Summary

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Published: September 26, 2011
doi:

Summary

量化血浆中HIV – 1 RNA水平和测序单一的HIV – 1病毒载量低于检测限(50-75拷贝/ ml)与个人的基因组是困难的。在这里,我们描述了如何提取和量化使用实时PCR检测方法能够可靠措施,HIV – 1 RNA下降到0.3拷贝/ ml,以及如何放大由单一的基因组测序的病毒基因组病毒载量非常低的样品,血浆病毒RNA。

Abstract

,获得患者的洞察力病毒动态和病毒宿主相互作用,病毒基因放大和量化,在HIV – 1病毒载量低于检测标准检测的限制(低于50-75拷贝/ ml)感染者HIV – 1 RNA是必要的日志自然控制的感染和抗逆转录病毒联合疗法(CART)的那些。

在这里,我们介绍了如何由单一的基因组测序(SGS协议)13,19和如何准确地量化在与病毒载量低(单拷贝检测(SCA)协议)12,20的患者的HIV – 1 RNA病毒的基因组扩增。

单拷贝检测的实时PCR检测的灵敏度取决于被检测血浆量。如果一个单一的病毒基因组是7毫升血浆中检测到,然后报道RNA拷贝数为0.3拷贝/ ml。该检测RNA提取效率的内部控制测试,并可能扩增DNA或污染控制。测量患者样本一式三份。

,现在已经广泛使用,被视为非劳动密集的3,7,12,14,15的单基因组测序法(SGS)的,是一个限制的稀释检测,其中端点cDNA产物稀释96孔蔓延板。根据泊松分布,不到1 / 3的水井时给予的产品,有一个80%的机会的PCR产物被放大的结果,从一个单一的基因分子。 SGS已超过克隆没有受到重采样和PCR引入重组19偏颇的优势。然而,SCA和SGS的扩增成功取决于引物的设计。两种检测为HIV – 1 B亚型,但可以适应不断变化的引物,探针和标准的其他亚型和基因组的其他地区。

Protocol

1。从大量的血浆提取的RNA 在图1和2提供了一个单拷贝检测(SCA)的概述和单基因组测序(SGS)的协议。 要获得7毫升血浆,旋转约14毫升的血液收集在15毫升EDTA,15分钟,在2600 XG(肝素)无制动收集管。移液器(一定要避免巴菲涂层)为15毫升管的血浆。 如果RNA是单拷贝检测(SCA),秒杀Rous肉瘤病毒内部的病毒粒子控制(RCAS)30000份血浆中提取。 RCAS病毒前准?…

Discussion

艾滋病毒1感染者抗逆转录病毒药物联合治疗(车)或自然控制感染病毒载量非常低,通常约1副本每毫升血浆4,11,12,17,18。自然控制患者的病毒载量,往往个人设置1,2,17点左右波动。检测的灵敏度,此处所描述的高度依赖的血浆输入量。一般来说,我们已与7毫升血浆,但取得了积极的成果,可以从少量的血浆,以及根据实际血浆病毒载量。此处所描述的RNA提取方法“,这?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者要感谢的患者参加的HIV – 1的许多研究。

江铃汽车的FM柯比基金会的支持下与美国癌症协会的研究教授。

Materials

Name of Reagent Company Catalogue number Comments
Random hexamers (500 ug/ml) Promega C118A  
Taqman buffer A Applied Biosystems 4304441  
RNAsin (40 U/ul) Promega N211B  
RT enzyme (200 U/ul) Strategene 600107-51  
Amplitaq Gold DNA polymerase Applied Biosystems 4311814 6-pack
Amplitaq 10XGold PCR buffer II Applied Biosystems 4311814 comes with the enzyme
Magnesiumcloride 25 mM Applied Biosystems 4311814 comes with the enzyme
1M Tris-HCl buffer, pH 8.0, 5 mM Invitrogen AM9855G  
Superscript III reverse transcriptase enzyme, 200 U/ul Invitrogen 18080-044  
Superscript III 10X buffer Invitrogen   comes with the enzyme
DTT 100 mM Invitrogen   comes with the enzyme
Platinum Taq DNA polymerase High Fidelity 5 U/ul Invitrogen 11304-102  
10X High Fidelity PCR Buffer Invitrogen 11304-102 comes with the enzyme
Proteinase-K 20 mg/ml Ambion 2546  
Guanidinium Thiocyanate solution 6M FlukaBiochemica 50983  
Glycogen 20 mg/ml Roche 10901393001  
Isopropanol Sigma-Aldrich 19516  
Ethanol 70% Sigma-Aldrich E702-3  
Molecular grade water Gibco/Invitrogen 10977-015  
dNTPs (25 mmol each) Bioline BIO-39025  
Name of Euipment Company Catalogue number Comments
Easy Seal Centrifuge Tubes (16x73mm) Seaton Scientific 6041  
White Delrin crowns Seaton Scientific 4020 Caps for the Easy Seal Tubes
Tube Rack for Optiseal Bell-Top Tubes, 8.9 ml Beckman 361642  
Hex driver ½ inc opening Seaton Scientific 4013  
cap removal tupe Seaton Scientific 4014  
5 ml serological pipettes Costar 4051  
Pipetboy any    
15 ml Transfer pipettes ISC Bioexpress P-5005-7  
5.8 ml Dispossable transfer pipettes, fine tip VWR 14670-329  
15 ml centrifuge tubes Falcon    
1 ml centrifuge tubes any    
Tris buffer Saline tablets Sigma-Aldrich T5030-100TAB  
Ultracentrifuge and rotor Sorval 90SE and T-1270  
96-well PCR plates Biorad HSS-9601  
50 ml Reagent reservoir Costar 4870  
Microamp optical 96-well reaction plate Applied Biosystems N801-0560  
Optical plate covers Applied Biosystems 4333183  
MicroAmp Optical Compression Pad Applied Biosystems 4312639  
Microseal A film Biorad MSA-5001  
2 ml centrifuge tubes Any    
1% agarose gels (E-gel, invitrogen) Invitrogen G-7008-01  
E-base (Invitrogen) Invitrogen EB-M03  

EDTA Collection tubes any brand

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Citazione di questo articolo
Mens, H., Kearney, M., Wiegand, A., Spindler, J., Maldarelli, F., Mellors, J. W., Coffin, J. M. Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays. J. Vis. Exp. (55), e2960, doi:10.3791/2960 (2011).

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