Materials (
Roche Diagnostics, Indianapolis, USA)A) HIV-1 monitor specimen preparation reagents (Roche Diagnostics, Indianapolis, USA)
1. HIV-1 monitor lysis reagent (LYS) containing Tris-Hydrchlorate (HCL) buffer, 68% guanidine thiocyanate, 3% Dithiothreitol, 1% Glycogen.
2. HIV-1 quantitation standard (QS) containing Tris-HCl buffer, 0.001% non-infectious invitro transcribed microbial RNA containing HIV-1 primer binding sequences and a unique probe binding region, 0.005% recombinant RNA polymerase (poly rA RNA) Ethylene diamine tetraacetic acid (EDTA), Amaranth dye, 0.05% sodium azide.
3. HIV-1 monitor specimen diluents (DIL) containing Tris-HCl buffer, 0.005% poly rA RNA, EDTA, 0.005% sodium azide.
B) HIV-1 Monitor control reagents (Roche Diagnostics, Indianapolis, USA)
1. Negative plasma control (NHP), containing human plasma non reactive for antibodies to HIV-1/2, HIV-1 P24 antigen, antibody to HCV and HBsAg.
2. HIV-1 monitor (-) control containing tris HCl buffer, EDTA, 0.05% sodium azide.
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3. HIV-1 monitor Low (+) control containing tris-Hcl buffer, <0.001% non infectious in vitro transcribed RNA (microbial) containing HIV-1 sequences, < 0.005% poly rA RNA, EDTA, 0.05% sodium azide.
4. HIV-1 monitor High (+) control containing tris-HCl buffer, <0.001% non infectious in vitro transcribed RNA (microbial) containing HIV-1 sequences, < 0.005% poly rA RNA, EDTA, 0.05% sodium azide.
C) HIV-1 Monitor amplification reagents (Roche Diagnostics, Indianapolis, USA) 1. 1 HIV-1 monitor Master Mix (MMX) containing Bicine buffer, glycerol, <0.01%
recombinant Thermus thermophilus DNA polymerase(rTth pol), <0.07%dATP, dCTP, dGTP, dTTP, dUTP, <0.001% biotinylated SK145 and SKCC1B primers, <
0.01% AmpErase (uracil-N-glycosylase) enzyme, 0.05% sodium azide
2. HIV-1 manganese solution containing <2% manganese, Acetic acid, Amaranth dye, 0.05% sodium azide
D) HIV-1 monitor detection reagents (Roche Diagnostics, Indianapolis, USA)
1. HIV-1 monitor Microwell plate (MWP) coated with HIV-1 specific DNA probe SK 102 (Rows A to F) and QS-specific probe CP35 (Rows G and H)
2. Monitor denaturation solution (DN) containing 1.6% sodium hydroxide, EDTA, Amaranth dye.
3. Monitor hybridization buffer (HYB) containing sodium phosphate solution, 25%
sodium thiocynate, 0.2% solubilizer
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4. Avidin-Horseradish peroxidase conjugate (AV-HRP), Bovine gamma globulin, 0.1%
phenol, 0.1% proclin®150 preservative.
5. Substrate A containing citrate solution ,0.01% hydrogen peroxide, 0.1%
proclin®150 preservative
Other materials (Roche Diagnostics, Indianapolis, USA) 1. Disposable gloves
2. Propylene screw cap tubes 2ml 3. Propylene screw cap tubes 1.5ml 4. Ethanol 95%
5. Isopropyl alcohol
6. Pipettes 12.5µl, 25 µl, 100 µl, 200 µl, 400 µl, 600 µl, 800 µl and 1000 µl.
7. Microcentrifuge
8. Refrigrated ultracentrifuge and fixed angle rotor 9. Vortex mixer.
10. GeneAmp PCR system thermal cycler 9600 11. Microwell plate washer
12. Microwell plate reader 13. Disposable reagent reservoir 14. Distilled water
15. Incubator 370c 16. Graduated vessels
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Preparation of reagents specimen and control (Roche Diagnostics, Indianapolis, USA) 1. Working master mix was prepared by adding 100µl HIV-1 Mn2+V1.5 to 1 vial of
HIV-1 MMX version 1.5.
2. Ethanol 70% was prepared by mixing 11ml 95% ethanol and 4ml distilled water.
3. The 2ml screw capped tubes for each patient specimen, HIV-1 neg, HIV-1 L (+) and HIV-1 H(+) control were labelled.
4. Working lysis reagents was prepared by adding 100 µl of HIV-1QS,V 1.5 to one bottle of HIV-1 LYS.
Assay Procedure
1. HIV-1 QS, V 1.5 was vortexed for 5-10 seconds, and 100µl of it was then added to one bottle of HIV-1 LYS and was thoroughly mixed to form the working lysis reagent.
2. The Working lysis reagent (600µl) was added to each of the labeled tubes
3. HIV-1 negative, HIV-1 L(+) and HIV-1 H(+) control and NHP were vortexed for 5 seconds and 50µl of each was then added into their designated tubes containing the Working lysis reagent. The mixture was then vortexed for 5 seconds.
4. Patients’ specimens (200µl) were added to the appropriately labeled tubes containing standard working reagents. The tubes were then capped and vortexed for 5 seconds.
5. The control and specimen tubes were then incubated for 10 minutes at room temperature.
6. Isopropyl alcohol (800µl) was added into each specimen and control tubes. The tubes were recapped and vortexed for 5 seconds.
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7. An orientation mark was placed on each tube. The tubes were then placed in the microcentrifuge with the orientation marks facing outwards and centrifuged at 12500xg for 15minutes at room temperature.
8. The supernatant was carefully removed and discarded from each tube using a fine tip disposable transfer pipette.
9. Ethanol 70% (1ml) was then added to each tube, recapped and vortexed for 5 seconds.
10. The tubes were placed into the microcentrifuge with the orientation mark facing outwards and centrifuged for 5 minutes at a speed of 12500xg. The supernatant was carefully removed without disturbing the pellets.
11. HIV-1 DIL (400 µl) was then added to each tube. The tubes were recapped and vortexed for 10 seconds and taken to the amplification area before moving to the master mix area.
12. At the master mix area 50µl working MMX was added into each reaction tube using a pipettor with reaction barrier. The Micro Amp tray containing appropriate number of reaction tubes were then placed in resealable bag and sealed securely and then moved to the amplification area.
13. At the amplification area 50µl each of the processed specimen and control were added to the appropriately labeled reaction tubes containing working MMX using a pipettor with aerosol barrier. The tubes were capped and sealed with Micro Amp cap installing tool. The position of the controls and specimen were recorded and
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amplification was commenced within 45minutes of adding specimen and MMX together.
14. At the amplification bench, the retainer assembly was placed into the thermal cycler block.
15. The Applied Biosystem Gene Amp PCR System 9600 thermal cycler for the Amplicor HIV-1 Monitor test was programmed as follows;
HOLD programme 2min at 500c HOLD programme 30 min at 60 0c
CYCLE programme (8 cycles) 10 seconds at 95 0c, 10 seconds at 52 0c, 10 seconds at 72 0c
CYCLE programme (23 cycles) 10 seconds at 90 0c, 10 seconds at 55 0c, 10 seconds at 72 0c
HOLD programme 15 minutes at 72 0c
The programme was then started and ran for about 1 hour 30minutes.
16. The specimens were then removed from the thermal cycler within 15minutes from the start of the final HOLD programme, the cap of the reaction tubes were removed carefully and 100 µl of Monitor DN was added immediately to each tube using a multichannel pipettor. The content of the tubes were mixed by pipetting up and down 5 times and then carried to the detection area.
17. At the detection area, working wash solution was prepared by adding 1 volume 10x WB to 9 volume of deionised water.
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18. The MWPs were allowed to warm to room temperature before being removed from the foil pouch. Monitor HYB 100 µl was then added to well on the MWP.
19. Denatured amplicon 25 µl was added to row a of the MWP and mixed by pipetting up and down 10 times with 12-channel pipettor with aerosol barrier tips. A fivefold serial dilutions in rows B through F using programme 3.
20. Denatured amplicon 25 µl was added to QS wells in row G of the MWP. It was mixed at least 15times with amplicor electronic pipettor programme 3. 25 µl was transferred from row G to row H and mixed as before, then 25µl was removed from row H and discarded.
21. The MWP was covered with its lid and incubated for 1 hour at 370c. The MWP was then washed using an automated washer.
22. AV-HRP 100µl was then added to the MWP covered and incubated for 15 minutes at 370c. The MWP was washed again with automated washer
23. Working substrate solution was prepared by mixing 12ml SUB A and 3ml SUB B before use. 100µl of the solution was added to each well and allowed to develop colour at room temperature, in the dark.
24. Stop solution 100µl was then added to each well. The optical density was measured at 450nm within 10minutes of adding stop solution. The absorbance value for each well was recorded.