Originally Published IVD Technology October 2003
Tools for molecular diagnostics
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| TThe AutoGenprep 965 by AutoGen Inc. (Holliston, MA) is a completely automated 96-well-format robotic system for whole blood DNA extraction. |
The discovery of DNA’s double helix structure by Watson and Crick 50 years ago has had a great influence on modern medicine. One of the more significant resulting developments was molecular diagnostics. The first commercial nucleic acid test came into being about 20 years ago, and now molecular diagnostics is revolutionizing the IVD industry much as antibody-based immunoassays did in the 1970s.
Although currently only 5% of the IVD market, molecular diagnostics is the fastest-growing segment of the industry. At least 80% of molecular tests are performed in the centralized laboratories of larger hospitals, and in specialized laboratories throughout the developed world, particularly North America, Western Europe, and Japan. FDA has approved 42 molecular tests produced by 14 companies worldwide. All but three of them are commercially available in the United States.
More than 80% of molecular tests performed today address infectious-disease detection and management. The test menu for infectious diseases includes human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), cytomegalovirus (CMV), Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycobacterium tuberculosis. Other molecular tests are applied in forensic medicine, paternity testing, tissue typing, oncology, and food and beverage testing.
The molecular diagnostic market is expected to grow 15–17% annually in the short term. Revenues would reach $1.8 billion worldwide by 2005. Anticipation of such steady growth is based on expectations that maturation of the technology, the development of diagnostic applications, and expansion of the market into developing countries will lead to increased acceptance and market penetration of the tests. Continuing simplification of the technology and instrumentation used in molecular diagnostic tests holds the key to this phase of development.
The growth of molecular diagnostics beyond 2005, however, will rely on the commercialization of rapid, user-friendly, and economical high-quality testing systems, and on the discovery of gene-based therapeutics that will individualize disease management.
Nucleic Acid Tests
The field of molecular diagnostics is represented primarily by the nucleic acid test (NAT), which is used to detect genomic DNA or RNA sequences and to characterize genomic aberration or sequence polymorphisms. For the past 20 years, NATs have been mainly hybridization-based assays, with detection a function of label molecules coupled to the probe sequence. Some commercial NATs are based on direct hybridization detection of ribosomal RNA. The high sensitivity of the assay is attributable partly to the fact that there are thousands of ribosomal RNA molecules per cell. To be able to detect less-abundant RNA or DNA molecules at increased sensitivity usually requires some form of amplification to generate a detectable signal.
Amplification strategies vary, but techniques can be categorized as target, probe, or signal amplifications. Target amplification employs various sequences of enzymatic reactions to amplify the targeted nucleic acid fragment—that is, to produce multiple copies of this target sequence. Most notable of the target amplification tests is polymerase chain reaction (PCR), developed by Roche Diagnostics Corp. (Indianapolis). Also worth mentioning are strand-displacement amplification (SDA) from BD (Franklin Lakes, NJ), transcription-mediated amplification (TMA) from Gen-Probe Inc. (San Diego), and nucleic acid sequence–based amplification (NASBA) from Organon Teknika Corp. (Durham, NC).
Although it is also a sequence amplification event, probe amplification, as exemplified by Abbott’s (Abbott Laboratories; Abbott Park, IL) ligase chain reaction (LCR), produces many copies of ligated probes as a result of the presence of specific target sequences. Both target and probe amplifications can enhance the detection of specific target sequences by at least four to six orders of magnitude.
Signal amplification, on the other hand, is a process of amplifying the detection signal after the targeted DNA or RNA has formed a stable hybridization complex with the probe sequence. Examples of signal amplification techniques include the branch DNA (bDNA) method of Bayer Diagnostics (Tarrytown, NY) and the hybrid capture method developed by Digene Corp. (Gaithersburg, MD). They can boost signal intensity by at least three orders of magnitude, with multiple label molecules being associated with each targeted molecule.
All of the hybridization-based assays may use chromogenic, fluorescent, or optical reporter labels to produce the measurable light units detected with an optical readout device. Like immunoassay in its infancy, each of the NAT methods has strengths and limitations.
Today, PCR is the gold standard for molecular assays, owing to its intrinsic versatility and flexibility. Roche’s PCR-based product lines, which have application strictly in the infectious diseases area, have captured 47% of the molecular diagnostics market. This circumstance will not change until molecular assays for cancer and heritable diseases become part of the routine test menu.
The objective of molecular test manufacturers is to develop homogeneous closed-tube molecular test systems that are suited for diagnostic applications. A number of labeling technologies for homogeneous molecular assays have been developed that employ various configurations of probe and fluorescence label, and incorporate different versions of fluorescence resonance energy transfer (FRET). In a FRET-based assay, a fluorescent-tagged molecular probe is excited by a laser. The fluorophore responds by emitting energy that is transferred to a second, quenching, fluorophore. If these two fluorophore molecules are very close to each other, the fluorescence is quenched.
The desire to find better ways to design and manufacture these labels and probes, so as to achieve better performance and a lower cost per test, drives the development of new labeling methods in this area. Now, homogeneous-label methods are widely used by molecular laboratories in in-house PCR tests for infectious diseases, heritable diseases, and cancer. Several IVD manufacturers are incorporating these methods in their own molecular assays. Although homogeneous assays often provide the best sensitivity in detection, the limited number of different fluorophores available for simultaneous detection of multiple analytes from a single sample makes this assay platform less attractive for use in multiplex assays.
NAT Automation
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| The MagneSil Blood Genomic, Max Yield System by Promega Corp. (Madison, WI) is a high-throughput system for DNA purification from blood. |
Automation remains a significant challenge for NAT manufacturers. However, recent advances in bioinformatics, computer science, and assay miniaturization have begun to be integrated into molecular tests. A generation of instruments also has been developed that can perform real-time quantitative or end-point qualitative PCR detection.
A new generation of fully automated and integrated NAT instruments has appeared on the horizon. Most notably, Gen-Probe has announced successful completion of clinical trials with the first fully automated NAT system. Named Tigris, the patented system performs specimen preparation, amplification, and detection in the same instrument without operator attention. The patented GeneXpert system from Cepheid (Sunnyvale, CA) is another automated instrument. It is a portable device designed to perform the entire procedure of detecting DNA in biological specimens, including sample preparation, amplification, and detection. Across the entire NAT industry, efforts are under way to bring fully integrated NAT automation systems into the marketplace—particularly sample preparation systems, including extraction and purification steps, for most commercial NATs now being performed manually or semiautomatically.
Taking sample preparation and probe labeling together, in 2000, clinical laboratories worldwide spent more than $40 million performing in-house molecular assays. Annual growth in this area alone is predicted to be more than 20% before 2005. Total revenues for reagent manufacturers that provide molecular assays are estimated to reach $1 billion by that year.
Gene-Based Diagnostics
The market potential for oncological and genetic-disease molecular testing is smaller in the near term than that for infectious-disease testing. For the foreseeable future, in-house molecular tests, offered as analyte specific reagents (ASRS), will remain an integral part of the testing in this field. Only an estimated 25% of the 4000 known hereditary diseases can be analyzed by means of molecular testing techniques today. Additional factors, including technical, patent, and ethical issues, may further restrict the development and market penetration of genetic diagnostic tests.
In this molecular medicine era, it has become widely accepted that almost every disease process results from the interaction of the environment and individual lifestyle with a specific genetic predisposition. There will be no more “one drug fits all,” and pharmaceutical medicines will be prescribed to targeted patient populations case by case on the basis of the individual’s genomic profile. Furthermore, with early detection and intervention available, most of the historically life-threatening diseases will become merely chronic disease conditions that require long-term management.
Pharmacogenomic testing for patient drug susceptibility, dose response, and drug resistance thus holds exciting potential for molecular diagnostics in years to come. According to a presentation at a recent industry conference, 370 biopharmaceutical drugs are in late stages of clinical development. Management of several medical conditions, including diabetes, asthma, autoimmune disease, and cardiac disease, will greatly benefit from these gene-based diagnostics and therapeutic interventions.
Looking Ahead
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| A membrane engineered for the high-throughput preparation of DNA is contained in a 384-well DNA binding plate by Whatman Inc. (Clifton, NJ). |
The scope of molecular diagnostics in molecular medicine could be expanded well beyond current nucleic acid testing. Gene expression profiling, and modifications at the protein levels, will be critical to cancer classification and staging. Lately, demand for multiplexing platforms that perform parallel nucleic acid or protein analyses in diagnostic applications has been steadily increasing.
Although generally perceived as a method for testing thousands of gene fragments with low sensitivity and reproducibility—the GeneChip from Affymetrix Inc. (Santa Clara, CA) is an example—advanced microarray platforms are being devised for diagnostic ends. New platforms, like the Flow-Thru Chip developed by MetriGenix Inc. (Gaithersburg, MD), promise high assay performance, amenability to automation, and the flexibility to handle low- to high-throughput diagnostic applications. These microassays will be targeting tens to hundreds of analytes rather than thousands. More important, they will combine bioinformatic and microfluidic technologies with the microarray in order to miniaturize and simplify the tests.
The first commercially available microarray, the AmpliOnc I from Abbott, was developed from a subsidiary’s Vysis GenoSensor microarray system. It is designed to detect abnormal increases in gene copies for 58 different genes. A number of biochip makers are pursuing collaborative arrangements with IVD manufacturers and drug manufacturers to exploit the possibility of their microarray technologies being used in clinical product development.
In addition to the microarray platform, bead-based technologies are also gaining recognition for their potential utility in clinical multiplex testing. Each bead incorporates a different label dye and thus has a different spectral address. One can be distinguished from another by its unique wavelength. If each bead is associated with only one specific analyte, then multiplex assays of nucleic acid or protein can be performed.
The flexibility of the multiplex format of bead-based assays is the biggest advantage these assays have over the microarray platform. The LabMAP system from Luminex Corp. (Austin, TX) is an example. However, this system carries a disadvantage in that it is limited to fewer than 100 elements per test.
Other technologies such as mass spectroscopy and denaturing high-pressure liquid chromatography have also recently been making their way into the molecular diagnostics field.
The involvement of thousands and millions of genetic markers in specific diseases and physiological pathways has just begun to be biologically validated. Development of biological assay content in this field will take much longer than technology development. But anticipation is high that, with the large-scale gene-discovery efforts being undertaken by many government and private institutions, molecular diagnostics will come to maturity in the next 5 to 10
years.
Eric Eastman, Andrew J. O’Beirne, Helen Schiltz, Adam Steel, MetriGenix Inc. (Gaithersburg, MD)
Copyright ©2003 IVD Technology
