Name: amplifying and quantifying hiv-1 rna in hiv infected individuals with viral loads below the limit of detection by standard clinical assays
Uploaded: 2011-09-26T12:00:00
Description: Watch this Scientific Journal Video about Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays at JoVE.com

The authors wish to acknowledge the patients who participated in the many studies of HIV-1.
J.M.C. was a Research Professor of the American Cancer Society with support from the F.M. Kirby Foundation.

1. Extraction of RNA from large volumes of plasma
An overview of the single copy assay (SCA) and the single genome sequencing (SGS) protocol are provided in Figures 1 and 2.
<ol>
 <li>To obtain 7 ml of plasma, spin approximately 14 ml of blood collected in 15 ml EDTA (not heparin) collection tubes at 2,600 xg for 15 minutes without braking. Pipette plasma (making sure to avoid the buffy coat layer) into 15 ml tubes.</li>
 <li>If RNA is being extracted for the single copy assay (SCA), spike t...

<ol>
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HIV-1 infected individuals on combination antiretroviral treatment (cART) or who naturally control the infection have very low viral loads, typically around 1 copy per ml of plasma4, 11, 12, 17, 18. Viral loads in patients with natural control, often fluctuate around an individual set point1, 2, 17. The sensitivity of the assays described herein is highly dependent on the input volume of plasma. Generally, we have been working with 7 ml of plasma, but positive results can be obtained from smaller am...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Random hexamers (500 ug/ml)</td>
			<td>Promega</td>
			<td>C118A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Taqman buffer A</td>
			<td>Applied Biosystems</td>
			<td>4304441</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNAsin (40 U/ul)</td>
			<td>Promega</td>
			<td>N211B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RT enzyme (200 U/ul) </td>
			<td>Strategene</td>
			<td>600107-51</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amplitaq Gold DNA polymerase</td>
			<td>Applied Biosystems</td>
			<td>4311814</td>
			<td>6-pack</td>
		</tr>
		<tr>
			<td>Amplitaq 10XGold PCR buffer II</td>
			<td>Applied Biosystems</td>
			<td>4311814</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>Magnesiumcloride 25 mM</td>
			<td>Applied Biosystems</td>
			<td>4311814</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>1M Tris-HCl buffer, pH 8.0, 5 mM</td>
			<td>Invitrogen</td>
			<td>AM9855G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III reverse transcriptase enzyme, 200 U/ul </td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III 10X buffer</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>DTT 100 mM</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>Platinum Taq DNA polymerase High Fidelity 5 U/ul</td>
			<td>Invitrogen</td>
			<td>11304-102</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X High Fidelity PCR Buffer</td>
			<td>Invitrogen</td>
			<td>11304-102</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>Proteinase-K 20 mg/ml</td>
			<td>Ambion</td>
			<td>2546</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Guanidinium Thiocyanate solution 6M</td>
			<td>FlukaBiochemica</td>
			<td>50983</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycogen 20 mg/ml</td>
			<td>Roche</td>
			<td>10901393001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>19516</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Sigma-Aldrich</td>
			<td>E702-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Molecular grade water </td>
			<td>Gibco/Invitrogen</td>
			<td>10977-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dNTPs (25 mmol each) </td>
			<td>Bioline</td>
			<td>BIO-39025</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<td>Name of Euipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Easy Seal Centrifuge Tubes (16x73mm)</td>
			<td>Seaton Scientific</td>
			<td>6041</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>White Delrin crowns </td>
			<td>Seaton Scientific</td>
			<td>4020</td>
			<td>Caps for the Easy Seal Tubes</td>
		</tr>
		<tr>
			<td>Tube Rack for Optiseal Bell-Top Tubes, 8.9 ml</td>
			<td>Beckman</td>
			<td>361642</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hex driver &frac12; inc opening</td>
			<td>Seaton Scientific</td>
			<td>4013</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>cap removal tupe</td>
			<td>Seaton Scientific</td>
			<td>4014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ml serological pipettes</td>
			<td>Costar</td>
			<td>4051</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipetboy</td>
			<td>any</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml Transfer pipettes </td>
			<td>ISC Bioexpress</td>
			<td>P-5005-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5.8 ml Dispossable transfer pipettes, fine tip</td>
			<td>VWR</td>
			<td>14670-329</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml centrifuge tubes</td>
			<td>Falcon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml centrifuge tubes</td>
			<td>any</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris buffer Saline tablets</td>
			<td>Sigma-Aldrich</td>
			<td>T5030-100TAB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge and rotor </td>
			<td>Sorval</td>
			<td>90SE and T-1270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-well PCR plates</td>
			<td>Biorad</td>
			<td>HSS-9601</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml Reagent reservoir </td>
			<td>Costar</td>
			<td>4870</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microamp optical 96-well reaction plate</td>
			<td>Applied Biosystems</td>
			<td>N801-0560</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optical plate covers </td>
			<td>Applied Biosystems</td>
			<td>4333183</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Compression Pad</td>
			<td>Applied Biosystems</td>
			<td>4312639</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microseal A film</td>
			<td>Biorad</td>
			<td>MSA-5001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 ml centrifuge tubes </td>
			<td>Any</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1% agarose gels (E-gel, invitrogen)</td>
			<td>Invitrogen</td>
			<td>G-7008-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E-base (Invitrogen)</td>
			<td>Invitrogen</td>
			<td>EB-M03</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
EDTA Collection tubes any brand

amplifying and quantifying hiv-1 rna in hiv infected individuals with viral loads below the limit of detection by standard clinical assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Watch this Scientific Journal Video about Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays at JoVE.com

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...