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The AMPLICOR� method for the quantitation of HIV-1 involves a number of precise steps:

Taking a blood sample

The doctor takes a small sample of blood (approximately 5�10ml) which is collected into in a special tube that contains chemicals to preserve the sample and separate the plasma from the whole blood cells. The sample is refrigerated until the laboratory is ready to run the test.

The Roche AMPLICOR HIV-1 Monitor� test has the advantage of not requiring that the sample be frozen. It can be stored and transported in refrigerated conditions or in wet ice containers. This allows samples to be drawn at outlying clinics and easily transported to the testing laboratory.

Once the sample has reached the laboratory, the following procedures are carried out.

Reagent and specimen preparation

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.

A special PCR solution, known as "Master Mix", needs to be prepared 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 by PCR.

PCR reaction (amplification)

First, the single-stranded RNA must be converted to DNA. This process is called reverse transcription. The DNA strands are then amplified 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 further information see the PCR tutorial.

Detection and measurement of PCR products

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 is �pre-lined� with RNA capture probes that are bound to the plastic surface of the well. The probes have binding sites that are complementary to the target sequence of interest. After the DNA Amplicons have been added to the wells, an alkaline solution is added to separate them into two single strands. This is called denaturation and it results in the exposure of the target sequence. These biotinylated strands are then hybridised onto the capture probes.

A peroxidase conjugate is then added which binds to the biotin on the target sequences. After washing to remove the unbound conjugate, Tetramethylbenzidine (TMB) is added to the wells, which turns blue in the presence of the peroxidase. 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.

If follows that the greater the quantity of Amplicons present, the more biotin present, the more peroxidase is attached, 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 measured using an instrument called a photometer. From this result, the RNA copies/ml of blood plasma can be calculated and printed out.

It has been estimated that in a modern, well-run laboratory viral load testing should take around 6 to 7 hours from starting to prepare the sample to obtaining the result.