Name: subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)
Uploaded: 2016-10-30T12:30:00
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. 바이오 안전성 조건을 고려하여 안전한 작업 환경을 준비 <ol><li> 미생물 작업을위한 관련이있는 실험실 및 안전 규정에 익숙해집니다. 대부분의 인간의 병원성 미생물, 바이오 안전성 수준에서 살모넬라 엔테의 혈청 형의 Typhi 등이 조건에 있지만, 일부를 처리 바이오 안전성 레벨을 www.cdc.gov/biosafety에 액세스 할 수있는 각 병원체를 처리 수준 3. 정보를 요구해야합니다. </li><li> 에 관계?...

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MSPP 방식의 설립에서 가장 중요한 단계는 바이오 마커 이온 정체성의 명확한 유전 적 결정이다. 그것은 틀림없이 바이오 마커를 식별 할 수 없다면, 이것은 반응식 13에서 제외한다. C. jejuni와의 SSP. doylei 방식은 14 개 바이오 마커 이온을 포함한다. 이 5 이하 C. 비교입니다 검출 가능한 C. 사이 jejuni와의 SSP. jejuni와 MSPP 방식 (1...

지난 십년 동안, 매트릭스 보조 레이저 탈착 이온화 비행 시간 형 질량 분석계 (MALDI-TOF MS)가 임상 미생물학 1,2 미생물 속과 종의 식별을위한 높은 값의 표준 방법으로 진행 하였다. 종 식별은 그대로 세포 또는 세포 용 해물의 작은 단백질 지문의 기록에 기초한다. 일상적인 임상 미생물에서 사용되는 질량 분석 장치의 일반적인 질량 범위는 2-20 kDa 이상이다. 또한, 결과적인 스펙트럼은 다음 종의 균주 및 아종 이하 레벨 3을 구별하는데 사용될 수있다. 초기 선구적인 연구는 캄 필로 박터 jejuni와 4 균주의 특정 하위 그룹, 클로스 트리 디움 디피 5, 살모넬라 엔테 SSP에 대한 특정 바이오 마커 이온을 확인했다. 엔테의 혈청 형 Typhi (6), 황색 포도상 구균 7-9, 및 E12 - scherichia 10 대장균. 대립 유전자 아형에 해당하는 여러 변수 바이오 마커 대중의 조합은 더 깊은 하위 유형에 대한 옵션을 제공합니다. 이전에, 우리는 성공적으로 C.에 질량 분석 기반 phyloproteomics (MSPP)라는 의미와 재현 phyloproteomic 관계로 대량 프로필에서 이러한 변화를 변환하는 방법을 구현 jejuni와의 SSP. jejuni와 격리 컬렉션 13. MSPP는 multilocus 시퀀스 입력 (MLST)과 같은 DNA 서열을 기초 subtyping이 기술에 질량 분석 당량 사용할 수있다. 캄 필로 박터 종은 세균성 위장염의 주요 원인 전세계 14, 15이다. 캄 필로 박터 감염 후 후유증의 결과, 즉, 길랑 바레 증후군, 반응성 관절염 및 염증성 장 질환 (16)를 발생할 수있다. 감염의 주요 소스는닭, 칠면조, 돼지, 소, 양, 오리, 우유, 표면의 물 (15), (17)로부터 오염 된 가축의 고기. 따라서, 식품 안전의 맥락에서 일정한 역학 감시 연구가 필요하다. MLST는 캄 필로 박터 종 (18)에 대한 분자 입력의 &quot;황금 표준&quot;입니다. MLST 방법을 기반으로 생거 시퀀싱은 노동 집약적, 시간과 상대적으로 고가이기 때문에, MLST 입력은 상대적으로 작은 분리 집단에 제한됩니다. 따라서, 저렴하고 빠른 하위 유형의 방법이 필요하다. 이 필요 MSPP 같은 질량 분석 방법에 의해 충족 될 수있다. 이 논문은 캄 필로 박터 jejuni와 SSP의 컬렉션을 사용하여 MSPP-입력에 대한 자세한 프로토콜을 제시한다. doylei 분리 및 MLST와 잠재력의 비교.

<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 MS는 종 및 아종 수준 그러나 심지어 아래 균주 수준에서뿐만 아니라 일부 박테리아를 차별화 할 수있는 가능성을 제공한다. 검출 가능한 바이오 마커의 대립 이온 이성체 분리는 특정 질량 변화 될. 질량 분석 기반 phyloproteomics (MSPP)를 참조 균주 염기 서열을 게놈에 비해 분리 특정 질량 변화에서 phyloproteomic 관계의 공제를 허용하는 방식으로 질량 분석 검출 바이오 마커의 질량을 결합하는 새로운 기술이다. 추론 된 아미노산 서열은 MSPP 기반 dendrograms을 계산하는 데 사용된다. 여기서 우리는 캄 필로 박터 jejuni와의 SSP를 입력하여 MSPP의 흐름을 설명합니다. 일곱 균주의 분리 수집을 doylei. 모든 일곱 균주는 유전 적 다양성을 보여 인간의 기원과 multilocus 시퀀스 입력 (MLST)의했다. MSPP-입력 충분히 자신의 PHY를 반영 일곱 가지 MSPP 시퀀스 유형의 결과logenetic 관계. C. jejuni와는 SSP. MSPP 방식은 2 ~ 11 kDa의 질량 범위에서 14 개 바이오 마커 이온, 주로 리보솜 단백질을 포함 doylei. MSPP 원칙적으로, 확장 된 질량 범위와 다른 질량 분석 플랫폼에 적용 할 수있다. 따라서,이 기술은 변형 된 미생물 레벨 입력을위한 유용한 도구가 될 가능성이있다.

이전에, 우리는 성공적으로 C.에 대한 MSPP 체계를 확립 jejuni와의 SSP. 13 jejuni와. 여기서, 우리는 C. 형제를 아종하는 방법을 확장하는 목적 jejuni와의 SSP. doylei. 이 특정 설정에서 일곱 C. jejuni와의 SSP. doylei 분리는 / 연구소 미생물학 UGent BCCM / LMG 겐트, 벨기에의 미생물의 벨기에 컬렉션에서 획득 하였다. 우리의 분...

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

질량 분석 기반 phyloproteomics (MSPP)는 캄 필로 박터 jejuni와 SSP의 모음을 입력하는 데 사용되었다. doylei는 multilocus 시퀀스 입력 (MLST)에 비해 변형 수준에서 분리합니다.

의 하위 유형<em&gt; 캄 필로 박터 jejuni와</em&gt; SSP.<em&gt; doylei</em&gt;은 질량 분석 기반 PhyloProteomics를 사용하여 분리 (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

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