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The tools available for molecular diagnostic assays have expanded considerably since the first non-amplified nucleic acid test (NAT) to confirm Legionnaires’ disease received FDA approval in 1985. It took another eight years before the first amplified test was approved, allowing detection of a target nucleic acid directly from a clinical sample. Amplification offered the benefit of increased sensitivity while simultaneously decreasing the time required to obtain a result, since culturing to enrich the target was no longer required. Another advance in molecular diagnostics testing is the ability to perform quantitative tests through the use of technologies such as real-time PCR.

The scope of molecular diagnostic testing includes a diverse range of infectious-disease agents, and is now expanding into the oncology field. As the genetic components of drug efficacy and predisposition to various diseases become more understood, new areas such as pharmacogenomics and disease susceptibility are being entered as well.

Although the use of molecular diagnostic testing is growing, several issues continue to prevent its wider acceptance. One is that nearly all molecular IVD tests have been classified as highly complex by FDA, meaning that the tests are 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.

Other issues such as cost per test and, especially when a test uses a new or unique technology, reluctance to adopt unproven technology are also factors preventing more wide-scale utilization of molecular diagnostics.

Overview of Molecular Diagnostics

Two broad categories of molecular diagnostic tests exist; commercially available FDA-approved tests and laboratory developed tests. The former are provided as kits that include all critical reagents needed to perform the test, often including any required calibrators and controls. Manufacturers providing these assays must provide data supporting the performance claims of the test that has been reviewed and approved by the FDA. Instrumentation integral to the performance of the tests requires FDA-approval as well. The instrumentation may be assay specific—that is, designed to run a specific company’s own IVD technology—such as the fully automated Tigris DTS System from Gen-Probe Inc. (San Diego), or generic and able to accommodate various products, such as the LightCycler real-time PCR instrument manufactured by Roche Applied Sciences GmbH (Mannheim, Germany).

Alternatively, a clinical laboratory can develop a generic, or home-brew molecular diagnostic test. Such a test is not reviewed or approved by the FDA, but the performance characteristics must be validated by the laboratory prior to use for testing and reporting clinical specimen results.

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 (DNA or RNA) while preventing sample matrix, cellular debris, and/or other interfering substances from affecting the sensitivity and specificity of the test. Nucleic acid-based diagnostics are sequence specific, therefore the test must have sufficient specificity to discriminate non-target nucleic acid (i.e., DNA or RNA in cellular debris) from the desired target sequence, 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 is often required. Detection of the target, whether amplified or not, is typically accomplished through the use of automated 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 test results may 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 and 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), are also becoming increasingly available.

Sample Preparation Tools

A wide range of biological sample types are used for molecular diagnostic assays. 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 be used for oncology tests. Blood is most often used for genomics tests, but saliva or cheek cells 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 (e.g., cellular debris, matrix, proteins, and so on) must be eliminated or minimized because of the 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 false negative or inconclusive results. The nucleic acid in the sample is usually concentrated at this initial step in order to increase sensitivity.

Chemical lysis is the most widely used method of releasing the nucleic acid from the sample. Other methods include sonication, which is employed in the GeneExpert system by Cepheid (Sunnyvale, CA). 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, 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 Applied Sciences, the BioRobot EZ1 workstation from Qiagen Sciences Inc. (Germantown, MD), and the NucliSens easyMAG platform from bioMérieux SA (Marcy l’Etoile, France) are systems that utilize magnetic-bead technology and are suitable for the automated nucleic acid purification of tens of samples simultaneously. Instruments that can process a thousand or more samples daily are also available.

Target Amplification

The first NATs were not amplified, using 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 produce a result directly from a patient sample, 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 instrument capable of both heating and cooling during the process. TMA, SDA, and NASBA are all isothermal processes that require only a single set temperature be maintained. This reduces considerably the complexity of the instrumentation required for amplification.

There is usually a large excess of non-target nucleic acid present in a sample, so amplification is performed using target specific oligonucleotide primers. 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, such as the warfarin metabolism NAT from Nanosphere Inc. (Northbrook, IL). This test identifies specific single-nucleotide polymorphisms related to drug dosing and is thus concerned only with whether a specific DNA sequence is present; the number of copies of the target sequence in the sample is irrelevant.

The need for infectious-disease NATs to be able to quantify the titer of target present, such as 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. Real-time target quantification can also used with other amplification technologies such as TMA and SDA.

Target quantification 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 proportionally. Because amplification of target is exponential during the early cycles of amplification, 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. At least 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 applicable), 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 reflects/scatters it. 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 non-amplified fluorescent in situ hybridization, or 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. Alternatively, the probe can serve as an intermediate in the detection method. 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. There are many variations on these themes. One, the Hybrid Capture technology of Qiagen 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 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 branched DNA (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 reagent chemistry developed by Third Wave Technologies Inc. (Madison, WI) is another quantitative, non-amplified technology. With this method, 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.

Conclusion

Molecular diagnostics are indispensable in infectious-disease screening, diagnosis, and monitoring, and are also becoming important tools in these aspects of the oncology field. The expansion of NATs into pharmacogenomics and oncology 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 IVD NATs to move from the dedicated diagnostics laboratory to the physician’s office.