In this video, methods are demonstrated for quantifying and sequencing single HIV genomes from individuals with viral loads of less than 50 copies per milliliter of plasma. First, A tube containing seven milliliters of plasma is centrifuge to palate. The virus RNA is then extracted to quantify the level of HIV one.
A single copy assay is performed in which the RNA is reverse transcribed, loaded onto a 96 well plate with standards and controls and subjected to quantitative real-time PCR. The level of viremia is then calculated based on the input volume of plasma. As few as 0.3 copies per milliliter can be detected to characterize the viral population.
A single genome sequencing assay is performed in which CDNA transcribed from viral RNA is spread over a 96 well plate to reach an endpoint dilution. Then nested PCR is performed to amplify viral genomes. If 30%or fewer of the PCR reactions are positive for a given sample, amplification of a single copy of CDNA is indicated.
Those samples are then sequenced to reveal the nucleotide sequence of individual viral genomes. The high sensitivity of these techniques enables us to monitor the level of Ian patients with undetectable HIV one RNA by the standard assays and understand the mechanisms of natural control. Though we use these methods to study HIV with different primers and an internal ion standard, the same method can be used to study other viruses as well.
Generally, this method is challenging because it is difficult to isolate virus from large volumes of plasma, and it takes a DNA free environment to avoid false positives. Begin this procedure by extracting RNA from patient plasma samples. Transfer seven milliliters of plasma to a clean 15 milliliter tube.
If RNA is to be extracted for the single copy assay, 200 microliters of the internal virion control containing 30, 000 palatable copies of RCAs virus is added at this point. Next to avoid lipids and cellular debris which can interfere with RNA quantification free-spin the plasma at 2, 600 times G for 15 minutes After the free-spin mark, one side of a settin easy seal centrifuge tube with a water resistant marker avoiding any cellular debris and or lipids. Use a pipette to transfer the plasma to the marked settin eae tube.
Then record the input volume of plasma. Fill the tubes with tris buffered saline to the bottom of the threaded neck. Make sure there are no bubbles.
Cap the tubes. Place the samples with the marking facing out in a pre-cool ultracentrifuge rotor. Spin at 170, 000 times G at four degrees Celsius for 30 minutes.
After centrifugation, the pellet will not be visible. Remove the sina, then add 90 microliters of molecular grade water and 10 microliters of protein, ACE K to the viral pellet. Next, ensuring that the tube is at an angle with the pellet immersed in the protein ACE K water mixture.
Place the tube in a 55 degree Celsius water bath and incubate it for 30 minutes. After the incubation, briefly spin the tube to bring the sample to the bottom of the tube. Then following the addition of 10 microliters of 20 milligrams per milliliter glycogen, and 315 microliters of six molar guad dium thiocyanate solution vortex.
Lightly transfer the contents of the tube to a new 1.5 milliliter micro centrifuge tube containing 495 microliters of molecular grade isopropanol vortex for 10 seconds. Then centrifuge at 21, 000 times G for 30 minutes at room temperature, discard the supernatant and add 500 microliters of 70%ethanol to the tube vortex for 10 seconds and centrifuge at 21, 000 times G for 15 minutes. At room temperature using a large transfer pipette, remove the ethanol spin again and remove any residual ethanol and then air dry the pellet for 10 minutes.
If a single copy assay will be performed, dissolve the pellet in 55 microliters of RNA buffer and place the tube on ice. If single genome sequencing will be performed. Dissolve the RNA in 40 microliters of tris hydrochloride at pH 8.0 and place the tubes at room temperature to perform a single copy assay.
The HIV one and RCAs standard curves are prepared by performing half log serial dilution of HIV one and RCAs RNA transcripts in RNA buffer. After labeling the 1.5 milliliter tubes, add 54 microliters of RNA buffer to each of the tubes. Then dilute down to 0.3 RNA copies for 10 microliters of HIV one and 100 RNA copies per 10 microliters for RCAs.
Mix by vortexing between each dilution. After 54 microliters of RNA buffer is added to each tube. Begin with then adding 25 microliters of the transcript to the first tube vortex pulse, spin and transfer to the next tube consecutively and continue down to the last labeled tube.
Once the dilution are prepared, combine the reverse transcriptase or RT cocktail for CD NA synthesis. Make enough for 96 R-T-P-C-R reactions, including eight no reverse transcriptase or NRT control reactions to control for amplification from DNA. Next, set up an optical 96 well plate, which will hold eight patient samples including standards and controls.
Add 20 microliters of the NRT reaction mixture to the eight NRT designated wells and add 20 microliters of RT reaction mixture to the remaining 88 wells. After adding the RT and NRT mixtures to the plate, add 10 microliters of water to wells labeled to indicate that they are the negative controls. Then add 10 microliters of HIV transcripts to the wells labeled HIV in concentrations shown on the accompanying written protocol, two wells labeled RCAs.
Add 10 microliters of RCAs transcripts in the desired concentrations in the eight sets of three adjacent wells labeled sample on the plate, add 10 microliters of the alluded sample. Also add 10 microliters of this sample to the eight wells labeled NRT then to the wells labeled internal control. Had five microliters of sample and five microliters of water when complete.
The 96 well plate is set up as shown in this figure. Seal the plate and run on a thermocycler as follows, 25 degrees Celsius for 15 minutes. 42 degrees Celsius for 40 minutes, 85 degrees Celsius for 10 minutes, 25 degrees Celsius for 30 minutes and hold at five degrees Celsius.
Prepare the PCR master mixes for HIV and RCAs while the CDNAs being synthesized. When the CDNA synthesis is complete. Take the plate to an area outside the clean room designated for handling CDNA there.
Add 20 microliters of H-I-V-P-C-R master mix for QPCR to each of the wells shown in green in this figure. Then add 20 microliters of RCAs PCR master mix to the well shown in gray in this figure. Spin the plate at 2, 600 times g following the spin.
Place the plate in a realtime PCR instrument and run the QPCR using the following conditions, 95 degrees Celsius for 10 minutes. Then 45 cycles of 95 degrees Celsius for 15 seconds and 60 degrees Celsius for one minute. Once the results have been obtained, separate analysis is required for RCAs and HIV to perform single genome sequencing.
Begin by synthesizing CD NA using RNA suspended in tris hydrochloride at pH 8.0 as a template. First in a 96 well PCR plate. Combine five microliters of 10 millimolar DN NTPs five microliters of the two millimolar gene specific primer, either pole or en and 40 microliters of the extracted RNA seal the plate with a micros eal a film from BioRad.
Then incubate the reaction for 10 minutes at 65 degrees Celsius to denature. The RNA mixture. Make up the RT reaction mixture while the RNA is denaturing.
Note that this is a different RT reaction mixture than what is used for the single copy assay. After the denaturing step, place the PCR plate on ICE to keep the RNA in the denatured form. Then add 50 microliters of the freshly made RT reaction mixture to each sample and run the PCR plate on the thermocycler with the following settings, 45 degrees Celsius for 50 minutes, 85 degrees Celsius for 10 minutes and four degrees Celsius for hold.
During this step, the RNA is reverse transcribed into CD NA while the cd NA is being synthesized. Prepare the PCR reaction mixture with either P six through RT or envelope PCR primers. Note that this is a different PCR reaction mixture than what is used for the single copy assay at 8.0 microliters of the PCR reaction mixture, freshly prepared into each of 73 wells on a new 96 well PCR plate, including 70 samples and three negative controls.
When the CD NA synthesis is completed, take the plate to an area outside the clean room designated for handling CD NA.Next, add 40 microliters of tris hydrochloride at pH 8.0 to the CD NA sample to bring the final volume to 140 microliters. Then add 2.0 microliters of sample to each of the 70 wells and 2.0 microliters of water to each of the negative controls. Seal a PCR plate and run the PCR using the program shown here.
Next, perform nested PCR according to the instructions in the accompanying written text. Following the PCR run samples on a 1%agarro gel. Inspect the results.
If no more than 30%of the wells are positive, the majority of the PCR products are the result of a single molecule of CDNA. A single copy assay was performed as described in this video. This figure shows the QPCR results from some of the elite controllers.
As can be seen here, the amplification curves for samples and the diluted HIV standard look correct as seen here. The recovery of the internal virion standard was above 1500 copies for most samples except sample seven circled. Ensuring an efficient RNA extraction highlighted in red are the results from sample three.
The copy number in the three wells were zero, one and zero copies. The NRT control highlighted in orange and the negative control highlighted in yellow were both negative, confirming that amplification was not biased by contamination from other samples or from DNA with the positive and negative controls having passed, the copy number can now be calculated. The average copy number of 0.3 has to be multiplied with a factor of 5.5 to get the total copy number in the RNA elution 0.3 times 5.5 equals 1.65, and then divided by the total volume of plasma analyzed, which was 7.3 milliliters, giving an HIV one RNA of 0.2 copies per ml.
The single genome sequencing assay was performed as described in the video. This figure shows an AROS gel from one of the HIV one non controllers in the study. The negative controls in, well, H one and H two are both negative.
Less than one third of the wells have amplified ensuring that this is an endpoint dilution, and most of these amplifications are from a single copy of CDNA. An alignment of the sequences from the SGS is shown here. There are no ambiguity sites ensuring that amplification was from a single copy of CDNA While attempting RNA extraction from large volumes of plasma.
It's important to remember to perform de pre spin to avoid cells and lipids that can interfere with the efficiency of the assay After its development. This technique has been used to investigate the viral dynamics in a wide variety of patient groups with viral loads below the limit of detection by standard assays.
