Protocol for Quantification of Integrated HIV DNA
Authors: Sofie Rutsaert, Eva Malatinkova, Pawel Bonczkowski, Maja Kiselinova, Ward De Spiegelaere

MATERIALS

REAGENTS (Note 1)
DNA extraction kit (Qiagen DNeasy Blood & Tissue kit, cat. 69504/69506)
GoTaq® G2 DNA Polymerase (Promega, cat. M7845)
Nuclease-free water (Qiagen, cat. 129114)
dNTP mix (Promega, cat. U1511)
Primers (see Table 1, ordered at Integrated DNA Technologies)
LightCycler® 480 Probes Master, 2x concentrated (Roche Applied Science, cat. 04707494001)

EQUIPMENT
Thermal Cycler Applied Biosystems® 2720 (cat. 4359659)
LightCycler® 480 System II (Roche, cat. 05015278001)

Primers

Sequence

1st PCR (Alu-Gag PCR)

Forward primer Alu(1)

5’-GCCTCCCAAAGTGCTGGGATTACAG-3’

Reverse primer Gag(1)

5’-GTTCCTGCTATGTCACTTCC-3’

2nd PCR (qPCR)

Forward primer RU5 (R Forward)(2)

5’-TTAAGCCTCAATAAAGCTTGCC-3’

Reverse primer RU5 (U5 Reverse)(2)

5’-GTTCGGGCGCCACTGCTAGA-3’

Probe(3)

5'-CCAGAGTCACACAACAGACGGGCACA-3'
(1) Liszewski et al. 2009, (2) Yu et al. 2008, (3) Yun et al. 2002

PROCEDURE (download as PDF)

Isolate genomic DNA from the target cells using the DNA extraction kit. To estimate the volume of material used for the input in PCR reaction, one can either use a fixed number of input cell equivalents, e.g. 7500 equivalents per reaction as described by (1) Liszewski et al. 2009, , or adjust input to the amount of total HIV DNA, as estimated by a prior total HIV DNA quantification by real time or digital PCR (Note 2).

INTRODUCTORY INFORMATION

The Alu-HIV PCR is designed to quantify integrated proviral HIV DNA through a nested PCR setup of two consecutive PCR amplification rounds. In the first PCR forward primers are used for the human Alu repeat sequence in combination with a reverse primer, targeting an HIV specific sequence (fig. 1). This reaction only leads to a logarithmic amplification of HIV DNA when this DNA is integrated, and thus in the vicinity of an Alu sequence. Unintegrated linear or episomal HIV DNA is amplified in a linear faschion. The second PCR is composed of a quantitative PCR (qPCR) that amplifies an HIV specific sequence which is nested within the amplicon of the first PCR. To enable the differentiation of the background signal originating from unintegrated HIV DNA, a control is run in parallel using only an HIV reverse primer to mimic the kinetic PCR that would take place when unintegrated HIV DNA is present.
Workflow
Figure 1: Schematic illustration of the Alu-PCR workflow. Showing the nested Alu-HIV PCR and the HIV-only PCR (background control).

  1. Depending on the origin of the DNA for analysis (patient samples or cell cultures), the input for the 1st PCR need to be selected. For patient samples, the reaction works best if the input is in the range of 1-2 HIV DNA copies/well (Note 3).

  2. To maximize the power of the analysis, 40 replicates for the Alu-Gag primers combination and minimally 20 for Gag only primer (background) are performed. If enough material is present, an equal number of Gag-only and Alu-Gag replicates is preferred (Note 4).

 THE FIRST PCR REACTION

  1. Prepare the mix for the appropriate number of replicates consisting of:

PROMEGA GoTaq

Alu-gag mix
µl/reaction

gag-only mix
µl/reaction

Buffer (5X)

 4 4

dNTP's (10mM)

 0.4 0.4

H2O

 4 4.2

GoTaq

 0.2 0.2

Forward primer (nM)

 0.2

Reverse primer (nM)

 1.2 1.2
The total volume adds up to 10 µl per reaction

  1. Distribute the mix in a 96 well plate (10 µl per well) taking the amount of replicates of both Alu-Gag and Gag only into account, add 2 wells of each for the positive and negative controls.

  2. Add 10 µl of the DNA solution to each well except for the controls. In the controls, add 9 µl of water and 1 µl of control DNA (from the integration standard) to the positive controls, and 10 µl of water to the negative controls.

  3. Seal the plate and run the PCR protocol in the Thermal Cycler, using the following cycling conditions:

Reaction details

Step

 °C

time
1X initial denaturation 95 02:00
denaturation 95 00:15
40X annealing 50 00:15
  elongation 70 03:30


THE SECOND PCR REACTION

  1. The relative quantification is performed with a qPCR on the PCR product from the first PCR step

  2. Prepare the qPCR mix consisting of:

 

µl/reaction

PCR mastermix

5

H2

 2

Probe (10 µM)

 0.2

Forward primer (10 µM)

 0.4

reverse primer (10 µM)

 0.4
The total volume adds up to 10 µl per reaction

  1. Distribute the mix in a 384 well plate, 8 µl per well.

  2. To each of the wells, add 2 µl of the 1st PCR solution from a corresponding well. Maintain the number of replicates, use each replicate only once.

  3. Run the reaction on LightCycler® 480 System II (Roche).

  4. The cycling conditions are:

Reaction details

Step

 °C

time
1X initial denaturation 95 05:00
45X denaturation 95 00:15
  annealing 60 01:00


ANALYSIS

To assess the number of integrations per cell, the excel template (Fig. 2) can be used. The raw quantitative output of the Alu-HIV PCR as Cq values (Cycle of quantification, also termed Ct) should be inserted as described below.
template
Figure 2: the excel template composed for Alu-HIV PCR quantification: the colored cells in the lower left quadrant require data input, i.e. the correction factor as measured on the standard for calibration to assess the fold difference between the observed and expected number of integrations/well, the total number of Alu-HIV replicates (required for accurate error estimation) the number of cells per replicate reaction (assessed by a qPCR on a reference gene, e.g. RPP30), and the raw quantification cycle (Cq)* values of the Alu-HIV PCR and the HIV only PCR. Note that negative reactions should be left empty not to interfere with the analysis. After data input the results are shown in the shaded areas of the lower right quadrant,
*note that the Cq values are in some real-time PCR programs referred to as treshold cycle (Ct), crossing point (Cp) or take-off point (TOP)



REFERENCES

Liszewski MK, Yu JJ, O'Doherty U: Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR. Methods 2009;47:254-60. url

De Spiegelaere W, Malatinkova E, Lynch L, Van Nieuwerburgh F, Messiaen P, O'Doherty U, Vandekerckhove L: Quantification of Integrated HIV DNA by Repetitive-Sampling Alu-HIV PCR on the Basis of Poisson Statistics. Clin Chem 2014. url

Yu JJ, Wu TL, Liszewski MK, Dai J, Swiggard WJ, Baytop C, Frank I, Levine BL, Yang W, Theodosopoulos T, O'Doherty U: A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients. Virology 2008;379:78-86. url

Yun Z, Frederiksson E,Sönnerborg A: Quantification of human immunodeficiency virus type 1 proviral DNA by the TaqMan real-time PCR assay. Journal of Clinical Microbiology 2002;40:3883-3884. url

NOTES

  1. These reagents and materials are being used by us. An alternative method for Alu-HIV PCR has also been described by Liszewsky et al (2009)

  2. Using a fixed number of cell equivalents provides a standardized reaction. However, in patient samples with limited HIV DNA, most reactions become negative which may limit the quantitative power of the Poisson calculation afterwards. By estimating the number of HIV DNA templates before Alu-HIV PCR, the concentration of HIV DNA templates can be estimated to get an optimal Poisson quantification.

  3. We generally start with 10 DNA copies for cell cultures per reaction, considering the fact that cell culture generally have a higher concentration of unintegrated HIV DNA, compared to patient derived samples.

  4. We use a cut-off of five positive Gag-only PCR reactions to estimate the background. If this number is not reached, the amount of Gag-only PCR replicates should be augmented.