In order to detect the viral load, an amplification process must be used to bring the targeted nucleic acid to detectable levels. RT-PCR is one of the most widely used target amplification techniques for the detection of HIV nucleic acid. Because the PCR uses Taq as the enzyme, and RNA is not an efficient substrate for the enzyme, it must first be transcribed to complementary DNA (cDNA) which can then be amplified. Testing is performed on plasma obtained from EDTA samples, unlike other immunoassays that use whole blood or serum.
Steps
- RNA is released from cellular material through extraction. There are several methods available to extract RNA using either manual or automated technology.
- The sample with extracted material is added to a reaction mixing chamber containing the reverse transcriptase enzyme, primers specific for the HIV RNA and nucleotides.
- In HIV-positive samples, primers will bind to the extracted RNA strand.
- Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from the primer.
- The temperature is raised to 95o C, and the RNA/DNA strands are denatured.
- The temperatures are then lowered, allowing primers to anneal to the newly formed cDNA.
- Polymerase enzyme synthesizes a new DNA strand, extending from the primer.
- This process called cycling is repeated multiple times to increase the number of copies of DNA until detectable levels are formed.
RT-PCR can be performed as a one- or two-step procedure:
- In a one-step procedure, the reverse transcriptase is performed in the same reaction tube as the polymerase chain reaction.
- In a two-step procedure, transcription of the RNA to cDNA is performed first. Transcription occurs between 40o C and 50o C, depending on the properties of the reverse transcriptase enzyme utilized. Products of that reaction are then amplified in a separate reaction.
NAT procedures typically have viral load reportability around 20-10,000,000 copies of RNA/mL.
