Gen-Probe Inc. (San Diego)
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The Tigris DTS system by Gen Probe Inc. (San Diego).
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Continued growth in the rapidly expanding molecular portion of the diagnostic testing market will depend primarily on the discovery and development of cost-effective new markers or meaningful combinations of existing markers that are strongly correlated with either the pathogenesis or the prognosis of a particular disease state. In addition to these more traditional applications, molecular diagnostic assays are also being used in the emerging field of theranostics. Here, the assays may be employed to monitor novel treatments, including the use of therapeutic vaccines. And studies are demonstrating that molecular diagnostic assays may be used successfully to monitor patient therapy and also to identify new biological markers associated with a particular disease or disease state. Ultimately, successful assays of this type will contribute directly to therapies that are more cost-effective and more timely and, ultimately, to more-successful therapeutic outcomes.
How Molecular Diagnostic Assays Work
Molecular diagnostic assays can be performed with many types of samples, including blood, urine, tissue, and sputum. But molecular technology is certainly not limited to traditional clinical applications, as samples from the environment also can be tested. For example, both GE Infrastructure Water & Process Technologies (Trevose, PA) and Millipore Corp. (Billerica, MA) have announced alliances with Gen-Probe Inc. (San Diego) to collaborate on developing tests for bacterial contamination of selected industrial and pharmaceutical water applications.
Regardless of the sample source or the sample matrix, the first steps in any molecular diagnostic assay protocol involve preparation and isolation of the nucleic acid for testing. Sample preparation should reduce or remove potential assay inhibitors from the matrix. The preparation methods have a strong influence on test accuracy and assay reproducibility, particularly with quantitative tests, which are sensitive to the consistency of aliquot volume and target nucleic acid concentration. These steps in assay processing typically do not contribute to improving the specificity of a molecular diagnostic assay.
When combined with nucleic acid amplification technology, molecular diagnostic assays can be extremely sensitive. Either the signal or the target may be amplified. Target amplification generates many copies of the nucleic acid, each of which serves as the basis for additional rounds of replication. As a result, the most commonly used amplification procedures proceed exponentially. These include the polymerase chain reaction (PCR), transcription-mediated amplification (TMA), and nucleic acid sequence–based amplification (NASBA) technologies. Each method uses enzyme-catalyzed synthesis and target-specific primer oligonucleotides to make copies of the target DNA or RNA, but the reaction intermediates and procedures differ from one to another. The designers of amplified-assay protocols should consider the requisite limit of detection for each target and should include precautions to minimize the risks of cross-contamination.
Detection of the analyte in a molecular diagnostic assay can be performed using various strategies. Typically, a nucleic acid probe is made with a sequence that is complementary to the analyte and labeled with a reporter molecule. Hybridization of this probe results in a positive signal that can be quantitative or qualitative. The design of the molecular probe determines its specificity for the target. For example, assays for infectious disease may be required to detect all strains of a particular pathogen. In other cases, it may be important to detect certain subtypes selectively. Other tests, such as genetic screens, might be required to distinguish a sequence with a single nucleotide polymorphism (SNP) from a relatively diverse population. Most commonly, probes to the target nucleic acid are labeled with fluorescent or chemiluminescent reporter molecules, and the light emitted during detection is measured to determine a positive result. In an amplified assay, the binding of probe can be measured during the course of the reaction (real-time detection) or after the completion of amplification (end-point detection). Products that include an internal control (often an RNA transcript or DNA oligonucleotide) in each reaction must be designed to discriminate this control signal from that of the analyte.
Complete Systems for Molecular Diagnostics
The development of a molecular diagnostic assay with complementary instrumentation requires a significant investment of time and capital. To date, only the Tigris DTS system from Gen-Probe is available as a fully integrated, FDA-cleared platform that automates all the steps of a molecular diagnostic assay as just described. A new and exciting entry into the market for fully automated molecular testing systems is Cepheid’s (Sunnyvale, CA) GeneXpert, which targets low-volume environments.
Other IVD systems are available that automate one or multiple steps in a molecular diagnostic assay. For example, the Viper system of BD Diagnostic Systems (Sparks, MD) automates the transfer of processed samples to 96-well reaction plates for amplification and detection. The Cobas AmpliPrep and the Cobas Amplicor systems from Roche Molecular
Diagnostics (Indianapolis) also automate sample preparation, amplification, and detection; however, these systems require operator intervention to transfer the reactions between modules.
Automating Sample Preparation
Not all molecular diagnostic instrumentation is found in complete systems that include hardware, software, and assays. Some instruments are available to automate particular steps of an assay protocol. For example, fluid-handling instrumentation for sampling and transfer is offered by several manufacturers, including Stratec Biomedical Systems AG (Birkenfeld, Germany), Tecan Group AG (Zürich, Switzerland), and Hamilton Co. (Reno, NV).
Methods for the isolation of nucleic acids in a prepared sample aliquot typically involve adsorption of the target molecules to a solid matrix, such as silica, in the presence of chaotropic salts. The advantage of this process is that it can be readily applied to any potential nucleic acid analyte, regardless of the sequence. The disadvantage is that this will indiscriminately yield DNA or RNA from any source present in the original sample. For example, this method alone will not separate host genomic DNA from that of a pathogen. Various combinations of reagents and enzymes can be used to selectively isolate DNA or RNA.
Once the nucleic acid is adsorbed, it can be washed and physically separated from the sample matrix by a number of methods, the choice depending on the properties of the solid phase. These methods are centrifugation, filtration, and capture of paramagnetic beads, processes that can easily be automated. Available instrument platforms include the Magna Pure LC DNA isolation kit from Roche Molecular Diagnostics, the NucliSens easyMAG platform of bioMérieux (Marcy l’Etoile, France), and the BioRobot M48 workstation produced Qiagen (Venlo, The Netherlands). Some systems, such as the Vidiera NSP from Beckman Coulter Inc. (Fullerton, CA), perform sample handling, nucleic acid isolation, and transfer to reaction tubes for subsequent amplification. Important things to consider when evaluating instrument platforms are the instrument’s footprint and throughput, and the ability of a system to accept primary sample tubes and to scan bar codes.
Tools for Nucleic Acid Amplification
Many new molecular products measure the presence of analyte, in real time, during the course of a nucleic acid amplification reaction. The technology for real-time nucleic acid amplification and detection is now 10 years old and has matured such that convenient hardware and software tools are now available to the molecular diagnostic assay developer. Most common real-time methods use fluorescence resonance energy transfer (FRET) to quench the emission of a fluorescent probe. As copies of the target (technically called the amplicon) are synthesized, binding or cleavage of the FRET probe physically separates the quencher from the emitter, allowing the release of fluorescent light. Various analyte-specific probes, each with a fluorophore emitting at a different wavelength, can be used to perform multiplex diagnostic analyses in a single reaction tube.
Nucleic acid amplification methods based on PCR require precise control of temperatures and incubation times during thermocycling. This can be achieved using a circulating-convection-air instrument such as the Rotor-Gene 6000 rotary analyzer from Corbett Robotics Inc. (Mortlake, Australia), which uses six light-emitting-diode (LED) light sources to capture multiple channels of fluorescence data from each reaction tube at 150-ms intervals. Peltier-based devices also can be used. Examples of these are the ABI 7500 system from Applied BioSystems (Foster City, CA) and the Chromo4 system made by BioRad Laboratories Inc. (Hercules, CA). Alternatively, isothermal amplification techniques such as NASBA and TMA can be performed in constant-temperature water baths or convection-air incubators.
An important consideration in the purchase of instruments for nucleic acid amplification is the reproducibility and consistency of heating. Other key factors are the signal-to-noise characteristics and the number of available channels for real-time detection optics and electronics.
Advances in Analyte Detection
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The Infiniti Analyzer by AutoGenomics Inc. (Carlsbad, CA).
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Offering an approach different from the real-time detection of synthesized amplicon, several highly specific end-point detection methods are available for use with amplified or nonamplified molecular diagnostic assays. For example, the Infiniti system marketed by AutoGenomics Inc. (Carlsbad, CA) is a self-contained automated system whose capabilities include sample handling, reagent management, hybridization, detection, and analysis of results. The system analyzes nucleic acids or protein by processing proprietary hydrogel matrices, called biofilm chip arrays, or microarrays, that can be configured with specific biomarkers and are scanned automatically with an integrated confocal microscope. Following analysis, results are presented in a graphical format for interpretation.
Another method for the automated preparation of microarrays employs the NanoChip400 analyzer from Nanogen Inc. (San Diego). This benchtop device uses electric charge to create addressable arrays of amplification products on a streptavidin gel–coated chip that can subsequently be queried with fluorescent probes.
Invader technology, which was developed by Third Wave Molecular Diagnostics (Madison, WI), is an example of a method in which the signal, but not copies of the analyte itself, is amplified. The assay is composed of two simultaneous reactions. The primary reaction is designed to detect SNPs, insertions, deletions, and changes in gene and chromosome number. The secondary reaction is used for generic readout and signal amplification. In the first reaction, two oligonucleotides associate with a specific region of DNA or RNA to generate a one-base overlapping structure querying the nucleotide of interest. If the variation in question is present, an overlapping structure is created and cleaved by means of proprietary enzymes in order to release a 5’ flap oligonucleotide. The released 5’ flaps are able to participate in a second cleavage reaction on labeled FRET probes, which generates a fluorescent signal. Multiple probe molecules are cleaved for each target molecule, resulting in approximately 10-million-fold amplification of the signal during a standard 4-hour reaction.
Opportunities for Diagnostics Development
The earliest molecular diagnostic assays were developed to detect infection by human pathogens. These assays have evolved into sensitive tests to quantitate or genotype disease-causing agents or genes. For example, the recognition that certain subtypes of human papilloma virus are the likely causative agents of cervical cancer drives the call for screening tests that will discriminate patients with ubiquitous and benign infections from high-risk cases. The role of such tests could expand in the future to include monitoring the course of infection or preventing cancer in a patient following immunization with new and potentially therapeutic vaccines.
Traditional medicine must accommodate the fact that some fraction of subjects in every clinical trial may not realize any benefit, or may even suffer harm, from the therapy under study. Personalized medicine, on the other hand, has the potential to match therapeutic methods efficiently with the right patients to maximize successful outcomes. Molecular tests can guide physicians in prescribing patient-specific antiviral drugs to patients infected with drug-susceptible strains of HIV. Cocktails of chemotherapeutic drugs could someday be custom-tailored to the genetics of a particular cancer and could also moderate the active metabolic clearance pathways in each individual patient. This potential will obviously be limited by the degree to which science may have characterized the positive predictive value of each marker.
The technology needed to develop sensitive and specific molecular diagnostic assays for these applications exists today. The clinical relevance and market potential needed to justify the development of such assays will have to be evaluated on a case-by-case basis.
Conclusion
Twenty years of molecular diagnostic assay development have led to a battery of tests for infection and genetic disease. And application of the technologies will continue to expand beyond medical uses into environmental and industrial testing. The addition of automation to assay protocols will inevitably continue to improve reproducibility and reduce the level of technical expertise required for performing these assays. New technologies for the automated preparation of microarrays may finally deliver on the promise of cost-effective, high-throughput detection of multiple analytes in the IVD format. In the era following the complete sequencing of the human genome, manufacturers will certainly use these tools to capitalize on newly discovered links between nucleic acid sequence and human illness.
