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. Forbered en sikker arbejdsplads ved at overveje Biosafety Betingelser <ol><li> Bliv fortrolig med laboratorie- og sikkerhedsregler, som er relevante for at arbejde med mikroorganismer. De fleste humane patogene mikroorganismer skal håndteres på biosikkerhed niveau 2 betingelser, men nogle, såsom Salmonella enterica serovar Typhi, kræver biosikkerhedsniveauet 3. Oplysninger om niveau håndtere hver patogen kan tilgås ved www.cdc.gov/biosafety. </li><li> Uanset den biohazard klassificeringen af ​​de...

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Den mest kritiske skridt i etableringen af ​​en MSPP ordningen er den utvetydige genetiske bestemmelse af biomarkør ion identiteter. Hvis det ikke er muligt at identificere en biomarkør utvivlsomt, så det bør være udelukket fra ordningen 13. C. jejuni ssp. doylei Ordningen omfatter 14 forskellige biomarkør ioner. Disse er 5 mindre i forhold til C. jejuni ssp. jejuni MSPP ordning 13 .Den væsentligste forskel mellem den påvi...

I det sidste årti, har matrix-assisteret laser desorption ionisering tid-of-flight massespektrometri (MALDI-TOF MS) frem for at være en højt værdsat standardmetode for mikrobiel slægt og identifikation art i klinisk mikrobiologi 1, 2. identifikation Arter er baseret på registrering af små protein fingeraftryk intakte celler eller cellelysater. Den typiske masse interval for et massespektrometer anvendes i rutinemæssig klinisk mikrobiologi er 2-20 kDa. Derudover kan den resulterende spektre anvendes til at skelne stammer på den nedenfor arter og under-underarter niveau 3. Tidlige banebrydende studier har identificeret specifikke biomarkør ioner for en særlig undergruppe af stammer i Campylobacter jejuni 4, Clostridium difficile 5, Salmonella enterica ssp. enterica serovar Typhi 6, Staphylococcus aureus 7-9, og Escherichia coli 10-12. Kombinationen af ​​flere variable biomarkør masser, der svarer til allele isoformer giver mulighed for dybere subtypning. Tidligere vi med succes gennemført en metode til at konvertere disse variationer i masse profiler i meningsfulde og reproducerbare phyloproteomic relationer kaldes massespektrometri baseret phyloproteomics (MSPP) på en C. jejuni ssp. jejuni isolat samling 13. MSPP kan anvendes en massespektrometrisk svarende til DNA-sekvens baseret subtypning teknikker som multilocus sekvens typebestemmelse (MLST). Campylobacter-arter er den hyppigste årsag til bakteriel gastroenteritis verdensplan 14, 15. Som følge af Campylobacteriose post-infektiøs følgesygdomme, nemlig Guillain-Barré syndrom, reaktiv arthritis og inflammatorisk tarmsygdom kan opstå 16. De vigtigste kilder til infektion erforurenet husdyr kød fra kylling, kalkun, svin, kvæg, får og ænder, mælk og overfladevand 15, 17. Derfor regelmæssige epidemiologiske overvågning studier i forbindelse med fødevaresikkerhed er nødvendige. MLST er &quot;gold standard&quot; i molekylær typning for Campylobacter arter 18. Fordi Sanger-sekventering baseret MLST fremgangsmåde er arbejdskrævende, tidskrævende og forholdsvis dyr, er MLST typning begrænset til relativt små isolere kohorter. Derfor er der et behov for billigere og hurtigere subtypning metoder. Dette behov kan opfyldes ved massespektrometriske metoder som MSPP. Denne artikel præsenterer en detaljeret protokol for MSPP-typning ved hjælp af en samling af Campylobacter jejuni ssp. Doylei isolater og sammenligning af dens potentiale med 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 giver mulighed for at skelne nogle bakterier ikke kun på niveau arter og underarter, men endog under, stammen være. Allele isoformer af de påviselige biomarkør ioner medfører isolat-specifikke masseskift. Massespektrometri-baserede phyloproteomics (MSPP) er en ny teknik, der kombinerer de massespektrometriske påviselige biomarkør masserne i en ordning, der giver mulighed for fradrag for phyloproteomic relationer fra isolere specifikke masseskift sammenlignet med et genom sekventeret henvisning stamme. De udledte aminosyresekvenser er derefter anvendt til at beregne MSPP-baserede dendrogrammer. Her beskriver vi arbejdsgangen af MSPP ved at skrive en Campylobacter jejuni ssp. Doylei isolere samling af syv stammer. Alle syv stammer var af human oprindelse og multilocus sekvens typning (MLST) viste deres genetiske mangfoldighed. MSPP-typning resulterede i syv forskellige MSPP sekvens typer, tilstrækkeligt afspejler deres grafilogenetic relationer. C. jejuni ssp. doylei MSPP Ordningen omfatter 14 forskellige biomarkør ioner, hovedsagelig ribosomale proteiner i massen området fra 2 til 11 kDa. MSPP kan i princippet tilpasses andre massespektrometriske platforme med en udvidet masse rækkevidde. Derfor er denne teknik har potentiale til at blive et nyttigt redskab for stamme mikrobiel skrive.

Tidligere vi med succes etableret en MSPP ordning for C. jejuni ssp. jejuni 13. Her har vi til formål at udvide metoden til søskende underart C. jejuni ssp. doylei. I dette specifikke indstilling, syv C. jejuni ssp. doylei isolater blev erhvervet fra den belgiske samling af mikroorganismer / Laboratorium for Microbiology UGent BCCM / LMG Gent, Belgien. Alle syv isolater anvendes til vores analyser var af human oprindelse....

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

Massespektrometri-baserede phyloproteomics (MSPP) blev anvendt til at skrive en samling af Campylobacter jejuni ssp. Doylei isolater på stamme i forhold til multilocus sekvens maskinskrivning (MLST).

subtypning af<em&gt; Campylobacter jejuni</em&gt; Ssp.<em&gt; doylei</em&gt; Isolater Brug massespektrometri-baserede PhyloProteomics (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

Forbedret polymerasekædereaktion-restriktionsfragmentlængde-polymorfisme Genotypebestemmelse af toksiske Pufferfish ved væskekromatografi / massespektrometri

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

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Procedure for Adaptive Laboratory Evolution af mikroorganismer Ved hjælp af en kemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

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Påvisning af Copy Number Ændringer Brug Single Cell Sequencing

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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Påvisning af mikroRNA ekspression i peritoneal membran af rotter ved hjælp af kvantitativ real-time PCR

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. ...

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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

Undersøgelse af RNA syntese ved hjælp af 5-Bromouridine mærkning og Immunoprecipitation

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

Kandidat gen test i kliniske kohorteundersøgelser med multipleksede genotypebestemmelse og massespektrometri

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

En Fast og kvantitativ metode til posttranslationel modifikation og Variant aktiveret kortlægning af peptider til genomer

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

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

Induktion og evaluering af indavl krydser ved hjælp af Ant, Vollenhovia Emeryi

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