Name: a method for targeted 16s sequencing of human milk samples
Uploaded: 2018-03-23T12:30:00
Description: Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

Vi vil gjerne takke Helty Adisetiyo, PhD og Shangxin Yang, PhD for utvikling av protokollen. Generell støtte for internasjonale mors Pediatric ungdom AIDS kliniske forsøk gruppe (IMPAACT) ble gitt av National Institute of Allergy og smittsomme sykdommer (NIAID) av den National Institutes of Health (NIH) under prisen tall UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC) og UM1AI106716 (IMPAACT lb), med co-finansiering fra Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) og National Institute of Mental Health (NIMH). Innholdet er ansvar forfattere og representerer ikke nødvendigvis den offisielle synet til NIH.

For alle protokollen trinnene, riktig personlig verneutstyr (PVU) må brukes, og strenge forurensning forebygging tilnærminger skal tas. Observere flyt av arbeid fra før forsterkning arbeidsområder til etter forsterkning arbeidsområder å redusere forurensning av eksempler. Alle rekvisita brukt er sterilt, gratis RNase, DNase, DNA og pyrogen. Alle pipette-spisser filtreres. Et flytskjema protokollen trinnene tilbys (figur 1).
1. sample Lysis
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Mock microbial communities Available from: <a target="_blank" href="https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1">https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1</a> (2017)</li></ol>

Målrettet neste generasjons sekvensering av 16S rRNA er en mye brukt, rask teknikk for microbiome karakterisering18. Men kan mange faktorer, inkludert satsvise effekter, miljøforurensning, prøve kryssforurensning, følsomhet og reproduserbarhet negativt påvirke eksperimentelle resultater og forvirre deres tolkning7,19 , 20. til beste rette robust 16S analyser, microbiome arbeidsflyter må innarbeide g...

De mikrobielle samfunnene som kolonisere mennesker antas å være kritisk viktig for menneskers helse og sykdom som påvirker metabolismen, immun utvikling, mottakelighet for sykdom, og svar til vaksinasjon og stoffet terapi1, 2. innsats for å forstå innflytelsen av bakterieflora på helse nå understreke identifikasjon av mikrober forbundet med definerte anatomiske rom (dvs., hud, gut, muntlig, etc.), samt lokaliserte områder innenfor rom3,4. Underbygger dette undersøkende arbeidet er rask fremveksten og økt tilgjengelighet av neste generasjons sekvensering (NGS) teknologier som gir et massivt parallelle plattform for analyse av mikrobiell genetisk innholdet (microbiome) av en prøve. For mange fysiologiske prøver, den tilknyttede microbiome er både komplisert og rikelig (dvs., avføring), men for noen prøver, microbiome er representert ved lav mikrobiell biomasse (dvs., morsmelk, nedre luftveier) der følsomhet, eksperimentelle gjenstander og mulig smitte bli store problemer. Felles utfordringer microbiome studier og riktig eksperimentell design har vært gjenstand for flere gjennomgang artikkel5,6,7,8.
Presenteres her er en robust NGS eksperimentelle rørledning basert på målrettet sekvensering av rRNA 16S V4 regionen9 å karakterisere microbiome for human melk. Microbiome analyse av morsmelk er komplisert ikke bare som en iboende lav mikrobiell biomasse10, men i tillegg av høye nivåer av menneskelig DNA bakgrunn11,12,13,14 og potensielle carryover PCR hemmere15,16 i utdraget nukleinsyre. Denne protokollen er avhengig av kommersielt tilgjengelig utvinning kits og semi-automatisert plattformer som reduserer variasjon over eksempel forberedelse bunker. Det har et veldefinert bakteriell narr samfunn som behandles sammen med eksempler som en kvalitet kontroll å validere hvert trinn i protokollen og gi en selvstendig beregning av rørledningen robusthet. Selv om protokollen som beskrevet er spesifikke for morsmelk prøvene, det er lett tilpasses andre eksempel typer inkludert avføring, rektal, vaginal, hud, areolar og muntlig vattpinner10,17, og kan tjene som et utgangspunkt for forskere som ønsker å utføre microbiome analyserer.

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">AllPrep RNA/DNA Mini Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">80204</td>
			<td style="width:395px;">DNA/RNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Eliminase</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:153px;">435532</td>
			<td>RNase, DNase, DNA decontaminant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Thermo Mixer</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td>temperature-controlled vortexer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Buffer RLT plus</td>
			<td style="width:115px;">Qiagen</td>
			<td>1053393</td>
			<td>guanidinium thiocyanate lysis buffer/ Part of Allprep kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#223;-Mercaptoethanol&#160;</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td>63689-25ML-F</td>
			<td>&#223;-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">LME Beads</td>
			<td style="width:115px;">MP Biomedicals</td>
			<td>116914050</td>
			<td>bead tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAgen TissueLyzer</td>
			<td style="width:115px;">Qiagen</td>
			<td>85300</td>
			<td>automated sample disruptor adapter set</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAshredder column</td>
			<td style="width:115px;">Qiagen</td>
			<td>&#160;79654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAgen RB tube</td>
			<td style="width:115px;"></td>
			<td></td>
			<td>manufacturer&#39;s microcentrifuge tube in kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAcube and related plasticware</td>
			<td style="width:115px;">Qiagen</td>
			<td>9001292</td>
			<td>automated DNA/RNA purification instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">DNA exitus plus</td>
			<td style="width:115px;">Applichem</td>
			<td>A7089</td>
			<td>non-enzymatic decontamination solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EB Buffer</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">19086</td>
			<td>elution buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAgility and related plasticware</td>
			<td style="width:115px;">Qiagen</td>
			<td>9001532</td>
			<td>robotic liquid handler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR water</td>
			<td style="width:115px;">MO BIO</td>
			<td>17000-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5PRIME HotMasterMix</td>
			<td style="width:115px;">Quantabio</td>
			<td>2200400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Barcoded reverse primers</td>
			<td style="width:115px;">Eurofin</td>
			<td style="width:153px;">No Catalog #&#39;s</td>
			<td>designed and ordered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">&#160;96 well PCR plate</td>
			<td style="width:115px;">USA scientific</td>
			<td style="width:153px;">1402-9708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tapestation 2200 and related plasticware</td>
			<td style="width:115px;">Agilent</td>
			<td>G2964AA</td>
			<td>automated DNA/RNA fragment analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D1000 reagents for Tapestation&#160;</td>
			<td style="width:115px;">Agilent</td>
			<td>5067-5585</td>
			<td>Sample buffer and ladder are part of this kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OneStep PCR Inhibitor Removal Kit&#160;</td>
			<td style="width:115px;">Zymo Research</td>
			<td style="width:153px;">50444470</td>
			<td>PCR inhibitor removal is done per the manufacturer&#39;s instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAquick PCR Purification Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">28104</td>
			<td>DNA clean up kit: silica-membrane-based purification of PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td style="width:153px;">Q32854</td>
			<td>dimethylsulfoxide-based dilution buffer and dye are part of this kit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Qubit Fluorometer</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NanoDrop</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td style="width:153px;"></td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MiSeq 300 V2 kit</td>
			<td style="width:115px;">Illumina</td>
			<td>15033624/15033626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MiSeq&#160;&#160;&#160;</td>
			<td style="width:115px;">Illumina</td>
			<td style="width:153px;">No Catalog #&#39;s</td>
			<td>next generation sequencer</td>
		</tr>
	</tbody>
</table>

a method for targeted 16s sequencing of human milk samples

Studier av mikrobielle samfunn har blitt utbredt med utviklingen av relativt billig, rask og høy gjennomstrømning sekvensering. Men som med alle disse teknologiene, avhenger reproduserbare resultatene en laboratorium arbeidsflyt som inkorporerer forsiktighetsregler og kontroller. Dette er spesielt viktig med lav biomasse eksempler der forurensende bakteriell DNA kan generere misvisende resultater. Denne artikkelen omhandler en semi-automatisert arbeidsflyt for å identifisere mikrober fra menneskelig bryst melk prøver med målrettet sekvensering av regionen 16S ribosomal RNA (rRNA) V4 på en lav og middels throughput skala. Protokollen beskriver eksempel forberedelse helmelk inkluderer: sample lysis, nukleinsyre utvinning, forsterkning av regionen V4 16S rRNA genet og biblioteket forberedelser med kvalitet kontrolltiltak. Viktigere, protokollen og diskusjon vurdere problemer som er fremtredende til forberedelse og analyse av lav-biomasse prøver inkludert aktuelle positive og negative kontroller, PCR hemmer fjerning, prøve forurensning av miljømessige, reagens, eller eksperimentell kilder og eksperimentelle rutiner for å sikre reproduserbarhet. Mens som er spesifikke for morsmelk prøver er det tilpasses mange lav og høy biomasse eksempel typer, inkludert samlet på vattpinner, frosset ryddig eller stabilisert i en bevaring buffer.

Protokollen presenteres her inneholder viktig kvalitetskontroll (QC) trinn for å sikre at data generert møte benchmarks for protokollen sensitivitet og spesifisitet forurensning kontroll. Protokollen QC steg følger PCR forsterkning av 16S V4 regionen (figur 2). En µL av PCR produktet fra hver prøve ble analysert av geleelektroforese å bekrefte at det var innen forventet størrelse 315-450 bp (figur 2, rød pil). Eksempler m...

Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

A Method for Targeted 16S Sequencing of Human Milk Samples

En semi-automatisert arbeidsflyt er presentert for målrettet sekvensering av 16S rRNA fra morsmelk og andre lav-biomasse prøven.

En metode for målrettet 16S sekvensering av morsmelk prøver

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

Research

JoVE Journal

Immunology and Infection

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In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

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The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

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Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Pinne Prøvetaking metode for påvisning av humant Norovirus på overflater

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

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Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

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