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. Prepare um local de trabalho seguro, considerando condições de biossegurança <ol><li> Familiarize-se com os regulamentos de laboratório e de segurança que são de relevância para trabalhar com microorganismos. A maioria dos microorganismos patogênicos humanos devem ser tratados a nível de biossegurança 2 condições, mas alguns, como a Salmonella enterica sorotipo Typhi, exigem biossegurança nível 3. Informações sobre nível de tratamento de cada agente patogénico pode ser acessado no www.cdc...

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O passo mais crítico no estabelecimento de um regime de MSPP é a determinação genética inequívoca de identidades biomarcador íon. Se não for possível identificar um biomarcador, sem dúvida, então ele deve ser excluído do regime 13. A C. ssp. doylei esquema jejuni inclui 14 diferentes íons de biomarcadores. Estes são menos 5 em comparação com o C. jejuni ssp. esquema jejuni MSPP 13 .A diferença mais significat...

Durante a última década, laser assistida por matriz dessorção ionização espectrometria de tempo-de-voo de massa (MALDI-TOF MS) tem avançado para ser um método padrão altamente valorizado por género microbiana e identificação de espécies em microbiologia clínica 1, 2. A identificação da espécie é baseada no registo de impressões digitais pequenas proteínas de células intactas ou de lisados ​​celulares. A faixa de massa típica para um espectrómetro de massa usado em microbiologia clínica de rotina é 2-20 kDa. Além disso, o espectro resultante pode ser utilizado para discriminar estirpes com as espécies e abaixo-abaixo-subespécies de nível três. Estudos pioneiros iniciais identificaram iões de biomarcadores específicos para um subgrupo particular de estirpes de Campylobacter jejuni 4, 5 Clostridium difficile, Salmonella enterica ssp. enterica serovar Typhi 6, Staphylococcus aureus 7-9, e Escherichia coli 10-12. A combinação de várias massas de biomarcadores para variáveis ​​correspondentes isoformas alélicas oferece a opção de subtipagem mais profunda. Anteriormente, foi implementado com sucesso um método para converter essas variações nos perfis de massa em relações phyloproteomic significativos e reprodutíveis chamados massa phyloproteomics baseados espectrometria (MSPP) em um C. ssp jejuni. coleção isolado jejuni 13. MSPP pode ser usado uma espectrometria de massa equivalente a técnicas de subtipagem baseada na sequência de DNA como digitação sequência multilocus (MLST). Espécies de Campylobacter são a principal causa de gastroenterite bacteriana em todo o mundo 14, 15. Como consequência da Campilobacteriose sequela pós-infecciosa, ou seja, síndrome de Guillain Barré, artrite reactiva e doença inflamatória intestinal pode surgir 16. As principais fontes de infecção sãocarne de gado contaminado de frango, peru, suínos, bovinos, ovinos e patos, leite e água de superfície 15, 17. Portanto, estudos de vigilância epidemiológica regulares no contexto da segurança alimentar são necessários. MLST é o &quot;padrão ouro&quot; na tipagem molecular de espécies Campylobacter 18. Uma vez que a sequenciação de Sanger-base MLST método é um trabalho intensivo, que consome tempo e relativamente cara, é restrito MLST tipagem de relativamente pequenas coortes isolados. Por conseguinte, existe uma necessidade de métodos de subtipagem barata e mais rápida. Essa necessidade pode ser satisfeita através de métodos de espectrometria de massa como MSPP. Este artigo apresenta um protocolo detalhado para MSPP-digitação usando uma coleção de Campylobacter jejuni ssp. Doylei isolados e comparação de seu potencial com 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 oferece a possibilidade de diferenciar algumas bactérias não só ao nível de espécies e subespécies, mas ainda abaixo, a nível da estirpe. isoformas alélicas dos íons de biomarcadores detectáveis ​​resultar em deslocamentos de massa específica do isolados. phyloproteomics à base de espectrometria de massa (MSPP) é um nova técnica que combina as massas espectrometria de massa de biomarcadores detectáveis ​​em um esquema que permite a dedução de relações phyloproteomic de deslocamentos de massa específicos do isolado em comparação com uma estirpe de referência do genoma sequenciado. As sequências de aminoácidos deduzidas são então usados ​​para calcular dendrogramas baseados em MSPP. Aqui nós descrevemos o fluxo de trabalho de MSPP digitando um ssp Campylobacter jejuni. Doylei coleção isolado de sete cepas. Todas as sete cepas eram de origem humana e multilocus digitação sequência (MLST) demonstraram a sua diversidade genética. MSPP-digitação resultou em sete tipos de seqüência MSPP diferentes, refletindo suficientemente sua phyrelações logenetic. A C. jejuni ssp. doylei esquema MSPP inclui 14 diferentes íons de biomarcadores, proteínas principalmente ribossômicas na faixa de massa de 2 a 11 kDa. MSPP pode, em princípio, ser adaptado para outras plataformas de espectrometria de massa com uma faixa de massa estendida. Portanto, esta técnica tem o potencial para se tornar uma ferramenta útil para nível de tensão tipagem microbiana.

Anteriormente, nós estabelecemos com sucesso um esquema MSPP para C. ssp jejuni. jejuni 13. Aqui, o objetivo foi estender o método para o irmão subespécie C. ssp jejuni. doylei. Neste cenário específico, sete C. jejuni ssp. doylei isolados foram adquiridos a partir da coleção belga de microorganismos / Laboratório de Microbiologia UGent BCCM / LMG Ghent, Bélgica. Todos os sete isolados utilizados...

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 à base de espectrometria (MSPP) foi usado para digitar uma coleção de Campylobacter jejuni ssp. Doylei isola a nível da estirpe em comparação com digitação sequência multilocus (MLST).

subtipagem de<em&gt; Campylobacter jejuni</em&gt; Ssp.<em&gt; doylei</em&gt; Isolados Utilizando PhyloProteomics baseados em espectrometria de massa (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

Melhoria Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Genotipagem de Pufferfish Tóxico por Cromatografia Líquida / Espectrometria de Massa

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

Genética

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Processo para o Adaptive Laboratório Evolução de microrganismos utilizando um quimiostato

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

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Detecção de Copy Number Alterações Usando Sequencing única célula

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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Detecção da expressão de microRNA na Membrana Peritoneal de Ratos Usando PCR Quantitativa em Tempo Real

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

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Análise do Promotor de Ligando C-KIT Usando Imunoprecipitação de Cromatina

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

Etiquetando metabólica e caracterização das RNAs de transferência usando Macroarrays

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

Investigação da síntese do RNA usando 5-Bromouridine rotulagem e imunoprecipitação

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

Gene candidato teste em estudos de coorte clínica com genotipagem multiplexada e espectrometria de massa

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

Um método rápido e quantitativo para a modificação pós-traducional e variante habilitado para mapeamento de peptídeos de genomas

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

Indução e avaliação de cruzes de endogamia utilizando o 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 ...