰染色、触酶、氧化酶等进行初步分类,然后通过手工生化试验或自动鉴定系统等来完成鉴定[1,2] ...
... 一、MALDI-TOF MS目前的应用现状传统的细菌鉴定流程通常是:根据细菌在不同培养基上的生长情况和菌落形态,应用一些简便、快速的试验如革兰染色、触酶、氧化酶等进行初步分类,然后通过手工生化试验或自动鉴定系统等来完成鉴定[1,2] ...
... 该方法首先最重要的一步是将细菌从细胞组分中分离出来,FERRONI等[3]在样本中环节做了很大的改进,血培养瓶报阳性后,应用温和去污剂裂解细胞膜,这一步只需要几分钟,从而使整个鉴定过程缩短到不足30 min ...
. ):153-158
1 Service de Mycologie-Parasitologie, H&pital Saint-Louis AP-HP, Universit& Paris Diderot Paris 7, Paris, France 2 Laboratoire de Mycologie-Parasitologie CHU, Nantes, France 3 Laboratoire de Mycologie-Parasitologie CHRU, Inserm U995, Lille, France 4 Service de Microbiologie, H&pital Necker-Enfants Malades AP-HP, Universit& Paris-Descartes, Paris, France 5 Laboratoire de Mycologie-Parasitologie CHU, Bordeaux, France 6 Laboratoire de Mycologie-Parasitologie CHU, Tours, France 7 Laboratoire de Mycologie-Parasitologie CHU, Poitiers, France 8 Laboratoire de Mycologie-Parasitologie H&pital Saint-Antoine AP-HP, Paris, France
Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas& and Bruker Biotyper&, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level ( for Andromas&;
for Bruker Biotyper&) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.
... 如在LACROIX等[4]的两种质谱技术在念珠菌鉴定的研究中,样本量达到1 383株,最终2种方法的鉴定正确率均为98 ...
... 90%[5,6] ...
... 90%[5,6] ...
... 而上述的处理流程是目前被证实较简便、快速且保证质谱鉴定准确的方法[6,7] ...
. , :231-235
... 而上述的处理流程是目前被证实较简便、快速且保证质谱鉴定准确的方法[6,7] ...
... 细菌性脑膜炎是临床最严重的感染之一,快速、准确的检测非常重要,目前通过MALDI-TOF MS直接检测脑脊液细菌的研究较少,有一份报道肺炎链球菌引起的脑膜炎,前期处理流程与尿液样本相似[8] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 有的研究认为提取细胞蛋白优于完整细胞分析,无碳颗粒的血培养瓶会得到准确的鉴定率,而含有碳颗粒的血培养瓶则结果不好[9,10,11,12] ...
... 总之,从血培养瓶中直接检测细菌和真菌可以显著地缩短鉴定时间[13,14],随着研究的深入,这些检测方法也会进一步改进,以获得更方便可行的操作和更高的敏感性,提高革兰阳性菌及念珠菌等病原菌的检出能力 ...
Background With long delays observed between sampling and availability of results, the usefulness of blood cultures in the context of emergency infectious diseases has recently been questioned. Among methods that allow quicker bacterial identification from growing colonies, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was demonstrated to accurately identify bacteria routinely isolated in a clinical biology laboratory. In order to speed up the identification process, in the present work we attempted bacterial identification directly from blood culture bottles detected positive by the automate. Methodology/Principal Findings We prospectively analysed routine MALDI-TOF identification of bacteria detected in blood culture by two different protocols involving successive centrifugations and then lysis by trifluoroacetic acid or formic acid. Of the 562 blood culture broths detected as positive by the automate and containing one bacterial species, 370 (66%) were correctly identified. Changing the protocol from trifluoroacetic acid to formic acid improved identification of Staphylococci , and overall correct identification increased from 59% to 76%. Lack of identification was observed mostly with viridans streptococci, and only one false positive was observed. In the 22 positive blood culture broths that contained two or more different species, only one of the species was identified in 18 samples, no species were identified in two samples and false species identifications were obtained in two cases. The positive predictive value of bacterial identification using this procedure was 99.2%. Conclusions/Significance MALDI-TOF MS is an efficient method for direct routine identification of bacterial isolates in blood culture, with the exception of polymicrobial samples and viridans streptococci. It may replace routine identification performed on colonies, provided improvement for the specificity of blood culture broths growing viridans streptococci is obtained in the near future.
... 总之,从血培养瓶中直接检测细菌和真菌可以显著地缩短鉴定时间[13,14],随着研究的深入,这些检测方法也会进一步改进,以获得更方便可行的操作和更高的敏感性,提高革兰阳性菌及念珠菌等病原菌的检出能力 ...
... -内酰胺分子及其降解产物的特异峰[15,16] ...
... -内酰胺分子及其降解产物的特异峰[15,16] ...
. ):193-204
1.Chinese Academy of Sciences National Chromatographic R & A Center, Dalian Institute of Chemical Physics Dalian 116023 People’s Republic of China Dalian 116023 People’s Republic of China
Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) is widely used in a variety of fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range for obtaining molecular weights and for structural characterization of macromolecules. In this article we summarize recent developments in matrix additives, new matrices, and sample-pretreatment methods using off-probe or on-probe techniques or nanomaterials for MALDI-TOF-MS analysis of biological samples.
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
. ):e31676
Rapid detection of carbapenem-resistant Acinetobacter baumannii strains is critical and will benefit patient care by optimizing antibiotic therapies and preventing outbreaks. Herein we describe the development and successful application of a mass spectrometry profile generated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) that utilized the imipenem antibiotic for the detection of carbapenem resistance in a large series of A. baumannii clinical isolates from France and Algeria. A total of 106 A. baumannii strains including 63 well-characterized carbapenemase-producing and 43 non-carbapenemase-producing strains, as well as 43 control strains (7 carbapenem-resistant and 36 carbapenem-sensitive strains) were studied. After an incubation of bacteria with imipenem for up to 4 h, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. The presence and absence of peaks representing imipenem and its natural metabolite was analyzed. The result was interpreted as positive for carbapenemase production if the specific peak for imipenem at 300.0 m/z disappeared during the incubation time and if the peak of the natural metabolite at 254.0 m/z increased as measured by the area under the curves leading to a ratio between the peak for imipenem and its metabolite being A. baumannii clinical isolates, showed a sensitivity of 100.0% and a specificity of 100.0%. Our study is the first to demonstrate that this quick and simple assay can be used as a routine tool as a point-of-care method for the identification of A. baumannii carbapenemase-producers in an effort to prevent outbreaks and the spread of uncontrollable superbugs.
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
J. Proteome Res.. ):79&84
Gero P. Hooff
Jeroen J. A. van Kampen
Roland J. W. Meesters
Alex van Belkum
Wil H. F. Goessens
Theo M. Luider
+ Department of Neurology, Laboratory of Neuro-Oncology and Clinical and Cancer Proteomics, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands
? Department of Microbiology and Infectious Diseases, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands *G. P. Hooff, Laboratory of Neuro-Oncology and Clinical and Cancer Proteomics, Department of Neurology, University Medical Center Rotterdam (Erasmus MC), Dr. Molewaterplein 50, Room Ae-307, 3015 GE Rotterdam, The Netherlands. Phone: + . Fax: + . E-mail: g.hooff@erasmusmc.nl .
Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in Gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry.
... -内酰胺酶尤其是碳青霉烯酶的方法已经应用于一些常规实验室[17,18,19,20,21] ...
... 这种方法可以在微生物实验室用于vanB阳性屎肠球菌的快速检测[22] ...
. , :81-86
... 用MALDI-TOF MS区分耐甲氧西林金黄色葡萄球菌和甲氧西林敏感金黄色葡萄球菌时有一定挑战,尽管有相关研究,但方法需要进一步优化和验证[23] ...
. 2012, :-
1. Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Worts Causeway, Cambridge, CB1 8RN, UK
2. Cancer Genomics Laboratory, Centre Hospitalier Universitaire de Québec, 2705 Laurier Boulevard, T3-57, Quebec City, QC, Canada
3. Genetics and Population Health Division, Queensland Institute of Medical Research, 300 Herston Rd, Herston, Brisbane, QLD 4006, Australia
143. INSERM U1052, CNRS UMR5286, Université Lyon 1, Cancer Research Center of Lyon, 28 rue La?nnec, Lyon, 69373, France
4. Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Hospitalier Universitaire de Lyon/Centre Léon Bérard, 28 rue La?nnec, Lyon, 69373, France
5. Section of Genetic Oncology, Dept. of Laboratory Medicine, University and University Hospital of Pisa, Via Roma 57, 56125, Pisa, Italy
6. Department of Oncology, Lund University Hospital, Lund, Sweden
7. Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden
8. Department of Oncology, Karolinska University Hospital, Stockholm, Sweden
9. Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden
10. Department of Oncology, Sahlgrenska University Hospital, Gothenburg, Sweden
11. Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
12. Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland
13. Department of Genetics and Pathology, Pomeranian Medical University, Szczecin and Postgraduate School of Molecular Medicine, Warsaw Medical University, Warsaw, Poland
14. Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre, Spanish Network on Rare Diseases (CIBERER), Madrid, Spain
15. Institute of Biology and Molecular Genetics, Universidad de Valladolid (IBGM-UVA), Valladolid, Spain
16. Oncology unit. Hospital clinico Universitario “Lozano Blesa”, Zaragoza, Spain
17. Human Genetics Group and Genotyping Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Centre, Spanish Network on Rare Diseases (CIBERER), Madrid, Spain
18. Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
19. Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The Netherlands
20. Department of Clinical Genetics, Academic Meical Center, Amsterdam, The Netherlands
21. Department of Clinical Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands
22. Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands
23. Department of Clinical Genetics and GROM, School for Oncology and Developmental Biology, MUMC, Maastricht, The Netherlands
24. Department of Clinical Genetics and GROM, School for Oncology and Developmental Biology, MUMC, Maastricht, The Netherlands
25. Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
26. Department of Clinical Genetics, Family Cancer Clinic, Erasmus University Medical Center, Rotterdam, The Netherlands
27. Department of Clinical Genetics, Family Cancer Clinic, Erasmus University Medical Center, Rotterdam, The Netherlands
28. Department of Medical Genetics, University Medical Center Utrecht, PO Box 8 AB, Utrecht, The Netherlands
29. Department of Genetics, University Medical Center, Groningen University, Groningen, The Netherlands
31. Genetic Medicine, Manchester Academic Health Sciences Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK
32. Clinical Genetics, Guy’s and St. Thomas’ NHS Foundation Trust, London, UK
33. Oncogenetics Team, The Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Kragujevac, UK
34. Yorkshire Regional Genetics Service, Leeds, UK
35. Ferguson-Smith Centre for Clinical Genetics, Yorkhill Hospitals, Glasgow, UK
36. West Midlands Regional Genetics Service, Birmingham Women’s Hospital Healthcare NHS Trust, Edgbaston, Birmingham, UK
37. Sheffield Clinical Genetics Service, Sheffield Children’s Hospital, Sheffield, UK
38. Department of Clinical Genetics, East Anglian Regional Genetics Service, Addenbrookes Hospital, Cambridge, UK
39. Institute of Genetic Medicine, Centre for Life, Newcastle Upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, UK
40. Department of Clinical Genetics, Royal Devon & Exeter Hospital, Exeter, UK
41. Medical Genetics Unit, St George’s, University of London, Kragujevac, UK
42. Northern Ireland Regional Genetics Centre, Belfast Health and Social Care Trust, and Department of Medical Genetics, Queens University Belfast, Belfast, UK
43. Oxford Regional Genetics Service, Churchill Hospital, Oxford, UK
44. All Wales Medical Genetics Services, University Hospital of Wales, Cardiff, UK
45. Clinical Genetics Department, St Michael’s Hospital, Bristol, UK
46. North West Thames Regional Genetics Service, Kennedy-Galton Centre, Harrow, UK
47. Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA
48. Clinical Molecular Genetics Laboratory, Fox Chase Cancer Center, Philadelphia, PA, USA
144. Unité INSERM U830, Institut Curie, Paris, France
145. Université Paris Descartes, Faculté de Médecine, Paris, France
49. Service de Génétique Oncologique, Institut Curie, Paris, France
146. Université Paris Descartes, Faculté de Pharmacie, Paris, France
50. Service de Génétique Oncologique, Institut Curie, Paris, France
51. Service de Génétique Oncologique, Institut Curie, 26 rue d’Ulm, Paris, France
52. Service de Génétique Oncologique, Institut Curie, Paris, France
53. INSERM U1052, CNRS UMR5286, Université Lyon 1, Centre de Recherche en Cancérologie de Lyon, Lyon, France
54. Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Hospices Civils de Lyon/Centre Léon Bérard, Lyon, France
55. Service de Génétique, Institut de Cancérologie Gustave Roussy, Villejuif, France and INSERM U946, Fondation Jean Dausset, Paris, France
56. Consultation de Génétique, Département de Médecine, Institut de Cancérologie Gustave Roussy, Villejuif, France
57. Département Oncologie génétique, Prévention et Dépistage, INSERM CIC-P9502, Institut Paoli-Calmettes/Université d’Aix-Marseille II, Marseille, France
58. Centre Antoine Lacassagne, Nice, France
59. Service de Génétique Clinique Chromosomique et Moléculaire, Centre Hospitalier Universitaire de St Etienne, St Etienne, France
60. Laboratoire de Génétique Chromosomique, H?tel Dieu Centre Hospitalier, BP 1125, Chambéry, France
61. Service de Génétique, Centre Hospitalier Universitaire Bretonneau, Tours, France
63. Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, UT, 84112, USA
64. Division of Population Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA
65. Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Surgery, Harvard Medical School, 27 Drydock Avenue, Boston, MA, 02210, USA
66. Department of Epidemiology, Columbia University, New York, NY, USA
67. Centre for Molecular, Environmental, Genetic and Analytic (MEGA) Epidemiology, Melbourne School of Population Health, Level 1, The University of Melbourne, 723 Swanston Street, VIC 3010, Kragujevac, Australia
68. Department of Epidemiology, Cancer Prevention Institute of California, 2201 Walnut Avenue, Suite 300, Fremont, CA, 94538, USA
69. Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Kragujevac, Australia
70. Department of Dermatology, University of Utah School of Medicine, 30 North 1900 East, SOM 4B454, Salt Lake City, UT, 84132, USA
71. Dept of OB/GYN and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria
72. Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
73. Department of Pathology, Landspitali, University Hospital, Reykjavik Iceland and Faculty of Medicine, University of Iceland, Reykjavik, Iceland
74. Epidemiology Research Program, American Cancer Society, Atlanta, GA, USA
75. Department of Environmental Medicine, NYU Cancer Institute, New York University School of Medicine, New York, NY, USA
76. Clinical Cancer Genetics Laboratory, Memorial Sloane Kettering Cancer Center, New York, NY, USA
77. Statistical and Data Center, Roswell Park Cancer Institute, Buffalo, NY, USA
78. Australia New Zealand (ANZGOG), Westmead Hospital, Sydney, Australia
79. Ohio State University, Columbus Cancer Council, Columbus, OH, USA
80. Evanston CCOP - NorthShore University Health System, University of Chicago, Chicago, IL, USA
81. Southern Pines Women’s Health Center, P.C., University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
82. Sarasota Memorial Healthcare, Tufts Medical Center, Sarasota, Florida, USA
83. Department of Molecular Virology, Immunology and Medical Genetics and Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
84. Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV - IRCCS, Padua, Italy
85. U.O.C. di Oncologia, ULSS5 Ovest Vicentino, Kragujevac, Italy
86. Laboratory of Molecular Oncology, N.N. Petrov Institute of Oncology, St.-Petersburg, Russia
87. Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA
88. Latvian Biomedical Research and Study Centre, Kragujevac, Latvia
89. Genetic Counselling Unit, Hereditary Cancer Program, IDIBELL-Catalan Institute of Oncology, Barcelona, Spain
90. Molecular Diagnostic Unit, Hereditary Cancer Program, IDIBELL-Catalan Institute of Oncology, Barcelona, Spain
91. Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
92. Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA
93. Women’s Cancer Program at the Samuel Oschin Comprehensive Cancer Institute at Cedars-Sinai Medical Center, Los Angeles, CA, USA
94. Department of Molecular Genetics, National Institute of Oncology, Budapest, Hungary
147. University Malaya Cancer Research Institute, University Malaya Medical Centre, Kuala Lumpur, Malaysia
95. Cancer Research Initiatives Foundation, Sime Darby Medical Centre, Kragujevac, Malaysia
96. Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, CA, USA
97. UCSF Cancer Risk Program, University of California, San Francisco, CA, USA
98. Cancer Genetics Laboratory, Department of Genetics, University of Pretoria, Kragujevac, South Africa
99. Oncogenetics Laboratory. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital, Barcelona, Spain
100. The Hong Kong Hereditary Breast Cancer Family Registry, The Universtiy of Hong K Cancer Genetics Center, Hong Kong Sanatorium and Hospital, Kragujevac, Hong Kong
101. Centre of Familial Breast and Ovarian Cancer, Department of Gynaecology and Obstetrics and Centre for Integrated Oncology (CIO), University hospital of Cologne, Cologne, Germany
102. Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany
103. Department of Gynaecology and Obstetrics, Division of Tumour Genetics, Klinikum rechts der Isar, Technical University Munich, Munich, Germany
104. Department of Gynaecology and Obstetrics, Ludwig-Maximilian University Munich, Munich, Germany
105. Department of Gynaecology and Obstetrics, University Hospital of Schleswig-Holstein, Campus Kiel, Christian-Albrechts University Kiel, Kiel, Germany
106. Institute of Human Genetics, University Hospital of Schleswig-Holstein, Campus Kiel, Christian-Albrechts University Kiel, Kiel, Germany
107. Department of Gynaecology and Obstetrics, University Hospital Düsseldorf, Heinrich-Heine University, Düsseldorf, Germany
108. Institute of Human Genetics, University of Münster, Münster, Germany
109. Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany
110. Institute of Human Genetics, Campus Virchov Klinikum, Charite Berlin, Germany
111. Department of Gynaecology and Obstetrics, University Hospital Ulm, Ulm, Germany
112. Centre of Familial Breast and Ovarian Cancer, Department of Medical Genetics, Institute of Human Genetics, University Würzburg, Würzburg, Germany
113. Institute of Human Genetics, Department of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany
114. Department of Gynaecology and Obstetrics, University Hospital Carl Gustav Carus, Technical University, Dresden, Germany
115. Institute of Human Genetics, University Regensburg, Regensbirg, Germany
116. Institute of Human Genetics, University Hospital Frankfurt a.M., Germany Molecular Oncology Laboratory, Hospital Clinico San Carlos, Madrid, Spain
117. Molecular Oncology Laboratory, Hospital Clinico San Carlos, Martin Lagos s/n, Madrid, Spain
118. Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Central Hospital, Biomedicum Helsinki, P.O. BOX 700, 00029 HUS, Helsinki, Finland
119. Faculty of Medicine - Medicine and Medical Specialties, Université de Montréal Hemato-oncology service, H?pital du Sacré-Coeur de Montréal, 5400 Gouin Blvd West Montreal, QC, Kragujevac, Canada
121. Department of Population Sciences, Beckman Research Institute of City of Hope, Duarte, CA, USA
122. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
123. Department of Medical Genetics, Mayo Clinic, Rochester, MN, USA
124. Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA
125. Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Preventive and Predicted Medicine, Fondazione IRCCS Istituto Nazionale Tumouri (INT), Milan, Italy
149. IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy
126. Unit of Medical Genetics, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale Tumouri (INT), Milan, Italy
127. Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia (IEO), Milan, Italy
128. Department of Experimental Oncology, Istituto Europeo di Oncologia, Milan, Italy
150. Consortium for Genomics Technology (Cogentech), Milan, Italy
129. Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico, IRCCS, Aviano (PN), Italy
130. Medical Genetics Unit, Department of Clinical Physiopathology, University of Florence, Firenze, Italy
131. Department of Molecular Medicine, “Sapienza” University of Rome, Rome, Italy
132. Clinical Genetics Branch, DCEG, NCI, Room EPS 7032, Rockville, MD, 20852, USA
133. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
151. Cancer Care Ontario, Departments of Molecular Genetics and Laboratory Medicine and Pathobiology, University of Toronto, ON, Kragujevac, Canada
134. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
152. Department of Laboratory Medicine and Pathobiology, University of Toronto, ON, Kragujevac, Canada
135. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
153. Department of Laboratory Medicine, and the Keenan Research Centre of the Li Ka Shing Knowledge Institute, St Michael’s Hospital, Toronto, ON, Canada
136. Ontario Cancer Genetics Network, Cancer Care Ontario, Toronto, ON, Canada
137. Department of Clinical Genetics, Odense University Hospital, Kragujevac, Denmark
138. Department of Clincial Genetics, Rigshospital and Copenhagen University, Kragujevac, Denmark
139. Department of Clinical Genetics, Skejby Hospital, Aarhus, Denmark
140. Department of Clinical Genetics, Vejle Hospital, Kragujevac, Denmark
141. Department of Laboratory Medicine and Pathology, Health Sciences Research, Mayo Clinic, Rochester, MN, USA
142. Cancer Genomics Laboratory, Centre Hospitalier Universitaire de Québec, Canada Research Chair in Oncogenetics, Department of Molecular Medicine, Faculty of Medicine, Laval University, 2705 Laurier Boulevard, T3-57, Quebec City, QC, Canada
... -内酰胺酶耐药决定基团的特异性峰值[24] ...
... 对革兰阴性杆菌外膜蛋白的蛋白组学和基因组学研究表明,膜孔蛋白、外排泵、脂多糖等细胞组分系统主要是通过对不同抗菌药物泵入和泵出的调节而表现耐药的[25] ...
... 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及2D电泳结合MALDI-TOF MS指纹图谱可以用来鉴定耐药和敏感菌株表达水平不同的外膜及周浆蛋白,建立一个综合的数据库来研究各种细胞组分,在耐药检测中发挥不同的作用[26,27] ...
... 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及2D电泳结合MALDI-TOF MS指纹图谱可以用来鉴定耐药和敏感菌株表达水平不同的外膜及周浆蛋白,建立一个综合的数据库来研究各种细胞组分,在耐药检测中发挥不同的作用[26,27] ...
... -内酰胺酶等[28,29] ...
. , :385-391
... -内酰胺酶等[28,29] ...
Anal. Chem.. ):
Po-Chiao Lin
Mei-Chun Tseng
Yu-Ju Chen
Chun-Cheng Lin
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan and Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan, and Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Functionalized magnetic nanoparticles (MNPs) were synthesized to serve as laser desorption/ionization elements as well as solid-phase extraction probes for simultaneous enrichment and detection of small molecules in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Two laser-absorbing matrices were each conjugated onto MNP to give MNP@matrix which provided high ionization efficiency and background-free detection in MS leading to unambiguous identification of target small molecules in a complex mixture. MNP@matrix was demonstrated to serve as a general matrix-free additive in MALDI-TOF MS analysis of structurally distinct small molecules. Also, MNP@matrix provides a simple, rapid, and reliable quantitative assay for small molecules by mass without either the use of an internal standard or an isotopic labeling tag. Furthermore, the affinity extraction of small molecules from complex biofluid was achieved by probe protein-conjugated MNP@matrix without laborious purification. We demonstrated that a nanoprobe-based assay is a cost-effective, rapid, and accurate platform for robotic screening of small molecules.
... LIN等通过方法学的改进,目前已成功检测出水杨酰胺等6种抗菌药物,未来这种方法有可能用于耐药机制及检测血样本中的微生物毒素和药物等[30] ...
质谱技术在临床微生物实验室中的应用前景}
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