CHAPTER TWO
2.2 SAMPLE COLLECTION AND LABORATORY METHODS
2.2.4 HIV-1 RNA-PCR Quantification (viral load)
The viral loads for this pilot study were initially performed at the Africa Centre Laboratory.
Due to the changes within the Africa Centre Department, the viral loads were no longer performed at the Africa Centre Laboratory. Continuation of the viral load testing was performed at the HPP Laboratory from November 2004 until the end of the study.
2.2.4.1 NucliSens
®: Lysis buffer, magnetic extraction reagents and NucliSens EASYQ HIV-1 version -1.2 (Biomerieux)
Principle: The Nucleic acid extraction method is based on magnetic silica particles. Nucleic acids bind to the silica particles under high salt concentrations. The silica acts as a solid phase and non-nucleic acid components are removed by several wash steps using the miniMAG.
The nucleic acids are eluted from the solid phase. NucliSens EasyQ HIV-1 v1.2 reagents are used for the nucleic acid amplification and detection assay for the quantitative determination of HIV-1 RNA in human plasma. The HIV-1 viral loads that can be measured in this assay range from 51 – 5,390,000 RNA copies/ml.
EASYQ HIV-1 NucliSens Magnetic Extraction:
200µl of plasma sample was added to a 2ml aliquot of NucliSens Lysis Buffer in a 15ml Falcon tube. This was vortexed and left to incubate for 10mins at room temperature. 220µl of elution buffer was then added to the calibrator (to reconstitute the lyophilized pellet) and this was vortexed thoroughly. 20µl of the reconstituted calibrator was added to the test sample and
vortexed followed by the addition of 50µl of vortexed silica. The tube was then vortexed thoroughly and incubated for 10mins at room temperature, thereafter centrifuged for 2mins at 1500g. The supernatant was then removed with a pastette and 400µl of wash buffer 1 was pipetted into the sample tube. The wash buffer was mixed with the pellet and this was then transferred to a sterile 1.5ml starsted tube using a sterile pastette. After 30 rotations on the miniMag, the supernatant was removed and the wash step was repeated. After 30 rotations, the supernatant was removed and two wash steps were done with 500µl wash buffer 2. After the supernatant was removed, the final wash was done with 500µl wash buffer 3. The supernatant was removed after 15 rotations on the miniMag. 25µl of the elution buffer was added to the tube and incubated at 600C for 5mins on a shaking incubator. This was followed by the removal of the silica using the miniMag. The extracted RNA was then transferred to a clean 1.5ml starsted tube.
Preparation of mastermix:
ENZYME: 45µl of Enzyme Diluent was added to the lyophilized pellet. After a gentle mix, the tube was left at room temperature for 15mins.
PRIMERS: 90µl of Primer Diluent was added to the lyophilized pellet and vortexed immediately.
Amplification and Detection:
5µl of the extracted RNA was aliquoted into the correctly labeled 8-tube strip. This was followed by the addition of 10µl of the primer. The strip was placed in the NucliSens EasyQ Incubator and the programmed for 1min at 650C, then 2mins at 410C. Thereafter 5µl of the enzyme solution aliquoted into the each of the caps of the strip, the strip was then capped and centrifuged (pulse spin) in the Mini-Strip Centrifuge. The strip was then transferred to the NucliSens EasyQ Analyser for amplification and detection. The results of the test samples were printed in a numerical format.
Internal quality control: The EASYQ HIV-1 NucliSens Magnetic Extraction is designed such that each run comprises of 8 tests. There were no individual control samples; however, the calibrator was incorporated with the test sample. If the calibrator failed then that particular test sample was repeated.
2.2.4.2 Cobas amplification HIV-1 Monitor test, version -1.5 (Roche Diagnostics)
Principle: The COBAS AMPLIFICATION HIV-1 MONITOR TEST, version-1.5 is an in vitro nucleic acid amplification test for the quantification of HIV-1 RNA in human plasma on the COBAS AMPLICORTM ANALYZER. The Test can quantitate HIV-1 RNA over the range of 50-750 000 copies/mL by using a combination of two specimen preparation procedures namely the Standard and the UltraSensitive procedures (Roche Diagnostics, Germany).
The Cobas amplification HIV-1 monitor test is based on five major processes:
· Specimen preparation
· Reverse Transcription (the target RNA is transcribed into cDNA)
· PCR Amplification (target cDNA used HIV-1 specific complimentary primers)
· Hybridization (of the amplified products to the oligonucleotide probes specific to the target)
· Detection (of the probe-bound amplified products by colorimetric determination)
Standard procedure (HIM test)
Preparation of mastermix (in pre-amplification room)
100µL of the HIV-1 Mn 2+ solution was added to one vial of the HIV-1 MMX solution and mixed well by inverting the tube (working HIV-1 MMX). 50µL of the working HIV-1 MMX was added to each of the 12 A-tubes of the A-ring. The A-ring was stored in the fridge (up to 4 hours) in a reseal-able bag, until use.
HIV-1 RNA Extraction from the controls and test samples (Performed in the HPP Virology Laboratory in a Biological Safety Cabinet)
A 2ml starsted tube was used for each of the three controls (Negative, Low Positive and High Positive Controls) and test samples processed. The precipitate that formed in HIV-1 LYSIS Buffer upon storage at 2-80C, was dissolved (by warming it up to room temperature). The working Lysis Buffer was prepared by adding 100µl of HIV-1 MONITOR Quantitation Standard (HIV-1 QS) to the vial of HIV-1 Monitor Lysis Reagent. The working Lysis Buffer was mixed well and 600µl was aliquoted into each tube. 200µL of Normal Human Plasma (NHP) was added to each of the control tubes and 200µL of patient’s sample to the respective tubes. 50µL of the controls were added to the respective control tubes.
All tubes were vortexed and incubated for 10 minutes. 800ul of 100% iso-propanol was added to each tube which was then vortexed and centrifuged for 20 minutes. The supernatant was discarded and the pellet was washed with 1ml of fresh 70% ethanol (Appendix 3). The tubes were vortexed and centrifuged for 5 minutes. The supernatant was discarded and the pellet was clearly visible. Thereafter 400µL of HIV-1 Monitor Specimen Diluent was added to each tube. 50µL of each processed controls and specimens were added to the appropriately labeled A- tube in the A-ring (which contained the working MMX solution).
Detection and Quantitation of HIV-1 RNA: The A-ring was transferred to the Roche Cobas Amplicor Machine where the process of reverse transcription, amplification, hybridization and detection was performed. The COBAS AMPLICOR HIV-1 MONITOR TEST KIT quantitates the HIV-1 viral RNA and utilizes the HIV-1 Quantitation Standard which was added to the test specimen of a known concentration. The results of the controls and test samples were printed in an exponential format.
Internal quality control: The COBAS AMPLICOR HIV-1 MONITOR TEST KIT is designed such that each run (12 tests) would include three controls (Negative, Low positive and High positive control) and nine test samples. All controls must be within the range stipulated by the COBAS AMPLICOR HIV-1 MONITOR TEST KIT. If any of the controls
failed to meet the stipulated criteria of the TEST KIT, the entire assay would have to be repeated.