|Hepatitis and Viral Load Testing|
Platforms for qualitative and quantitative viral testing, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV), were another key area of development. Roche launched its AMPLICOR® quantitative MONITOR assays for HIV and HCV outside of the United States in 1995. The HIV-1 assay was FDA approved in 1996; the HCV assay was not cleared until 2001. The HCV and HIV assays were cleared for use on the COBAS® AMPLICOR instrumentation in 2001 and 2005, respectively. In 2005, Roche launched its next generation platform, the AmpliPrep/COBAS® TaqMan®, outside the United States. This platform was designed for automated sample preparation, amplification, and quantitation of HIV, HBV, and HCV. FDA approval was obtained for HIV in 2007; HCV and HBV followed in 2008.Bayer Versant utilized branched chain DNA (bDNA) technologies for its assays. Their HIV viral load assay obtained FDA approval in 2002; both HBV and HCV received approval by 2003. Although by 2005, molecular diagnostics had begun to find its way into the routines of clinical diagnostic laboratories, for the most part, areas of testing were confined to specific applications that had been well developed and were available in FDA approved formats: sexually transmitted diseases, hepatitis testing, HIV monitoring, and in some labs, Mycobacterium detection and/or identification. Until this point, for other more easily maintained, cultivated, and identified pathogens, molecular methods, if available, had not found broad based acceptance and utilization.
|Categories of Methods|
Nucleic acid amplification (NAA) methods are now numerous and varied. In general, they can be categorized into two broad categories: target amplification and signal amplification. The chief difference between these two categories is that in target amplification methods, the nucleic acid sequence of interest is geometrically replicated into potentially millions of copies. Signal amplification methods do not increase the number of copies of the target sequence that are present in the sample. Large quantities of signal bind to the target sequence so that the signal becomes measurable. The following table summarizes some of the available techniques. This table is not intended to provide an exhaustive list of all possibilities. Target Amplification MethodsSignal Amplification MethodsPolymerase chain reaction (PCR)Probe hybridization assay (Digene)Ligase chain reaction (LCR)Branched DNA technologies (b-DNA)Transcription mediated amplification (TMA)Cleavage based signal amplification (Invader®)Strand displacement amplification (SDA)Reverse transcriptase-polymerase chain reaction (RT-PCR): Amplifies RNA targetsIn addition to NAA, fluorescence in situ hybridization (FISH) techniques, employing nucleic acid probes, represent another useful strategy. FISH assays do not involve amplification and are described in detail in a later section.Although many alternatives have been developed, PCR and PCR-derived techniques remain the most widely used, primarily because of their simplicity and flexibility. Of the target amplification methods, PCR is the principle for many assays. In some ways, the other methods could be considered "variations on the theme." Although it is beyond the scope of this course to describe in detail each and every technique, the essentials of PCR and RT-PCR will be reviewed.