Improving Health Care and Laboratory Medicine: The Past, Present, and Future of Molecular Diagnostics

In vitro diagnostics has many different disciplines. Histology tests are classic for diagnosis, followed by chemistry and serology tests. The newest in vitro diagnostic tools are molecular. These clinical tests are used to determine therapy type, duration, and dose; they are also used to determine if therapy has been successful. The choice of test depends upon the question being asked. As molecular diagnostics has evolved, it has demonstrated clear advantages over some traditional methods, although it does not completely replace other methods. Molecular methods are extremely sensitive, which is critical for the direct detection of viral nucleic acids. When a pathologist states that patients have undetectable virus, that does not mean that they have no virus in their systems. It means that the level of virus is below the limits of detection for the assay used. A robust and sensitive assay can provide more useful information. Other advantages are that molecular assays often require minimal sample volumes and do not require culture. Molecular methods can be very accurate, as they measure DNA or RNA sequences specific for the virus or mutation of interest. Finally, molecular diagnostics usually offers tremendous time savings in the laboratory, allowing clinicians earlier access to data and thus earlier treatment for patients. Nucleic acid amplification is the key to molecular diagnostics. A molecular test for a virus must detect a specific RNA or DNA sequence in the midst of a tremendous amount of other nucleic acids: human genomic DNA, mRNA, bacterial DNA, and RNA. This process is like pulling the proverbial needle out of the haystack. The power of a nucleic acid amplification technique is that it can amplify a specific sequence and do so in a predictable manner so that the number of molecules in the initial sample can be quantified. Polymerase chain reaction (PCR) is the gold standard for amplification processes in diagnostics. Since the technique was first published in 1985 (1), it has become the most widely used nucleic acid amplification technology. With PCR, a target sequence of nucleic acid is amplified through a repetitive series of reactions catalyzed by a single enzyme, a thermal stable nucleic acid polymerase. PCR can amplify both DNA and RNA. The process of PCR typically involves taking the sample through three different temperatures, each of which causes a different event. The sample is placed into a tube with the proper primers, thermal stable polymerase, nucleotides, and buffers. In the first step, denaturation by heating to 95°C causes the double-stranded DNA to form single strands. In the second step, the temperature is dropped to 55°C to 60°C, which allows the target specific primer to bind to only the intended nucleic acid sequence. The specificity of an assay depends on the quality of primer choice. Finally, in a third step, the temperature of the reaction is raised to about 72°C and the polymerase synthesizes an identical copy of the target sequence, called an amplicon. The cycle is repeated many times through the three different temperatures. Everything occurs rapidly in a closed tube in a thermocycling instrument. PCR causes exponential amplification of a target nucleic acid region. Starting with 1 copy of a molecule leads to 16 amplicons after 4 cycles. Starting with 2 copies leads to 32–4 copies after the first cycle, 8 after the second cycle, 16 after the third cycle, and finally 32 after the fourth. Clinicians do not know how many copies are present in a sample but use the result to extrapolate back. If they found 96 amplicons after 4 cycles, they could de-termine that the sample had 6 copies initially. Instrumentation now allows this to be done automatically. Roche has developed a portfolio of PCR business units based on disease areas. In six areas, an expertise is already present: virology, women's health, genomics, microbiology, blood screening, and oncology. This article discusses applications of molecular diagnostics in these fields.