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The Tigris System by Gen-Probe Inc. (San Diego). (Photo courtesy Gen-Probe Inc.)
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The tools available for molecular diagnostic assays have expanded considerably since the first nonamplified nucleic acid test (NAT) to confirm Legionnaires’ disease received FDA approval in 1985. Eight years after that, the first amplified test was approved, allowing detection of a target nucleic acid directly from a clinical sample. Amplification offered the additional benefit of increasing sensitivity while simultaneously decreasing the time required to obtain a result, since culturing to enrich the target was no longer required. The next major advance in molecular diagnostics was the transition from qualitative to quantitative tests through the use of technologies such as real-time polymerase chain reaction (PCR).
The scope of molecular IVD testing has expanded as well to include many different infectious-disease agents and is now moving into the oncology field. On the horizon, as the genetic components of drug efficacy and predisposition to various diseases become more clear, are new areas such as pharmacogenomics and disease susceptibility.
As a percentage of the entire diagnostics market, molecular diagnostics continues to exhibit strong growth in terms of both revenue and the number of tests performed. Infectious-disease testing is the largest market segment within molecular diagnostics and is growing at an annual rate of more than 7%. The molecular diagnostics market overall is expected to double to roughly $6 billion in five years, with genomics and oncology testing becoming increasingly important.
Although molecular IVD testing is growing, several issues prevent its wider acceptance. One is that, until recently, all molecular IVDs have been classified as highly complex by FDA, meaning that the tests were required to be performed by highly trained laboratory personnel. This issue is being addressed by the trend toward greater automation and all-in-one systems able to provide sample-to-answer functionality with minimal user intervention.
A second obstacle is the slower-than-anticipated progress in genomics in identifying and validating suitable biomarkers for detecting the presence of, or predisposition to, cancers and other diseases. This has been true also in the pharmacogenomics field, where highly informative markers predictive of drug response have been harder to identify than expected.
Overview of Molecular Technology
Two broad categories of molecular diagnostic tests exist. Commercially available FDA-approved tests are provided as kits that include all critical reagents needed to perform the test, including controls. These require FDA-approved instrumentation for running the test and analyzing the results. The instrumentation may be platform specific—that is, designed to run that company’s own IVD technology—such as the Tigris system from Gen-Probe Inc. (San Diego), or generic and able to accommodate various products. The LightCycler real-time PCR instrument manufactured by Roche Molecular Diagnostics (Pleasanton, CA) is an example of the latter.
Companies can alternatively provide analyte-specific reagents (ASRs), which a molecular diagnostic laboratory can use to develop its own in-house tests. ASR-based tests do not require FDA approval; however, the laboratory must be able to validate the performance of the test.
Molecular diagnostic tests have several common requirements. The sample containing the potential target of interest must be treated so as to release the nucleic acid—which can be DNA, RNA, or both—while preventing sample matrix and other interfering substances from affecting the sensitivity and specificity of the test. The test must be of sufficient specificity to discriminate the desired target sequence from nontarget sequences, and be able to detect the presence of the target at the required sensitivity. To meet demands for higher sensitivity and specificity, amplification of the targeted nucleic acid sequence or the detected signal often is required. Detection of the target is typically accomplished through the use of instrumentation because of the greater reproducibility and sensitivity possible. Here too, detection methods can be target specific, thereby affecting both sensitivity and specificity. Oncology and infectious-disease tests often additionally require quantification of the target sequences present in the original sample.
An increasing number of tests require multiple targets to be amplified or detected simultaneously, or multiplexed. Multiplexed tests are becoming more popular because they allow multiple diagnostic tests to be performed in parallel with a single sample. Depending on the test, multiplexing can be used to analyze several closely related targets, such as the multiple high-risk subtypes of the human papillomavirus, or unrelated targets, such as VKORC1 and 2C9 single-nucleotide polymorphisms for predicting warfarin metabolism. Multiplexed panel tests, such as the xTAG respiratory virus panel from Luminex Corp. (Austin, TX), also are becoming increasingly available.
Sample-Preparation Tools
A wide range of biological sample types are used as starting material for a molecular diagnostic assay. Body fluids such as plasma or urine are commonly used for infectious diseases, while tissue or biopsy material, as well as blood and body fluids, can serve as starting material for oncology tests. Blood is most often used as the starting material for genomics tests, but saliva may be used as well.
Sample preparation is a critical first step regardless of the sample type or downstream technology. The non–nucleic acid components in the sample (cellular debris, matrix, proteins, and so on) must be eliminated or minimized because of their potential to interfere with or inhibit downstream amplification or detection chemistries and negatively affect sensitivity or specificity. At the same time, excessive degradation or fragmentation of the extracted nucleic acid must be avoided in order to prevent negative or inconclusive results. The nucleic acid in the sample is usually concentrated at this initial step in order to increase sensitivity.
Release of the nucleic acid is typically followed by its adsorption to microbeads that may or may not be magnetic. Nonmagnetic beads are usually reserved for manual preparation methods, because centrifugation is needed to collect the beads during subsequent wash steps. Magnetic microbeads, while more expensive and complicated to manufacture, offer the advantage of allowing semi- or fully automated sample preparation through the use of magnets to collect and manipulate the beads. Not only does this reduce the requirement for hands-on operation; it also lowers the risk of operator error and potential for sample contamination. The MagNA Pure LC isolation kit from Roche Diagnostics GmbH (Mannheim, Germany), the BioRobot EZ1 workstation from Qiagen Sciences Inc. (Germantown, MD), and the NucliSens easyMAG platform from bioMérieux S.A. (Marcy l’Etoile, France), for example, are systems based on magnetic-bead technology that are suitable for the automated nucleic acid purification of tens of samples simultaneously. Other systems are available that can process a thousand or more samples daily.
Target Amplification
The first NATs were unamplified, and used abundant ribosomal RNA sequences as targets. The lack of amplification meant that those tests were confirmatory in nature, often requiring culturing of the sample to enrich the amount of target available. To detect directly from a patient sample the small amount of target in it, infectious-disease and oncology NATs require amplification of the specific nucleic acid target, the detection signal, or both.
Amplification of the desired nucleic acid target can be accomplished by several different methods, including PCR, transcription-mediated amplification (TMA), strand-displacement amplification (SDA), and nucleic acid sequence–based amplification (NASBA). PCR is the most common method in use. It requires a thermal cycler with precise temperature control, an accessory capable of both heating and cooling during the process. TMA, SDA, and NASBA are all isothermal processes: during performance of these processes, only a single set temperature must be maintained. This reduces considerably the complexity of the instrumentation required for amplification.
There is usually a large excess of nontarget nucleic acid present, so amplification is performed using oligonucleotide primers designed to be target specific. The specificity of these primers for the target is a major determinant of the specificity of a particular test, and a factor in the test’s sensitivity as well. Generally, signal is measured at the end of the reaction, which limits most assays to being able to provide only qualitative results. This is not an issue for genomic NATs, however. An example is Roche Molecular’s AmpliChip CYP450 assay. This test identifies specific single-nucleotide polymorphisms in drug-metabolizing enzymes and is thus concerned only with whether a gene sequence is present; the number of copies of the gene in the sample is irrelevant.
The need for infectious-disease NATs to be able to quantify the titer of target present—as, for example, human immunodeficiency viral load—led to the introduction of real-time PCR. Real-time PCR overcomes the qualitative limitations of PCR by performing amplification and detection simultaneously. This is accomplished by including a target-specific oligonucleotide probe in the amplification mixture that contains both a fluorophore and fluorescent quencher. In the absence of target, the probe self-hybridizes in such a manner that the fluorophore and quencher are brought into close proximity, quenching fluorescence through fluorescence resonance energy transfer (FRET) and resulting in low signal being detected. In the presence of target, the probe preferentially binds to it in a sequence-specific manner, changing conformation; the binding physically separates the fluorophore and target and unquenches the fluorophore. The amount of target increases during amplification, and the amount of fluorescence increases in proportion. Because amplification of target is exponential during the early cycles of amplification prior to the plateau, the amount of target present in the reaction—and thus the amount of fluorescent signal—is directly proportional to the number of targets originally present.
Although relatively difficult to develop, and requiring complicated instrumentation owing to the combination of fluorescent detection with thermal cycling, assays based on real-time PCR have moved into the mainstream of molecular diagnostic testing for infectious diseases. Nearly 20 FDA-approved infectious-disease molecular tests now on the market use real-time-PCR technology.
Detection Technologies
In combination with the specificity of the amplification step (if there is one), the sequence specificity of the detection probe determines the specificity of the test. Many different methods for detecting the presence of a particular target sequence exist. Typically, an oligonucleotide probe with a sequence complementary to the targeted sequence is used. This probe may be labeled with a reporter molecule that generates light through fluorescence or chemiluminescence or that scatters it colorimetrically. Alternatively, the probe can serve as an intermediate in the detection method. Using fluorophores with distinct excitation and emission spectra allows multiple targets to be detected in a single test. The Urovysion test for bladder cancer developed by Abbott Molecular Diagnostics (Des Plaines, IL) is a nonamplified fluorescent in situ hybridization (FISH) test in which four probes, each with a distinct fluorescent label, bind to target sequences present in a cellular sample fixed on a microscope slide. The primer extension technology of AutoGenomics Inc. (Carlsbad, CA) uses a sequence-specific probe that is extended by means of a polymerase. During the extension step, fluorescently labeled nucleotides are incorporated for detection.
The industry offers many variations on these themes. One, the Hybrid Capture technology of Qiagen Gaithersburg Inc. (Gaithersburg, MD), uses RNA probes in conjunction with antibodies that detect DNA-RNA hybrids. These antibodies are used both for target capture, when bound to a solid substrate, and for detection, when conjugated to alkaline phosphatase.
Several detection technologies do not rely on target amplification but rather on signal amplification alone. The Verigene ID system from Nanosphere Inc. (Northbrook, IL) relies on very high signal amplification involving the deposition of silver onto gold nanoparticle–labeled probes. Siemens Healthcare (Tarrytown, NY) uses a distinctive chemiluminescent approach in its bDNA technology: the target-complementary probe is an intermediate that serves as a target for additional branched probes containing alkaline phosphatase labels. An advantage of this technology is that it is also quantitative, making it useful for infectious-disease testing. The Invader chemistry developed by Third Wave Molecular Diagnostics (Madison, WI) also is a quantitative, nonamplified technology. Intermediate oligonucleotides are generated from probe-binding the unamplified target nucleic acid, and these intermediates bind to a FRET probe that is then cleaved, resulting in the generation of fluorescent signal.
Detection technologies that do not use light generation or scatter have been developed as well. The eSensor platform from Osmetech Molecular Diagnostics (Pasadena, CA) uses redox-reactive ferrocene derivatives as reporter molecules. Rather than light, in this system electron flow as current is measured. The use of ferrocene derivatives with different redox potentials allows multiple signals to be detected simultaneously. Because light-producing and light-gathering optics, as well as the associated mechanicals, are not required, a much simpler, all-solid-state design is possible.
Conclusion
Molecular diagnostics have become indispensable in the infectious-disease field for pathogen detection and for monitoring treatment progress, and are also becoming important tools for cancer detection and typing. The expansion of NATs into the pharmacogenomics and theranostics areas will have major implications for the pharmaceutical industry as they begin to play a role in identifying patient populations particularly suited for a given disease treatment. New technologies will have to deliver greater sensitivity, faster turnaround, and smaller platforms if they are to become part of disease treatment monitoring as well as diagnostics. If this can be achieved, it will also open the door for NATs to move from the dedicated diagnostics laboratory to the physician’s office.
