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. Preparare un ambiente di lavoro sicuro, considerando biosicurezza Condizioni <ol><li> Acquisire familiarità con le norme di laboratorio e di sicurezza che sono di rilevanza per l&#39;utilizzo di microrganismi. La maggior parte dei microrganismi patogeni umani devono essere gestite a livello di biosicurezza 2 condizioni, ma alcuni, come la Salmonella enterica sierotipo Typhi, richiedono livello di biosicurezza 3. Informazioni sul livello di gestione di ogni agente patogeno si può accedere a www.cdc.gov/bi...

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La fase più critica nella creazione di un sistema MSPP è la determinazione genetica inequivocabile di identità biomarker di ioni. Se non è possibile identificare un biomarker senza dubbio, allora dovrebbe essere escluso dal regime 13. Il C. ssp. doylei schema jejuni comprende 14 diversi ioni biomarker. Questi sono 5 meno rispetto al C. jejuni ssp. schema jejuni MSPP 13 .La differenza più significativa tra il rilevabile <...

Durante l&#39;ultimo decennio, matrix-assisted laser desorbimento di ionizzazione tempo di volo spettrometria di massa (MALDI-TOF MS) ha avanzato ad essere un metodo standard di grande valore per il genere microbica e l&#39;identificazione delle specie in microbiologia clinica 1, 2. identificazione di specie si basa sulla registrazione di piccole impronte proteici di cellule intatte o lisati cellulari. La gamma di massa tipico di uno spettrometro di massa usata nella routine microbiologia clinica è 2-20 kDa. Inoltre, gli spettri risultante può essere utilizzato per discriminare ceppi ai sotto-specie e sotto-sottospecie livello 3. I primi studi pionieristici hanno identificato specifici ioni biomarker per un particolare sottogruppo di ceppi di Campylobacter jejuni 4, 5 Clostridium difficile, Salmonella enterica ssp. enterica sierotipo Typhi 6, Staphylococcus aureus 7-9, ed Escherichia coli 10 - 12. La combinazione di diverse masse biomarker variabili corrispondenti alle isoforme alleliche offre la possibilità per il sottotipo più profondo. In precedenza, abbiamo implementato con successo un metodo per convertire queste variazioni nei profili di massa nelle relazioni phyloproteomic significative e riproducibili chiamate di massa phyloproteomics basate spettrometria (MSPP) su un C. jejuni ssp. collezione jejuni isolare 13. MSPP può essere utilizzato una spettrometria di massa equivalente alle tecniche basate sottotipizzazione sequenza del DNA, come la tipizzazione sequenza multilocus (MLST). Specie Campylobacter sono la principale causa di gastroenterite batterica in tutto il mondo 14, 15. Come conseguenza di Campilobatteriosi sequela post-infettive, cioè, sindrome di Guillain Barre, artrite reattiva e la malattia infiammatoria intestinale può sorgere 16. Le principali fonti di infezione sonodi carni animali contaminati da pollo, tacchino, suino, bovino, ovino e le anatre, il latte e di superficie 15, 17. Pertanto, sono necessari studi di sorveglianza epidemiologica regolari nel contesto della sicurezza alimentare. MLST è il &quot;gold standard&quot; nella tipizzazione molecolare per le specie Campylobacter 18. Poiché il Sanger-sequenziamento metodo MLST base è alta intensità di lavoro, che richiede tempo e relativamente costosa, MLST digitazione è limitata a relativamente piccole coorti isolare. Pertanto, vi è la necessità di metodi sottotipizzazione più economico e rapido. Questa esigenza potrebbe essere soddisfatta con metodi di spettrometria di massa come MSPP. Questo articolo presenta un protocollo dettagliato per MSPP-tipizzazione mediante una raccolta di Campylobacter jejuni ssp. Doylei isolati e il confronto delle sue potenzialità con 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 possibilità di differenziare alcuni batteri non solo a livello di specie e sottospecie ma anche al di sotto, a livello di ceppo. isoforme alleliche del ioni biomarcatori rilevabili provocano spostamenti di massa specifico per isolare. phyloproteomics spettrometria di massa a base di (MSPP) è una tecnica innovativa che combina le spettrometria di massa masse biomarcatori rilevabili in uno schema che consente la deduzione delle relazioni phyloproteomic da isolare specifici turni di massa rispetto a un genoma sequenziato ceppo di riferimento. Le sequenze di amminoacidi dedotta vengono poi utilizzati per calcolare dendrogrammi MSPP-based. Qui si descrive il flusso di lavoro di MSPP digitando un ssp Campylobacter jejuni. Doylei raccolta isolato di sette ceppi. Tutti e sette i ceppi erano di origine umana e multilocus sequenza di battitura (MLST) dimostrato la loro diversità genetica. MSPP-tipizzazione portato a sette diversi tipi di sequenze MSPP, sufficientemente che riflette la loro PHYrelazioni logenetic. Il C. jejuni ssp. doylei schema MSPP comprende 14 diversi ioni biomarker, proteine soprattutto ribosomiali nel range di massa di 2 a 11 kDa. MSPP può in linea di principio, essere adattato ad altre piattaforme di spettrometria di massa con una gamma di massa estesa. Pertanto, questa tecnica ha il potenziale per diventare uno strumento utile per la tipizzazione microbica livello di deformazione.

In precedenza, abbiamo stabilito con successo un sistema di MSPP per C. jejuni ssp. jejuni 13. Qui, abbiamo voluto estendere il metodo per il fratello sottospecie C. jejuni ssp. doylei. In questo contesto specifico, sette C. jejuni ssp. doylei isolati sono stati acquisiti dalla collezione belga di microrganismi / Laboratorio di Microbiologia UGent BCCM / LMG Gand, in Belgio. Tutti i sette isolati utilizzati per le nostre an...

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

Massa phyloproteomics spettrometria-based (MSPP) è stato utilizzato per digitare una collezione di Campylobacter jejuni ssp. Doylei isolati a livello di sforzo rispetto a tipizzazione sequenza multilocus (MLST).

sottotipizzazione di<em&gt; Jejuni Campylobacter</em&gt; Ssp.<em&gt; doylei</em&gt; Isolati Utilizzando PhyloProteomics Spettrometria di Massa-based (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

Migliorata la Polymerase Chain Reaction-restrizione frammento lunghezza polimorfismo genotipizzazione di Pufferfish Tossico per cromatografia liquida / spettrometria di massa

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