Human Immunodeficiency Virus (HIV-1) RNA PCR Procedure (Qiagen Artus HIV PCR Kit)

Human Immunodeficiency Virus (HIV-1) RNA PCR Procedure using Qiagen Artus HIV PCR Kit to detect the presence of HIV virus RNA...

Principle:
Pathogen detection by the PCR is based on the amplification of specific regions of the pathogen genome. The amplified product is detected via fluorescent dyes. These are usually linked to oligonucleotide probes which bind specifically to the amplified product. Monitoring the fluorescence intensities during the PCR run allows the detection and quantitation of the accumulating product without having to re-open the reaction tubes after the PCR run.


RNA Extraction Procedure:(See DNA Extraction using QIAmp DSP Virus Kit DNA/RNA Extraction Procedure)

Master Mix Preparation(pre-PCR): 
  • Please use the following pipetting scheme:

number of samples
1

HIV-RG Master A 
12 ul 
Prep. of
HCV-RG MasterB
18 ul
Mastermix
HCV-RG IC
  2 ul

Total volume
32 ul
   


Prep of
Mastermix
30 ul
PCR assay
Sample
20 ul

Total volume
50 ul
















  • Mix the solution by pipetting up and down. Close the PCR tubes and ready to load the tubes in Rotorgene 6000 for amplification.

Interpretation of Results: 
  • A signal is detected in fluorescence channel Cycling A.FAM. 
          The result of the analysis is positive: The sample contains HIV-RNA. 
  • In fluorescence channel Cycling A.FAM no signal is detected. At the same time, a signal from the internal control appears in the Cycling A.ROX channel. 
          In the sample no HIV RNA is detectable. It can be considered negative. 
  • No signal is detected in the Cycling Green or Cycling A.FAM channels, or in the Cycling Orange or the Cycling A.ROX channels.
          No result can be concluded. Presence of PCR inhibitions are suspected, for repeat extraction. 


Reference Values: 
  • HIV- RNA >72 IU/ml: The result is within the determined test range. 
                                              The detection probability of HIV RNA is >95%.
                                                     
                                              The positive result is statistically ensured.
  • HIV- RNA <72 IU/ml : The result is outside the determined test range. 
                                              The reproducibility of the positive result is not assured.
  • HIV- RNA Negative: No HIV RNA was detected.

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