测序外周血中细菌菌群：我们的经验与艾滋病毒感染者
Summary
我们的实验将展示如何执行转位HIV阳性患者的外周血中的细菌种类的测序分析。
Abstract
生理健康的胃肠道是殖民地的影响共生微生物的体液免疫和细胞的粘膜免疫系统1,2的发展的种类繁多。
屏蔽的微生物是通过一个强大的黏膜屏障的免疫系统。感染和抗生素被称为改变正常的消化道障碍和居民的细菌组成，这可能会在可能的免疫异常导致3。
艾滋病毒会导致违反与胃肠黏膜免疫和全身血液循环进入细菌的生物制品，如内毒素和细菌的DNA片段，这有助于全身免疫激活4-7泄漏的渐进破坏屏障。在艾滋病毒/艾滋病的免疫病理及治疗 4,8微生物易位是牵连。
我们的目的是表征转位HIV感染患者的外周血中的细菌组成。为了追求我们的目标，我们成立了ribosomial panbacteric 16S基因测序分析PCR反应。
简单地说，艾滋病毒感染者和正常人全血使用。在这些患者中预计，鉴于目前健康的个人正常的肠道动态平衡没有菌群易位。静脉穿刺的全血和血浆分离收集，DNA是从血浆中提取和使用执行范围广泛的PCR反应ribosomial panbacteric 16S 基因 9 。执行下面的PCR产物纯化，克隆和测序分析。
Protocol
对感染艾滋病毒的血液样本的处理需要一些重要的建议。 所有血液标本必须在强大的防漏容器运送。采集标本时，必须小心，以避免污染容器的外观和附带任何手续的标本。 所有处理受感染的血液的人必须戴手套。手套必须改变和标本的处理完成后洗净双手。 艾滋病毒感染者的血液样品的处理，必须做到在II级生物危害柜罩。 应采用机械吹打艾滋病。 必须加以限制，其中…
Discussion
<p class="jove_content"在此，我们显示的pcr ><p class="jove_content">最可靠的结果是使用EDTA含管收集血浆时获得。采血后，血浆应通过离心分离2 / 3小时内，以避免溶血和DNA片段退化。第一样本储存在-20 ° C，约5天，然后在-80 ° C应避免多个冻融循环，这可能会导致细菌的DNA损失。</p><p class="jove_content">血浆样品应解冻后12小时内使用。</p><p>…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们非常感谢詹尼Scimone与视频制作的优秀援助。
Materials
| Name of Reagent | Company | Catalogue Number | Comments (optional) |
|---|---|---|---|
| Easty-DNA Kit | Invitrogen | K 180001 | |
| Chloroform | Sigma-Aldrich | C2432 | |
| Ethanol | Sigma-Aldrich | E7203 | |
| UltraPure Waer | Invitrogen | 10977049 | |
| Lysozyme | Fluka | 62970 | 10 μg/mL in Distillated Water |
| AmpliTaq Gold | Applied Biosystem | 26478701 | |
| Microcon 100 | Millipore | 42413 | |
| PCR primers | Invitrogen | ||
| Agarose | Eppendorf | C1343 | |
| DNA ladder | Invitrogen | 15628019 | |
| Purelink PCR micro kit | Invitrogen | K310050 | |
| LB Agar, powder | Invitrogen | 22700025 | |
| Bactoagar | Invitrogen | ||
| Ampicillin | Invitrogen | 11593027 | 10 mg/mL in Distillated Water |
| X-gal | Invitrogen | 15520034 | 40mg/mL in DMF |
| IPTG | Invitrogen | 15529019 | 100mM in Distillated water |
| Topo TA cloning kit | Invitrogen | K450002 | |
| Purelink Quick Plasmid Miniprep kit | Invitrogen | K210010 | |
| Big dye | Applied Biosystem | 4337455 | |
| DyeEx 2.0 spin kit | Qiagen | 63204 |
Riferimenti
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Tags
SequencingBacterial MicrofloraPeripheral BloodHIV-infected PatientsGastrointestinal TractCommensal MicrobesMucosal Immune SystemMicrobiotaImmune AbnormalitiesBreach In The Gastrointestinal BarrierMucosal ImmunitySystemic CirculationBacterial BioproductsLipopolysaccharideBacterial DNA FragmentsSystemic Immune ActivationMicrobial TranslocationHIV/AIDS ImmunopathogenesisResponse To TherapyCharacterise The Composition Of Bacteria Translocating In Peripheral BloodPCR ReactionPanbacteric 16S Ribosomal GeneSequencing Analysis
Citazione di questo articolo
Merlini, E., Bellistri, G. M., Tincati, C., d’Arminio Monforte, A., Marchetti, G. Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients. J. Vis. Exp. (52), e2830, doi:10.3791/2830 (2011).