JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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免疫与感染

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

Published: July 11, 2016
doi:
10.3791/53176
, , ,
1Institut für Medizinische Mikrobiologie und Hygiene, Medizinische Fakultät Carl Gustav Carus,TU Dresden, 2Institut für Virologie, Medizinische Fakultät Carl Gustav Carus,TU Dresden

Summary

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

Abstract

有许多的这些报告导致特别是在免疫功能低下患者严重感染罕见的，因此，不能充分描述的细菌病原体。在大多数情况下只有很少的数据，主要是出版病例报告，提供其研究这些病原体的作用，作为一种传染性病原体。因此，为了阐明这些微生物的致病性质，有必要进行的流行病学研究，其中包括大量的这些细菌。在这样的监控研究中所用的方法必须满足以下条件：菌株的识别必须根据有效命名法准确，它们应该是易于处理（鲁棒性），经济的常规诊断和他们要产生可比不同实验室之间的结果。通常，有一种用于在常规设置识别细菌菌株三种策略：1）的表型鉴定表征Biochemica公司细菌的升和代谢性质，2）分子技术如16S rRNA基因测序和3）质谱作为一种新的蛋白质为基础的方法。因为质谱和分子的方法可用于鉴定了大量的各种细菌物种的最有前途的工具，描述这两种方法。使用这些技术时进步，局限性和潜在的问题进行了讨论。

Introduction

在常规诊断罕见病原体安全识别是由以下事实古典文化和生物化学方法是繁琐，有时可疑阻碍。此外，诊断微生物学实验室必须处理大量的病原体，范围从几百到几千，每天，这需要使用自动化系统。除了高每日吞吐量的管理，需要细菌物种的精确识别。因为他们在抗菌药物敏感性的模式不同，因此正确识别提供了选择适当的抗生素的基本信息（ 如， 肠球菌 ， 不动杆菌属）12,43医生这是必要的。

全自动微生物鉴定系统（AMIS）应用规范组酶促反应的表征细菌分离株的代谢特性<sup> 13,15,16,26,27。虽然在这些系统中使用的墨盒利用大量不同的生化反应的， 例如，47在本研究中52所用的AMI的GN卡，这种策略允许安全识别只为一组有限的细菌。此外，数据库，一个先进的专家系统，显然是集中在检测的医学重要性13,15,16,36相关的和高度相关的细菌。另外两个系统，广泛应用于实验室，也适用于细菌鉴定这种生化途径。最近的研究表明在该研究中使用的的AMI和竞争对手之一（93.7％和93.0％）之间的比较的识别精度，同时第三阿美族具有只有82.4％的识别精度上物种水平35。这种差异可能由底层识别数据的引用，包和软件，在麦太保差异的版本的质量来解释栓塞技术人员的35,36精通。

两条自动化MALDI-TOF MS系统（MALDI-TOF微生物鉴定系统，MMIS）主要使用。这些系统允许检测大量基于其蛋​​白质指纹质谱细菌种类的。例如，所用的MMIS的数据库包含6000参考光谱。基于质谱鉴定系统提供的种类繁多的微生物，包括致病菌罕见的快速11,48,51和可靠的检测。迄今只有少数直接比较是在本研究中使用的MMIS和其竞争者19,33之间提供。根据Daek 等人的这两个系统提供识别精度的类似的高率，但是在本研究中使用的MMIS似乎是在物种鉴定19更可靠。

同样，分子技术寻址非常保守，而且不同的基因（ <em>如16S rDNA全或的rpoB）允许一个明确的物种鉴定3,22,61。这其中，16S rDNA的是因为它在所有的细菌34存在的最广泛使用的持家基因。其功能保持不变，并且最后，用大致1500 bp的，这是足够长，以适合于生物信息14,34。许多研究者认为16S rRNA基因分析的“黄金标准”进行细菌鉴定21。这是由于这样的事实，即几个实验室使用DNA-DNA杂交技术迄今为罕见的或新的细菌14,34的鉴定。此外，越来越多的数据库可用，可用于16S rRNA基因分析50。但是，它必须考虑到，基于16S rDNA的检测系统相比，标准PCR方案有一个有限的敏感度。此外，该分子的方法是复杂的，耗时的，并且需要训练有素的人员，以及专门的实验室设施的，因此，不容易落实到日常诊断55。此外，已经表明，细菌鉴定至少两种不同的方法的组合导致高度精确的菌种鉴定。 MALDI-TOF MS和16S rDNA序列的结合允许大量以高精度不同细菌种类的识别。最近提出进行细菌鉴定流行病学研究的问题和罕见的病原体56 MALDI-TOF MS和16S rRNA基因分析的结合。

Protocol

1.细菌DNA的提取的PBS溶液的制备称量1.65克Na 2 HPO 4×2H 2 O，0.22克的NaH 2 PO 4×2H 2 O 8.80克NaCl在一烧瓶中，并用蒸馏水补至1000 ml的终体积。将pH调节至7.4。对于最终使用过滤通过防菌（0.22微米）过滤该溶液。 DNA提取革兰氏阴性菌条纹在合适的培养基（ 如哥伦比亚血琼脂）病人资料，识别和隔离潜在的病原体。 …

Representative Results

MALDI-TOF MS是一种新型的，用于微生物常规诊断快速和廉价的方法。通过MALDI-TOF MS的菌种鉴定产生光谱主要由核糖体蛋白，还包括其他“与受影响到环境条件的最小程度看家功能非常保守的蛋白”的这个MMIS的17 .The数据库包含一大组参考光谱的甚至目前在临床分离株很少发现细菌能够安全地识别7,56,57。得分值显示所识别的物种的可靠性。上述2.300分数代表，…

Discussion

两个MALDI-TOF MS和16S rRNA基因测序提供识别大量不同的细菌的可能性。 MALDI-TOF MS是一个快速和廉价的方法，这是易于处理和细菌质谱大型数据库是可用的。出于这个原因，MALDI-TOF MS是进行集中在罕见的细菌病原体17,20,39,51筛选研究一种快速，成本有效和可靠的方法。在前瞻性研究中比较MALDI-TOF MS和其他表型的鉴定方法，诚等人证明成本效益和MALDI-TOF MS 59和Tan的速度等人?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to thank Prof. Enno Jacobs for his continuing support.

Materials

CHROMASOLV, HPLC grade water, 1 L Sigma-Aldrich Chemie, München, Germany 270733
Tissue Lyser LT Qiagen, Hilden, Germany 85600 Oscillating Homogenizer
Glass-beads 1,0mm VWR International, Darmstadt, Germany 412-2917
Thermomixer 5436 Eppendorf, Hamburg, Germany 2050-100-05
QIAamp DNA Mini Kit (250) Qiagen, Hilden, Germany 51306
Taq PCR Core Kit (1000 U) Qiagen, Hilden, Germany 201225
Forward Primer TPU1 (5´-AGA GTT TGA TCM TGG CTC AG-3’) biomers.net GmbH, Ulm, Germany  –
Reverse Primer RTU4 (5´-TAC CAG GGT ATC TAA TCC TGT T-3´) biomers.net GmbH, Ulm, Germany  –
Mastercycler  Eppendorf, Hamburg, Germany -  Thermocylcer
Reaction tube 1.5 mL SARSTEDT, Nümbrecht, Germany 72,692
Reaction tube 2 mL SARSTEDT, Nümbrecht, Germany 72,693,005
PCR 8er-CapStrips Biozym Scientific, Hessisch Oldendorf, Germany 711040X
PCR 8er-SoftStrips Biozym Scientific, Hessisch Oldendorf, Germany 711030X
Sharp R-ZV11  Sharp Electronics, Hamburg, Germany – Microwave
Titriplex III (EDTA Na2-salt dehydrate; 1 kg) Merck, Darmstadt, Germany 1084211000
SeaKem LE Agarose Biozym Scientific, Hessisch Oldendorf, Germany 849006
(2 x 500 g)
SmartLadder SF – 100 to 1000 bp Eurogentec, Lüttich, Belgium MW-1800-04
Bromphenol blue (25 g) Sigma-Aldrich Chemie, München, Germany B0126
Xylene cyanol FF (10 g) Sigma-Aldrich Chemie, München, Germany X4126
ComPhor L Maxi  Biozym, Hessisch Oldendorf, Germany –
Ethidium bromide solution 1 %(10 mL) Carl Roth, Karlsruhe, Germany 2218.1
Gel Doc 2000 Bio-Rad Laboratories, München, Germany – Gel-documentation system 
ExoSAP-IT (500 reactions) Affymetrix UK, Wooburn Green, High Wycombe, United Kingdom 78201
Buffer (10 x) with EDTA  Life Technologies, Darmstadt, Germany 402824
BigDye Terminator Kit v1.1 Life Technologies, Darmstadt, Germany 4337450
Hi-Di formamide (25 mL) Life Technologies, Darmstadt, Germany 4311320
DyeEx 2.0 Spin Kit (250) Qiagen, Hilden, Germany 63206
3130 Genetic Analyzer Life Technologies, Darmstadt, Germany – Sequenzer
MicroAmp optical 96-well reaction plate with barcode Life Technologies, Darmstadt, Germany 4306737
3130 Genetic Analyzer, plate base 96-well Life Technologies, Darmstadt, Germany 4317237
3130 Genetic Analyzer, plate retainer 96-well Life Technologies, Darmstadt, Germany 4317241
3130 Genetic Analyzer, well plate septa Life Technologies, Darmstadt, Germany 4315933
3130 Genetic Analyzer, POP-7 Polymer, 7 mL Life Technologies, Darmstadt, Germany 4352759
3130 Genetic Analyzer, 4-Capillary Array, 50 cm Life Technologies, Darmstadt, Germany 4333466
Sequencing Analysis Software 5.4 Life Technologies, Darmstadt, Germany –
microflex (the MALDI TOF MS maschine) Bruker Daltonik, Bremen, Germany –
MALDI Biotyper (the MALDI TOF MS system) Bruker Daltonik, Bremen, Germany – our mMIS
VITEK MS  bioMérieux, Nürtingen, Germany  2nd mMis 
flexControl 3.4 (control software) Bruker Daltonik, Bremen, Germany –
Biotyper Realtime Classification 3.1 (RTC), (analysis software) Bruker Daltonik, Bremen, Germany –
α-cyano-4-hydroxycinnamic acid, HCCA, 1 g Bruker Daltonik, Bremen, Germany 201344
Peptide Calibration Standard II Bruker Daltonik, Bremen, Germany 222570
MSP 96 target polished steel Bruker Daltonik, Bremen, Germany 8224989
peqgreen  peqlab  37-5010
MALDI Biotyper Galaxy  Bruker Daltonik, Bremen, Germany Part No. 1836007 
Vitek 2  bioMérieux, Nürtingen, Germany  our aMis 
MicroScan  Beckman Coulter  2nd aMis 
BD Phoenix™ Automated Microbiology System BD 3rd aMis 
Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 25923™) ATCC  postive control for PCR 
DOWNLOAD MATERIALS LIST

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16S RRNA Gene SequencingMALDI-TOF MSRare Bacterial PathogensMyroidesOdoratimimusBacterial DNA Extraction16S RRNA PCRSanger SequencingMALDI-TOF MS AnalysisMALDI Biotyper SoftwareBacterial IdentificationDiagnostic Microbiology

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Schröttner, P., Gunzer, F., Schüppel, J., Rudolph, W. W. Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS. J. Vis. Exp. (113), e53176, doi:10.3791/53176 (2016).
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