There are four commercially available HIV RNA viral load assays in common use:
Nucleic acid sequence-based amplification (NASBA), branched chain DNA amplification (bDNA), reverse transcription PCR (RT-PCR), and the ligase chain reaction (LCx). The NASBA assay ( Organon Technica)
utilizes the Boom method for the extraction of total RNA from a plasma sample and this method produces sufficient high-quality RNA that can be used for other investigations such as HIV genotyping. The major difficulty with NASBA is the cumbersome nature of the RNA extraction procedure and the low throughput of the method. Automation of the extraction procedure has in part addressed these difficulties but not eliminated the problem altogether. The NASBA assay utilizes three enzymes which operate at low annealing temperatures and this could result in nonspecific interactions of the primers. The absence of temperature-dependent cycling steps in NASBA reduces the extent to which the amplification process can be externally controlled and may lead to variation in amplification efficiency.
RT-PCR (Roche Amplicor) remains the most sensitive HIV viral load detection system. The new Ampliprep system provides the first fully automated system for the determination of HIV-1 viral load and this will eliminate potential variation in assay results introduced by human error. The Ampliprep system is also bar-coded and is optimized for high throughput of samples. Polymerase chain reaction-based approaches increase the possibility of introduction of contaminating RNA which may then be amplified resulting in false positive results. The reliance on one set of primers derived from the gag gene of HIV-1 may mean that quasi-species of HIV with multiple mutations in the 3' primer attachment sequences are inefficiently amplified leading to under-detection or not amplified at all leading to false negative results.
The bDNA method (Versant Diagnostics) amplifies signal and therefore it does not alter the number of target molecules present which reduces the effect that introduction of contamination into a sample may have .The assay is well suited to high throughput of plasma specimens with as many as 168 samples being processed per assay run in the semiautomated 340 machine. The assay has no internal control within the sample and therefore there is no way of discerning if amplification was successful or whether the test was inhibited.
More recently, Abbott Diagnostics has launched the HIV-1 LCx ligase chain reaction assay which uses similar technology to the Roche Amplicor system.
Abbott has developed a fully automated robotic system for their assay which will reduce technical ''hands-on'' time and operator error.
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