Name: subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)
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Description: Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

We are grateful to Hannah Kleinschmidt for excellent technical support. This paper was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universita&#776;t Go&#776;ttingen.

1. Préparer un milieu de travail sécuritaire en considérant les conditions de biosécurité <ol><li> Se familiariser avec les règlements de laboratoire et de sécurité qui sont pertinentes pour travailler avec des micro-organismes. La plupart des micro - organismes pathogènes pour l&#39; homme doivent être traitées au niveau de biosécurité 2 conditions , mais certains, tels que Salmonella enterica sérotype Typhi, exigent de biosécurité de niveau 3. Des informations sur le niveau de traitement de ...

<ol>
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L&#39;étape la plus critique dans la mise en place d&#39;un régime MSPP est la détermination génétique sans équivoque des identités biomarqueur d&#39;ions. S&#39;il est impossible d&#39;identifier un biomarqueur sans aucun doute, alors il devrait être exclu du régime 13. C. ssp. doylei régime jejuni comprend 14 ions de biomarqueurs différents. Ceux - ci sont 5 moins par rapport à C. ssp jejuni. schéma jejuni MSPP <...

Au cours de la dernière décennie, laser assistée par matrice désorption ionisation spectrométrie à temps de vol de masse (MALDI-TOF MS) a avancé comme une méthode standard très apprécié pour le genre microbienne et l&#39; identification des espèces en microbiologie clinique 1, 2. L&#39;identification des espèces est basée sur l&#39;enregistrement des petites empreintes de protéines des cellules intactes ou des lysats cellulaires. La gamme typique de masse pour un spectromètre de masse utilisé en microbiologie clinique de routine est 2-20 kDa. En outre, les spectres résultant peut être utilisé pour distinguer les souches aux dessous-espèces et sous - espèces ci - dessous-niveau 3. Les premières études pionnières ont identifié des ions de biomarqueurs spécifiques pour un sous - groupe particulier de souches de Campylobacter jejuni 4, 5 Clostridium difficile, Salmonella enterica ssp. enterica sérotype Typhi 6, Staphylococcus aureus 7-9 et Escherichia coli 10-12. La combinaison de plusieurs masses de biomarqueurs variables correspondant aux isoformes alléliques offre la possibilité de sous-typage plus profond. Auparavant, nous avons mis en place avec succès une méthode pour convertir ces variations dans les profils de masse dans les relations phyloproteomic significatives et reproductibles appelés phyloproteomics basés spectrométrie de masse (MSPP) sur un C. ssp jejuni. collection isolat jejuni 13. MSPP peut être utilisé d&#39;une spectrométrie de masse équivalente à des techniques de typage à base de séquences d&#39;ADN comme le typage de séquence multilocus (MLST). Espèces de Campylobacter sont la principale cause de gastro - entérite bactérienne dans le monde entier 14, 15. En conséquence de sequela post-infectieuse campylobactériose, à savoir, le syndrome de Guillain - Barré, l&#39; arthrite réactive et la maladie inflammatoire de l&#39; intestin peut se produire 16. Les principales sources d&#39;infection sontla viande de bétail contaminé du poulet, de la dinde, du porc, des bovins, des moutons et des canards, le lait et l&#39; eau de surface 15, 17. Par conséquent, les études de surveillance épidémiologique régulière dans le contexte de la sécurité alimentaire sont nécessaires. MLST est le «gold standard» dans le typage moléculaire pour les espèces de Campylobacter 18. Parce que le Sanger séquençage méthode MLST base est un travail intensif, de temps et relativement coûteux, MLST typage est limitée à relativement petites cohortes isolées. Par conséquent, il existe un besoin de méthodes de typage moins coûteux et plus rapide. Ce besoin pourrait être satisfaite par des méthodes de spectrométrie de masse comme MSPP. Cet article présente un protocole détaillé pour MSPP-typage en utilisant une collection de Campylobacter jejuni ssp. Doylei isolats et la comparaison de son potentiel avec MLST.

<table border="0" cellpadding="0" cellspacing="0" width="1348">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">acetonitrile</td>
			<td style="width:663px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">34967</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoflex III TOF/TOF 200 system</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">GT02554 G201</td>
			<td>Mass spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">bacterial test standard BTS</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">BioTools 3.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>263564</td>
			<td>Software Package</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Bruker IVD Bakterial Test Standard</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">8290190</td>
			<td>5 tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8843</td>
			<td>ATCC 49349;IMVS 1141;NCTC 11951;strain 093</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9143</td>
			<td>Goossens Z90</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG7790</td>
			<td>ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9243</td>
			<td>Goossens N130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8871</td>
			<td>NCTC A603/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9255</td>
			<td>Goossens B538</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8870</td>
			<td>NCTC A613/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Columbia agar base&#160;</td>
			<td>Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1.10455 .0500</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Compass for FlexSeries 1.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>251419</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">defibrinated sheep blood&#160;</td>
			<td>Oxoid Deutschland GmbH, Wesel, Germany</td>
			<td>SR0051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">ethanol</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">02854 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">HCCA matrix</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Kimwipes paper tissue</td>
			<td style="width:663px;">Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MALDI Biotyper 2.0</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>259935</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Mast Cryobank vials</td>
			<td>Mast Diagnostica, Reinfeld, Germany</td>
			<td style="width:119px;">CRYO/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MSP 96 polished steel target</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>224989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Mini Kit&#160;</td>
			<td style="width:663px;">Qiagen, Hilden, Germany</td>
			<td style="width:119px;">51304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">recombinant human insulin</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">I2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">T6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">water, molecular biology-grade</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">W4502</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)

MALDI-TOF MS offre la possibilité de différencier certaines bactéries non seulement au niveau des espèces et sous-espèces, mais même au-dessous, au niveau de la souche. isoformes alléliques des ions de biomarqueurs détectables se traduisent par des changements de masse spécifiques isolés. phyloproteomics basée sur la spectrométrie de masse (MSPP) est une nouvelle technique qui combine les spectrométriques de masse détectables masses de biomarqueurs dans un système qui permet la déduction des relations phyloproteomic de isoler des changements de masse spécifiques par rapport à un génome séquencé souche de référence. Les séquences d&#39;acides aminés déduites sont ensuite utilisées pour calculer dendrogrammes à base de RRMS. Nous décrivons ici le flux de travail de MSPP en tapant un ssp Campylobacter jejuni. Doylei collection isolat de sept souches. Les sept souches étaient d&#39;origine humaine et multilocus sequence typing (MLST) ont démontré leur diversité génétique. MSPP-typage a donné lieu à sept types de séquence MSPP différents, reflétant suffisamment leur phyrelations logenetic. C. jejuni ssp doylei régime MSPP comprend 14 ions de biomarqueurs différents, la plupart du temps des protéines ribosomiques dans la gamme de masse de 2 à 11 kDa.. MSPP peut en principe, être adapté à d&#39;autres plates-formes de spectrométrie de masse avec une gamme étendue de masse. Par conséquent, cette technique a le potentiel de devenir un outil utile pour le typage microbien de niveau de la souche.

Auparavant, nous avons réussi à établir un régime MSPP pour C. ssp jejuni. jejuni 13. Ici, nous avons cherché à étendre la méthode à la fratrie sous - espèce C. ssp jejuni. doylei. Dans ce cadre spécifique, sept C. ssp jejuni. doylei isolats ont été acquis de la collection belge de micro - organismes / Laboratoire de microbiologie UGent BCCM / LMG Gand, Belgique. Les sept souches util...

Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass phyloproteomics la spectrométrie (MSPP) a été utilisé pour taper une collection de Campylobacter jejuni ssp. Doylei isolats au niveau de la souche par rapport à multilocus sequence typing (MLST).

typage de<em&gt; Campylobacter jejuni</em&gt; Ssp.<em&gt; doylei</em&gt; Les isolats utilisant PhyloProteomics basés sur la spectrométrie de masse (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

Research

JoVE Journal

Genetics

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

Amélioration de la Polymerase Chain Reaction restriction Fragment Length Polymorphism génotypage de Pufferfish Toxique par chromatographie liquide / spectrométrie de masse

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

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Génétique

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Procédure d&#39;adaptation Laboratoire de l&#39;évolution de Microorganismes Utilisation chémostat

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Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

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Détection de l&#39;expression de microARN dans la membrane peritoneale des rats en utilisant une PCR quantitative en temps réel

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

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Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Marquage métabolique et profilage des ARN de transfert à l’aide de macroréseaux

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Enquête de synthèse de l’ARN à l’aide de 5-Bromouridine étiquetage et immunoprécipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Gène candidat essais dans des études de cohortes cliniques avec génotypage multiplexé et spectrométrie de masse

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Une méthode rapide et Quantitative pour Modification post-traductionnelle et variante compatible cartographie des Peptides aux génomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

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L’induction et l’évaluation des croisements de consanguinité à l’aide de la fourmi, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...