PCR Tutorial

The AMPLICOR HIV-1 MONITOR™ test is a semi-automated process. By developing systems to carry out the tests, the time taken to do the test can be reduced, more tests can be done at the same time, the possibility of human error is reduced and valuable laboratory staff time is saved.

The laboratory environment is very strictly controlled to ensure that samples are correctly handled, stored and tested and that the AMPLICOR® test is carried out following exact instructions.

The laboratory activity can be thought of as two very distinct processes - Pre-PCR and Post-PCR. Clearly defined laboratory areas and equipment are used for the different processes. In this way contamination of the blood samples and the possibility of incorrect results are avoided. In both areas, laboratory staff wear protective clothing and gloves and work using special sterile areas and equipment.

Pre-PCR

The term Pre-PCR covers two steps – reagent and specimen preparation.

A special PCR solution, known as "Master Mix", needs to be prepared during reagent preparation, before PCR can begin. The "Master Mix" contains everything needed to support PCR. This includes all the chemicals, DNA components and enzymes necessary to "build" DNA copies.

Before the viral load can be measured the HIV virus particles in the blood plasma must be separated and the RNA inside the virus must be extracted during specimen preparation. Only the RNA of HIV-1 in the plasma of the blood can be measured. The RNA of HIV-1, which is present in the white blood cells (lymphocytes) or other sites in the body, for example the lymph nodes, can not be measured using the commercially available viral load tests.

Viral RNA is extracted from the blood by centrifuging (rapidly spinning) the sample and by using special chemicals to break open the virus particles and making the RNA clump together.

Post-PCR

The term Post-PCR covers two steps - amplification and detection.

When using PCR to detect HIV-1 RNA, the RNA must first be converted to DNA (a process called reverse transcription). This is then followed by PCR. This amplification process involves repeated cycles of denaturing ("unzipping" the target DNA), annealing (starting the process of building DNA) and extension (continuing to build the DNA strand), which result in amplification of the original RNA into many copies of complementary DNA.

For an detailed explanation of PCR view the PCR Tutorial

Once amplification is finished the products of PCR must be detected and measured.

This process begins by transfering the products of PCR, the DNA copies known as Amplicons, to small "wells" in a microwell plate.

Each well holds the PCR products from one blood sample. The Amplicons are attached to the bottom of the wells by using special chemical probes which attract the Amplicons.

An enzyme is then added to wells which binds to a small chemical "tag", biotin, on the end of each Amplicon. Every Amplicon will have a single enzyme attached. The "tag" is part of the primer which was used to start PCR and is now incorporated into each Amplicon.

A chemical is then added to the wells. The chemical contains hydrogen peroxide and Tetramethylbenzidine (TMB). The enzyme acts on TMB and, using the hydrogen peroxide present, causes a chemical reaction which turns the TMB blue. A "stop" solution is finally added to the wells of the microwell plates. This stabilises the contents and turns the final solution from blue to yellow.

The greater the quantity of Amplicons present, the more enzymes can attach to the biotin, and the greater the intensity of blue, and then yellow, colour. The intensity of colour is therefore directly related to the number of RNA copies originally present in the blood sample.

The intensity of the yellow colour is then measured using an insturment called a photometer. From this result the RNA copies/mL blood plasma can then be calculated and printed out.