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. Lag en trygg arbeidsplass ved Vurderer biosikkerhet betingelser <ol><li> Bli kjent med laboratoriet og sikkerhetsbestemmelser som er av relevans for å arbeide med mikroorganismer. De fleste humanpatogene mikroorganismer skal håndteres på biosikkerhetsnivå 2 forhold, men noen, som for eksempel Salmonella ente serovar typhi, krever biosikkerhetsnivå 3. Opplysninger om nivået håndtere hver patogen kan nås på www.cdc.gov/biosafety. </li><li> Uavhengig av biohazard klassifisering av den spesifikke mikr...

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Den mest kritiske trinnet i opprettelsen av en MSPP ordningen er utvetydig genetisk bestemmelse av biomarkør ion identiteter. Hvis det ikke er mulig å identifisere en biomarkør utvilsomt, så det bør være utelukket fra ordningen 13. Den C. jejuni ssp. doylei Ordningen omfatter 14 ulike biomarkører ioner. Dette er fem færre enn i C. jejuni ssp. jejuni MSPP ordningen 13 sikret viktigste forskjellen mellom påvisbare C. jejuni...

I løpet av det siste tiåret, har matrise-assistert laser desorpsjon ionisering time-of-flight massespektrometri (MALDI-TOF MS) avansert til å være en høyt verdsatt standard metode for mikrobiell slekten og artsbestemmelse i klinisk mikrobiologi 1, 2. Artsbestemmelse er basert på opptak av små protein fingeravtrykk av intakte celler eller cellelysatene. Den typiske masseområde for et massespektrometer som brukes i rutinemessig klinisk mikrobiologi er 2-20 kDa. I tillegg kan det resulterende spektra bli brukt til å diskriminere stammer ved under-arter og underarter under-nivå 3. Tidlige banebrytende studier har identifisert konkrete biomarkør ioner for en bestemt undergruppe av stammer i Campylobacter jejuni 4, Clostridium difficile 5, Salmonella ente ssp. ente serovar typhi 6, Staphylococcus aureus 7-9, og Escherichia coli 10-12. Kombinasjonen av flere variable biomarkør masser tilsvarende allele isoformer gir mulighet for dypere inndeling i undergrupper. Tidligere implementert vi en metode for å konvertere disse variasjoner i masse profiler i meningsfulle og reproduserbare phyloproteomic relasjoner kalles massespektrometri basert phyloproteomics (MSPP) på en C. jejuni ssp. jejuni isolat samling 13. MSPP kan brukes en massespektrometrisk tilsvarende DNA sekvens basert subtyping teknikker som multilocus sekvens typing (MLST). Campylobacter arter er den ledende årsak til bakteriell gastroenteritt verdensomspennende 14, 15. Som en konsekvens av Campylobacteriose post-infektiøs sekvele, nemlig, Guillain-Barré syndrom, reaktiv artritt og inflammatorisk tarmsykdom kan oppstå 16. De viktigste smittekildene erforurenset husdyr kjøtt fra kylling, kalkun, svin, storfe, sau og ender, melk og overvann 15, 17. Derfor vanlige epidemiologiske overvåking studier i sammenheng med mattrygghet er nødvendig. MLST er &quot;gullstandarden&quot; i molekylær typing for Campylobacter arter 18. Fordi Sanger-sekvensering basert MLST metode er arbeidskrevende, tidkrevende og relativt kostbart, er MLST typing begrenset til relativt små isolere kullene. Det er derfor et behov for billigere og raskere subtyping metoder. Dette behovet kan dekkes ved massespektrometri metoder som MSPP. Dette notatet presenterer en detaljert protokoll for MSPP-typing ved hjelp av en samling av Campylobacter jejuni ssp. Doylei isolater og sammenligning av sitt potensial 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 tilbyr muligheten for å skille enkelte bakterier ikke bare på arter og underarter nivå, men til og med under, ved belastningen nivå. Allele isoformer av de påvisbare biomarkør ioner resultere i isolat spesifikke masse skift. Massespektrometri-baserte phyloproteomics (MSPP) er en ny teknikk som kombinerer massespektrometri påvisbare biomarkør massene i en ordning som gjør fradrag for phyloproteomic relasjoner fra isolere bestemte masse skift i forhold til et genom sekvensert referansestamme. Den utledede aminosyresekvenser blir så brukt til å beregne MSPP-baserte dendrogrammer. Her beskriver vi arbeidsflyten av MSPP ved å skrive en Campylobacter jejuni ssp. Doylei isolat samling av syv stammer. Alle de syv stammene var av menneskelig opprinnelse og multilocus sekvens typing (MLST) viste deres genetiske mangfold. MSPP-typing resulterte i syv forskjellige MSPP sekvenstyper, tilstrekkelig gjenspeiler deres grafilogenetic relasjoner. Den C. jejuni ssp. doylei MSPP ordningen omfatter 14 forskjellige biomarkør ioner, for det meste ribosomale proteiner i massen området fra 2 til 11 kDa. MSPP kan i prinsippet tilpasses andre massespektrometri plattformer med en utvidet masse utvalg. Derfor, har denne teknikken potensial til å bli et nyttig verktøy for belastning nivå mikrobiell skrive.

Tidligere har vi opprettet en MSPP ordning for C. jejuni ssp. jejuni 13. Her har vi som mål å utvide metoden til søsken underarter C. jejuni ssp. doylei. I denne bestemt innstilling, syv C. jejuni ssp. doylei isolater ble kjøpt fra den belgiske samling av mikroorganismer / Laboratory of Microbiology UGent BCCM / LMG Ghent, Belgia. Alle de syv isolater som brukes for våre analyser var av menneskelig opprinnelse. Genomet...

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-baserte phyloproteomics (MSPP) ble brukt til å skrive en samling av Campylobacter jejuni ssp. Doylei isolerer på stammenivå i forhold til multilocus sekvens typing (MLST).

inndeling i undergrupper av<em&gt; Campylobacter jejuni</em&gt; Ssp.<em&gt; doylei</em&gt; Isolater Bruke massespektrometri-baserte 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 Polymerase Chain Reaction-rflp Genotyping av Toxic 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

Prosedyre for Adaptive Laboratory Utviklingen av mikroorganismer ved hjelp av en kjemostat

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

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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 ...

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Undersøkelse av RNA syntese med 5-Bromouridine merking 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

Kandidaten Gene Testing i klinisk Kohortstudier multiplex genotyperingteknologi 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 rask og kvantitativ metode for post-translasjonell modifikasjon og Variant aktivert tilordning av peptider å 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

Induksjon og evaluering av Inbreeding krysser ved hjelp av maur, Vollenhovia Emeryi

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