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Originally Published May 2000
Solid-phase nucleic acid extraction, amplification, and detection
Roy R. Mondesire, Diane L. Kozwich, Kristine A. Johansen, John C. Gerdes, and Shannon E. BeardThe use of molecular technologies for clinical diagnostics doesn't have to be synonymous with high-cost, high-complexity instrumented systems.
Since the introduction of the first FDA-approved clinical laboratory test kit for Legionella, by Gen-Probe Inc. (San Diego), molecular diagnostic methods have played an important role in the diagnosis of a variety of diseases.1,2 Although the market is dominated by infectious-disease diagnostics, molecular tests for cancer and genetic diseases are on the increase. The primary factors that have contributed to the rapid expansion of molecular diagnostic pathology include improvements in nucleic acid extraction techniques, the development of more-efficient DNA-sequencing methods in conjunction with the Human Genome Project, the growing diversity of nucleic acid amplification techniques, the accessibility of several commercially available molecular detection methods, and the introduction of semiautomated instrumentation for probe testing.In an effort to secure their share of the emerging nucleic acid diagnostics market, several companies are competing to develop new medical devices. Many such companies are developing products based on the relatively complex and instrumented technologies of DNA microarrays and related microfluidics.3–5 By contrast, our company is focusing on the development of relatively simple solid-phase extraction, isothermal amplification, and rapid detection by lateral-flow techniques. It is hoped that this approach will address the need for point-of-care (POC) testing in molecular diagnostics, eventually making it possible for clinicians to conduct rapid and inexpensive on-site testing for a variety of disease markers.
Extraction and Capture
In the method developed by Xtrana Inc. (Denver), the immobilization of nucleic acid target is achieved through the use of the company's solid-phase nucleic acid binding material trademarked as XtraBind. This material has a number of unique properties that provide for rapid nucleic acid capture and purification. It can irreversibly bind both single-stranded DNA and RNA. Under optimal buffer conditions, capture of the nucleic acid target occurs immediately even when starting with low copy numbers. DNA thus bound to XtraBind is quite stable and can be stored for later analysis for at least 18 months. Nucleic acid captured using this material can be amplified directly on the solid phase using a variety of amplification strategies, including those requiring single-strand initiation. The material is commercially available in a kit format for use with blood (the XtraAmp whole blood DNA-extraction kit), and is capable of capturing single copies of genes from as little as 10 µl of blood. Studies are under way to examine the use of this material for nucleic acid extraction from several other types of clinical samples.
| Xtrana Method | |||
| FSIS Method | Positive | Negative | Total |
| Positive | 5 | 0 | 5 |
| Negative | 0 | 17 | 17 |
| Total | 5 | 17 | 22 |
Table I. Results of a study comparing the binding properties of the Food Safety and Inspection Service (FSIS) method to Xtrana's solid-phase nucleic acid binding material, XtraBind.
One study investigated the binding properties of the XtraBind solid-phase surface as compared with the Food Safety and Inspection Service (FSIS) method. As its target molecule, the study used RNA from Escherichia coli 0157:H7 amplified by means of nucleic acid sequence-based amplification (NASBA). Results of the study showed complete agreement with the FSIS method, and indicated that there was no cross-reactivity with related gram-negative microorganisms (see Table I). Another study of the solid-phase technology demonstrated that, after extraction and amplification, it could detect RNA from as little as one Cryptosporidium parvum oocyst per liter of water (see Figure 1). Other pathogenic microorganisms successfully adapted to this method include Chlamydia trachomatis, Neisseria gonorrhea, Listeria monocytogenes, and Yersinia pestis.
Figure 1. Detection of Cryptosporidium parvum oocysts in raw water samples. The bars represent positive lateral-flow results following the spiking of oocysts, extraction of RNA, and amplification by NASBA.
To selectively capture nucleic acid targets where very few copies are present in complex specimens, users can perform hybridization target selection with the specific oligonucleotide-derivatized XtraBind where the target nucleic acid is to be captured. In this approach, total nucleic acid is released from the test specimen, then diluted with a hybridization buffer. Hybridization capture makes it possible to select for low concentrations of specific target, even in the presence of high levels of other molecules. Proteins and other potential inhibitors of the nucleic acid amplification reaction are removed by washing.
Amplification
The technology developed by Xtrana can be adapted to a variety of target amplification techniques, including the widely used polymerase chain reaction (PCR) and related methods. PCR is based on the ability of DNA polymerase to copy a strand of DNA by elongation of complementary strands initiated from a pair of oligonucleotide primers.
Reverse transcriptase-PCR is used to amplify RNA targets. In this process, the reverse transcriptase enzyme is used to convert RNA to complementary DNA (cDNA), which can then be amplified using PCR. This method has proven useful for the detection of RNA viruses.
Nested PCR employs two sets of amplification primers to conduct a dual amplification process that results in very high sensitivity. In multiplexed PCR, two or more pairs of primers for different targets are introduced into the same reaction mixture. Here, there is simultaneous amplification of more than one unique DNA sequence present in a sample.
Isothermal nucleic acid amplification systems include the transcription-based amplification system (TAS) and its derivatives. Methods based on TAS include self-sustaining sequence replication (3SR), NASBA, and transcription-mediated amplification (TMA). Another non-PCR isothermal system is strand-displacement amplification (SDA). The ligase chain reaction (LCR), a probe amplification technique, has also been successfully used in diagnostics.
Detection Chemistries
A variety of chemistries are available for the detection and measurement of amplified target molecules. When colorimetric methods are used, results can be assessed visually or with the aid of a spectrophotometer. Labeling of the target is commonly accomplished by using enzymes such as horseradish peroxidase. Two of the common chromogenic substrates for peroxidase are 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS).
Detection techniques based on the use of fluorescent labels are also useful in clinical diagnostics.6 Fluorometric assays are more sensitive than colorimetric ones and can be used to detect or measure lower levels of nucleic acid. However, fluorometric assays have the disadvantages that they involve more-complex procedures and must be assessed using instrumentation. The use of traditional fluorescent compounds is further complicated by the background interference that can be caused by light scattering and the intrinsic fluorescence of sample components. To overcome such limitations related to background interference, assay developers can design tests to make use of time-resolved fluorescence. The technique of time-resolved fluorescence is based on the principle that some lanthanides, such as europium (Eu3+), form fluorescent chelates with certain organic ligands. These fluorophores have very large Stokes' shifts and decay times (200 nm and >500 ns, respectively). Time-resolved fluorescence takes advantage of these long decay times and large Stokes' shifts, making them qualifiers for the fluorescent signal to be recorded. Thus, any short-lived fluorescent background signal or scattered excitation radiation can be readily filtered for elimination, and the desired fluorescent signals can be measured under conditions that are virtually without background interference.
In chemiluminescent assays, luminescent compounds emit light during the course of a chemical reaction. The labels used for such assays are commonly luminol derivatives or acridinium esters. The kinetics of assays using chemiluminescence are very fast, and light is emitted within seconds of substrate oxidation. In an electrochemiluminescence (ECL) technique, a ruthenium metal chelate and tripropylamine are utilized.7,8 Both of these molecules become oxidized at the surface of an electrode, where they react to form an excited state of ruthenium that decays, releasing a photon at 620 nm.
Detection techniques using biosensors are also gaining acceptance among the makers of nucleic acid diagnostics. Biosensors are analytical devices in which a molecule of biological origin serves as the chemical recognition element (e.g., an antibody or enzyme), and is measured when it comes into contact with a physical transducer or detector.9 A wide variety of such biosensors have been developed, including biocatalytic, electrochemical, optical, mass-detection, amperometric, pharmacological, and immunochemical sensors.10,11 In the field of clinical diagnostics, the application of immunological biosensors has gathered considerable impetus, with a rapid influx of new applications now under development. Some DNA biosensors utilize single-stranded DNA immobilized on quartz optical fibers, piezoelectric crystals, or amperometric electrodes. Unfortunately, such biosensor detection technologies are relatively complex, expensive, and require instrumentation in the vast majority of cases.
Lateral-Flow Detection
Although the detection and measurement technologies described above are suitable for use in nucleic acid assays, most require instrumentation that increases the overall costs of such testing. To eliminate this drawback, Xtrana has focused its efforts on developing a detection platform based on lateral-flow principles utilizing dyed microsphere labels. The method is sensitive, inexpensive, and easy to perform.
To perform the assay, the operator adds biotinylated and fluoresceinated oligonucleotide detection probes to the amplified reaction mixture. At this time, if the target is present, the two sets of probes will specifically hybridize with the same strand of the target DNA or RNA molecule. When mixed with streptavidin-coated dyed microspheres, the biotinylated portion of the probe will bind to the streptavidin. The resultant complexes are then applied to a nitrocellulose membrane containing an immobilized antifluorescein isothiocyanate antibody line.
Migration of the complexes occurs through the membrane until the antibody binds the fluoresceinated portion of the probe. This interaction arrests further migration of the microsphere-haptenized duplexes and rapid accumulation of these complexes occurs at the antibody line (see Figure 2). The unaided eye can easily detect this result. Figure 3 shows the correlation between agarose gel electrophoresis and the lateral-flow technique. The lateral-flow test is several logs more sensitive than gel electrophoresis, and the results are revealed within two minutes after the addition of reaction products to the membranes.
Figure 2. Schematic of Xtrana's lateral-flow method for detection of amplified single-stranded DNA intermediates following amplification. The migration of the microsphere labeled nucleic acid complexes are arrested at the antifluorescein isothiocyanate stripe, where the reaction is observed by the user.
Figure 3. Agarose gel electrophoresis (a) and detection by lateral flow (b) of specific RNA product after NASBA amplification of target captured using XtraBind. Lanes 1 through 7 represent 10-fold dilutions of product starting at a high copy of specifically amplified RNA. Lane 8 is a no-target control.
Lateral-flow detection methods are economical and do not require instrumentation. This translates into a drastic reduction in cost for nucleic acid–based testing. These methods are usually single-step assays that do not require multiple washing or long incubation steps.
Containment
As a containment vessel for its lateral-flow molecular assays, Xtrana has developed a closed, self-contained device called the self-contained integrated particle (SCIP), which combines all the steps required to perform DNA or RNA testing (see Figure 4). The sample is introduced into one end of the device and a series of simple manipulations moves the sample to other compartments of the device where extraction, amplification, and detection occur automatically. This closed system prevents contamination and eliminates the need for expensive equipment.
Figure 4. Prototype of the self-contained integrated particle (SCIP) device illustrating its essential components. Extraction, amplification, and detection are achieved in a self-contained fashion through a series of simple fluid-transfer processes.
The SCIP device integrates XtraBind for nucleic acid extraction with immunodiagnostic detection technology. Initial experiments using SCIP prototypes have produced detection sensitivities that meet currently available DNA testing requirements. This SCIP device, incorporating microsphere lateral-flow detection, removes many of the roadblocks of cost and complexity associated with the technological transfer and adaptation of nucleic acid–based testing to the end-user. Furthermore, it should produce a significant shift of certain types of diagnostic testing from centralized urban reference laboratories to rural settings or POC testing.
Conclusion
The current market for in vitro diagnostics is approximately $20 billion worldwide. The U.S. portion is estimated at approximately $8 billion, with clinical chemistry and immunoassay testing making up approximately 45%.12 However, molecular technologies are rapidly forging their way to the forefront of the industry, and with a current market share of 3%, DNA- and RNA-probe diagnostics constitute the fastest growing segment of the clinical diagnostics market.
Some analysts have suggested that today's competitive market will demand that new molecular technologies fulfill the need for a broad panel of assays, automated systems, and high throughput.13 However, the complexity and high cost of molecular systems designed to meet all such needs may prohibit their use in some laboratories. A formidable challenge for manufacturers in the rapidly expanding nucleic acid diagnostic market will be to produce easy-to-use, inexpensive devices with high-performance characteristics. The R&D efforts at Xtrana are directed toward achieving these requirements.
The development of molecular diagnostic technologies in this decade will be influenced by innovations in chemistry, molecular biology, immunology, automation, and data management. At the same time, small and inexpensive POC devices will continue to play a major role in providing accurate and objective results in doctors' offices as well as alternative-care settings. Manufacturers of molecular diagnostics are in an exciting early phase of this technology and have new challenges that offer the opportunity to assist in providing the highest quality medical care worldwide.
References
1. CS Hill, "Molecular Diagnostics for Infectious Diseases," Journal of Clinical Ligand Assay 19 (1996): 43–52.
2. YW Tang et al., "Molecular Diagnosis of Infectious Diseases," Clinical Chemistry 43 (1997): 2021–2038.
3. C Henke, DNA-Chip Technologies, part 1: Research Fundamentals and Industry Catalysts, IVD Technology 4, no. 5 (1998): 28–32.
4. C Henke, DNA-Chip Technologies, part 2: State-of-the-Art and Competing Technologies, IVD Technology 4, no. 7 (1998): 35–44.
5. C Henke, DNA-Chip Technologies, part 3: What Does the Future Hold? IVD Technology 5, no. 1 (1999): 37–48.
6. IL Hemmilä, Applications of Fluorescence in Immunoassays (New York City: Interscience: Wiley & Sons, 1991).
7. H Yang et al., "Electrochemiluminescence: A New Diagnostic and Research Tool," BioTechnology 12 (1994): 193–194.
8. F Jameison et al., "Electrochemiluminescence-Based Quantitation of Classical Clinical Chemistry Analytes," Analytical Chemistry 68 (1996): 1298–1302.
9. APF Turner et al., eds., Biosensors: Fundamentals and Application (Oxford: Oxford University Press, 1987).
10. MYK Ho, "An Introduction to Biosensors," in RM Nakamura et al., eds., Immunochemical Assays and Biosensor Technology for the 1990s (Washington, DC: ASM Press, 1992), 275–290.
11. PG Malan, "Immunological Biosensors," in D Wild, ed., The Immunoassay Handbook (New York City: Stockton Press, 1994), 125–134.
12. Molecular Diagnostics and Related Instrument Systems: Moving to Point-of-Care (Montclair, NJ: Genesis Group, 1999).
13. J Keefe, U.S. DNA/RNA Probe Diagnostic System Markets (Mountain View, CA: Frost & Sullivan, 1999).
Roy R. Mondesire is project leader, Diane L. Kozwich is a former vice president for operations, Kristine A. Johansen is a senior scientist, John C. Gerdes is vice president for research and development, and Shannon E. Beard is director of business development at Xtrana Inc. (Denver). This article is based on work supported by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under agreement nos. 99-33610-8149 and 98-33610-6345. Opinions, findings, conclusions, and recommendations expressed herein are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.
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