Molecular pathology is a relatively new division of laboratory medicine that detects, characterizes, and/or quantifies nucleic acids to assist in the diagnosis of human disease. Molecular assays augment classical areas of laboratory medicine by providing additional diagnostic data either in a more expeditious manner or by providing results that would not be obtainable using standard methodologies. For these reasons, molecular pathology is the most rapidly growing area in laboratory medicine. Molecular pathology can be categorized into five subdivisions that specialize in the diagnosis of diseases or conditions associated with (1) hematology/oncology, (2) solid tumors, (3) genetics, (4) pharmacogenetics, and (5) infectious diseases. Based on test volume, detection, and characterization of infectious diseases is currently the dominant subdivision of molecular pathology and is projected to continue to dominate the other areas of molecular pathology for the next several years.
Molecular methods used for detecting infectious agents have several advantages when compared to classical microbiology approaches. Molecular methods are highly sensitive and, therefore, can detect minute amounts of infectious agents. Because these methods generally do not require growth in culture media, various bacteria, viruses, and fungi, which are difficult or impossible to culture, can be readily identified (1-8). In addition, the sensitivity of these methods can allow the analysis of nonviable infectious agents (i.e., permitting the analysis of archived formalin-fixed tissue). The viral load and genotype of certain infectious agents that can facilitate treatment protocols can also be determined. These advantages are some of the reasons why infectious diseases are dominating the area of Clinical Molecular Pathology. As a result, these new molecular methodologies have placed the highest priority on the ability to equally detect and quantify genetic variants of various infectious agents.
Initially, polymerase chain reaction (PCR) was the primary molecular method used in clinical laboratories. However, numerous negative factors have limited its role in diagnostic testing laboratories. Some of these issues are PCR's requirement for (1) specialized instruments, (2) workflow, (3) specially trained personnel, (4) substantial quality assurance, and (5) overhead cost for intellectual properties. These issues, combined with the increase in the infectious diseases diagnostic market, have supported the development of numerous other molecular platforms. The success of these platforms in the infectious disease arena has resulted in a spillover into other areas of clinical molecular diagnostics, specifically genetic and solid tumor testing. Recent advances in genetic medicine is now requiring the development of methods that can detect and identify numerous genetic polymorphisms ranging from viral sequences to human single-nucleotide polymorphisms (SNPs) to whole genomic scanning (9). Various platforms have been developed to address these goals and include technologies that range from single-temperature-based assays to multiple-temperature assays that can either amplify a target nucleic acid or a reporter molecule. The focus of this chapter will be to provide an overview of some of these non-PCR-based detection systems for nucleic acid testing (NAT).
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