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MOLECULAR DIAGNOSTICS

Nucleic acid amplification technology has fueled the growth of molecular diagnostics, the fastest-expanding IVD industry segment. Molecular diagnostic products are rapidly transforming the healthcare industry by becoming a vital and indispensable part of the treatment process. The sensitivity, specificity, and flexibility of these products make them ideal for addressing both previously existing and emerging healthcare needs.

While the polymerase chain reaction (PCR) has long been the dominant technology for nucleic acid amplification, a variety of isothermal target amplification methods have been developed as alternatives to PCR. Isothermal amplification methods include strand displacement amplification (SDA),^1,2 transcription-mediated amplification (TMA),³ loop-mediated amplification (LAMP),⁴ rolling-circle amplification (RCA),⁵ and helicase-dependent amplification (HDA).⁶ These methods allow for the possibility of developing less-complicated and less-expensive machinery than is nec- essary for PCR. Other advantages are their potential for use in point-of-care settings and the avoidance of the expense involved in licensing the many PCR and real-time PCR (RT-PCR) patents. Isothermal amplification techniques increase competition and choice in the molecular arena without sacrificing performance.

This article describes isothermal amplification platforms, focusing on the most recent of them, HDA, and discusses their potential applications, especially as a screening and diagnosis tool for hospital-acquired infections.

Established Isothermal Methods

Two of the most well-known isothermal methods have been incorporated into large high-throughput machines, TMA in the Tigris system by Gen-Probe Inc. (San Diego) and SDA in the ProbeTec system from Becton Dickinson and Co. (Franklin Lakes, NJ). These machines are specifically designed to deal with the high volume of molecular testing performed in large clinical and reference laboratories. Both systems are capable of processing a very large number of tests daily, making them most suitable for those markets, such as testing for sexually transmitted diseases (STDs), that involve very high volumes of work. Therefore, many of the isothermal molecular tests now available on the high-throughput machines are tests for STDs. These tests do not require rapid turnaround; thus, samples can be processed off-site at large centrally located laboratories. Many of the clinical laboratories in middle-tier hospitals do not have the financial resources, nor the test volume, to justify investing in these high-throughput platforms.

And as the instruments required to automate these tests involve a significant capital expense, a high test volume is essential for commercial viability when a reagent lease model is used as a means of distribution.

Another isothermal method that has been commercialized for clinical use is an isothermal signal amplification and sequence detection method called Invader developed by Third Wave Technologies Inc. (Madison, WI).⁷ This chemistry uses the structure-specific recognition and cleavage enzyme Cleavase to cut a detection probe bound to double-stranded nucleic acids as a triplex invasion fork. This assay is compatible with several detection formats, ranging from matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectroscopy to fluorescence detection. The manufacturer has also developed an enhanced system, the Invader Plus, that preamplifies the target sequence with PCR in order to increase the sensitivity of the assay platform. Indeed, the Cleavase reaction allows for only a millionfold amplification when used alone, and thus falls short of the sensitivity of PCR. Interfacing Invader chemistry with another isothermal amplification assay system has the potential to make the enhanced system truly isothermal.

Close to 40 assays using this technology are being commercialized. In addition, products for human papilloma virus genotyping, human genotyping, and STD identification fill an aggressive development pipeline.

Several novel methods of isothermal amplification developed more recently have the potential to play a role in advancing the use of isothermal amplification techniques in diagnostic laboratories. One is the loop-mediated amplification platform (LAMP). This technology employs a strand-displacing polymerase and from four to six pairs of primers to selectively amplify sequences from either RNA or DNA, and targets as many as six distinct regions simultaneously.⁴ LAMP products can be detected using an RT-PCR machine and fluorescently labeled primers, or by means of a turbidity detector.⁸ Unlike with RT-PCR, which uses a probe to bind to the amplified product, LAMP method specificity is ensured by the use of multiple primer sets to target multiple regions. Kits for detection of food and environmental pathogens and research-use-only (RUO) detection kits based on the LAMP method are available from Eiken Chemical Ltd. (Tokyo).

A significant drawback of this method is that primer design remains complicated. This has prevented its widespread adoption for the development of in-house assays.

Ionian Technologies Inc. (Upland, CA) has developed a very rapid isothermal amplification technology for the detection of small DNA or RNA fragments generated directly from the target nucleic acid. The amplification process uses single-strand nicking enzymes to generate oligonucleotides that feed a primer extension reaction such that alternating cycles of nicking and extension lead to exponential amplification.⁹ Amplification products are detected by a variety of methods that include liquid chromatography–mass spectroscopy, real-time fluorescence, and capillary electrophoresis detection.

Although very quick, this system lacks the specificity of PCR and of other isothermal methods owing to the lack of a detection probe. Thus, it may be of limited utility in medical diagnostics. The speed of this amplification chemistry makes it ideal, however, for sensors for biodefense applications.

Platform Limitations

Expansion of molecular diagnostics beyond the small fraction of laboratories that currently employ the technology has been hindered by a combination of the high costs associated with its adoption and the shortage of available trained labor. This has resulted in less than 10% of the capable clinical laboratories choosing to practice molecular diagnostics. Many of these labs could potentially be interested in implementing molecular diagnostic techniques if the barrier to entry were lowered to an affordable level and the available assays were also simple to perform.

Isothermal amplification platforms may be well suited to address this market need in part because of the lack of complexity in instrumentation design; thermocycling is no longer an absolute necessity. This opens the door to the use of less costly instruments, which may increase the interest of previously reluctant laboratories in implementing molecular diagnostics.

While TMA and SDA have proven to be valuable diagnostic platforms and have paved the way for the acceptance of isothermal amplification methods in clinical and reference labs, those platforms have limitations. Mainly, they are closed platforms, meaning that clinical labs can purchase a handful of FDA-cleared and RUO kits but cannot purchase general-purpose reagents (GPRs) or analyte-specific reagents (ASRs) in order to develop home-brew assays for the targets of their choice. Gen-Probe and Becton Dickinson have not chosen to commercialize GPRs based upon TMA or SDA. Therefore, PCR remains the only commercially available platform for laboratory-developed tests.

As many of the clinical labs that practice molecular diagnostics develop and validate their own assays in-house, the lack of availability of an isothermal amplification platform has prevented them from adopting alternative molecular methods for these home-brew tests. Therefore, rapid and cost-effective molecular platforms still need to be developed in order to encourage additional laboratories to venture into the realm of molecular diagnostics. This is particularly important for dealing with hospital-acquired infections, which are discussed later in this article. A timely and inexpensive screening program upon patient entry could decrease the acquisition and spread of these deadly infections.

Helicase-Dependent Amplification

Figure 1. (click to enlarge) Helicase-dependent amplification (HDA) uses an enzyme called a helicase (red oval) to separate DNA, allowing two primers (P1 and P2) to bind and DNA polymerase (green oval) to bring about subsequent amplification.

BioHelix Corp. (Beverly, MA) has developed a novel proprietary isothermal technology platform known as helicase-dependent amplification (HDA).^6,10 HDA employs essentially the same reaction mechanism as PCR, except that a helicase enzyme, rather than heat, separates the double-stranded DNA or RNA (see Figure 1). Thus, it does not require a thermocycler for amplification.

The simple reaction scheme requires a single set of specific primers to amplify a target sequence from genomic DNA. In the first step of the HDA reaction, the helicase enzyme loads and traverses the target DNA, disrupting the hydrogen bonds that join the two strands. This helicase unwinding results in the exposure of the nucleotide sequence to the primers, which then anneal. A DNA polymerase can extend the 3'-ends of each primer using free deoxynucleotides to produce two DNA replicates. Similarly to PCR, the replicates will independently enter the next cycle of HDA, resulting in exponential amplification of the target sequence.

BioHelix has incorporated the isothermal HDA platform into two product lines, HDA-Inside and IsoAmp On Demand. The former is a GPR that sophisticated laboratories can use to develop home-brew IsoAmp assays based on the HDA platform just described. Product formation can be fluorescently detected with existing instrumentation such as real-time thermocyclers—the SmartCycler from Cepheid (Sunnyvale, CA) is an example—or fluorescence monitoring incubators—the LightScanner from Idaho Technology Inc. (Salt Lake City), for instance. HDA assays are compatible with a variety of probes, as well as with double-stranded DNA binding dyes like SYBR Green.

Even though HDA is performed isothermally and does not involve thermocycling, no isothermal machine capable of real-time fluorescence detection is commercially available. However, the BioHelix GPR can be used with the equipment that a diagnostic laboratory already possesses, primarily as a less expensive alternative to a PCR-based home-brew test. The HDA platform is also being utilized to develop IsoAmp assays that can be performed in a water bath and whose products can be detected with a disposable lateral-flow device.

HDA-Inside is the first isothermal nucleic acid amplification technology capable of use for home-brew assay development. Either other isothermal amplification technologies are too complex to implement, or manufacturers have chosen not to support such applications by not selling GPR based on their platforms.

Although home-brew assays are used for a variety of diagnostic applications, including lower-volume infectious-disease testing, they are particularly useful for esoteric human genotyping applications. Many of the human genetic tests typically being performed by clinical laboratories are not FDA-cleared assays, because insurance reimbursement policies or test volumes for these do not justify the expense associated with obtaining such regulatory clearance.

Laboratories using isothermal amplification rather than PCR to perform home-brew genetic assays can thereby increase their throughput owing to the fact that amplification reactions can be incubated in an inexpensive water bath instead of a thermocycler.

Unlike viral load monitoring, for genetic testing there is no advantage to monitoring the progress of the amplification reaction during incubation; therefore, real-time detection is not necessary. A laboratory using an Idaho Technology LightScanner and a water bath consequently could perform as many as 9600 genotyping assays per eight-hour shift using 384-well plates and including three controls for each assay.

Figure 2. (click to enlarge) Schematic diagram of the one-tube reverse-transcription HDA reaction. Step 1: Complementary primers (solid black arrow) bind to input RNA (dotted line). Step 2: A reverse transcriptase (rectangle) extends the primer and generates a complementary DNA (cDNA) strand (solid line). Step 3: A helicase unwinds the DNA-RNA hybrid, creating cDNA and RNA. Step 4: Complementary primers bind to the separated RNA and DNA. Step 5: The RNA strand reenters the reaction scheme (5-1), while a DNA polymerase extends the primer bound to the cDNA and generates a daughter strand (5-2). Step 6: The helicase unwinds the DNA strands. Steps 7–9: Primers bind to the DNA strands and a DNA polymerase copies the strands, resulting in exponential amplification.

The HDA platform was originally developed for the amplification of DNA. However, the platform has been modified for use with RNA as well, by including a thermostable reverse-transcriptase enzyme. The many-stage reaction scheme for reverse-transcriptase-based HDA, briefly defined, involves the reverse transcription of the input RNA into complementary DNA (cDNA), followed by the exponential amplification of the cDNA using HDA (see Figure 2). In both reactions, the same helicase is capable of unwinding the DNA-DNA duplexes as well as the RNA-DNA hybrid, allowing for continuous cycling of both the RNA transcription reaction and the DNA amplification reaction. By contrast with many of the RT-PCR assays, the reverse-transcription and amplification reactions can occur simultaneously in the same tube at a single temperature, simplifying and accelerating the full process.

Figure 3. (click to enlarge) Real-time reverse-transcription-based helicase-dependent amplification (RT-HDA) is fast. An Armored RNA of the Ebola virus from Asuragen Inc. (Austin, TX) was used as the template for the RT-HDA reactions in the presence of EvaGreen double-stranded DNA fluorescent dye. Assays were performed in the presence (+SSB) or absence (–SSB) of thermostable single-stranded binding proteins. Reactions were performed at 65°C in an ABI 7300 thermocycler from Applied Biosystems (Foster City, CA). The green line indicates the cycle threshold (Ct).

The inclusion of amplification enhancement proteins such as single-strand binding proteins (SSBs) from extreme thermophiles can greatly enhance the speed of HDA reactions. One example of the ability of these additives to accelerate HDA is provided by assays involving the amplification of a specific sequence that targets the Ebola virus, Asuragen Inc.’s (Austin, TX) Armored RNA (see Figure 3). In the presence of Primer Navigator SSBs developed by BioHelix, the cycle threshold (Ct) value—that is, the time at which the threshold in fluorescence is crossed—for the detection of 5000 copies of RNA is 20 minutes, whereas a Ct value of 40 minutes is obtained in the absence of SSBs.

Although the exact mechanism underlying the enhancement in reaction time due to the inclusion of the SSBs is not known, it is possible that the proteins increase the processivity of the enzymes to increase the efficiency of the reaction. The real-time HDA (RT-HDA) assays whose results are graphed in Figure 3 were performed in a slow thermocycler, the ABI 7300 from Applied Biosystems (Foster City, CA), and yet the reaction time required for detecting as few as 50 copies is still below 30 minutes—that is, as fast as RT-PCR performed in a LightCycler from Roche Diagnostics (Indianapolis) or a Cepheid SmartCycler.

HDA has been used to amplify targets from bacteria, viruses, and human DNA, and is compatible with such various detection formats as gel electrophoresis, fluorescence-based real-time detection, and lateral-flow detection. The HDA-Inside isothermal amplification technology works with fluorescence-based detection devices that are currently available in diagnostic labs. Fluorescence monitoring in real time, during the amplification reaction, allows the user to measure quantitatively input nucleic acids by using an external calibration series consisting of nucleic acid targets of known concentration, just as with RT-PCR. The HDA product is potentially a cost-effective alternative that high-complexity diagnostic laboratories can employ to develop assays for any target they choose.

Target Market: Hospital Infections

A target market that may benefit from advances in isothermal amplification methods is the screening and diagnosis of hospital-acquired infections. These infections are a growing problem in the United States and Europe. The overuse of antibiotics to treat infections has had the unfortunate consequence of developing a variety of strains of drug-resistant bacteria. The mortality rate for patients that develop a hospital-acquired infection is almost 13% per hospitalization, in comparison with a rate of 2.3% for patients without a hospital-acquired infection.¹¹

Methicillin-resistant Staphylococcus aureus (MRSA), a virulent strain of S. aureus (SA), alone is responsible for more than 100,000 hospitalizations and an estimated 10,000 to 15,000 deaths every year. Avoiding this infection can save hospitals more than $20,000 per case in estimated nonreimbursable expenses, in addition to potentially saving lives.¹² Traditional culture-based diagnostics can identify this strain inexpensively, but only after 48 hours, which is too long for either preventive screening or time-sensitive diagnosis.

More recently, several rapid molecular tests, such as the GeneExpert system by Cepheid, have been developed to reduce the MRSA identification time to less than two hours; however, the required equipment is expensive, particularly for small and middle-tier hospitals. This diminishes interest in the tests’ use as a screening tool. Dependence on the clinical laboratory already having a thermocycler, or being able to purchase one, is also a deterrent to initiating molecular screening. In addition, if the laboratory has not already implemented molecular testing, then specialized training for operators of the machines to perform the assays will be necessary.

At the same time, there is tremendous societal pressure surrounding the reporting of MRSA infections in hospitals that will greatly expand the need for MRSA testing. Sixteen states already have passed laws mandating reporting of hospital infection rates, and many other states have legislation pending. Therefore, an inexpensive and simple alternative for MRSA screening has the potential to be implemented by hospitals looking for a viable solution to the increasing MRSA problem.

The ability to perform molecular tests as soon as a sample arrives in the clinical laboratory is also particularly valuable in the case of the life-threatening illness Clostridium difficile–associated disease (CDAD), another hospital-acquired infection. C. difficile is one of the most common nosocomial infections encountered in hospitals. An estimated 10–12 million adults are infected with this organism each year in the United States, and about a third of them become symptomatic.¹³ The clinical spectrum of CDAD ranges from diarrhea to severe, life-threatening pseudomembranous colitis.

Although enzyme-linked immunoassays are available to detect toxicogenic strains of C. difficile, these have high false-negative rates, even in patients with severe clinical disease. The treating physician tries to compensate for this shortcoming by ordering several successive tests. However, as the toxin degrades at room temperature and may be undetectable within two hours after collection of a stool specimen, repeat testing may not always be productive.

A fecal cytotoxicity assay using cell culture also is available, but it requires a much longer processing time. Also, because it is so complex, most hospitals do not perform the assay in-house but instead ship samples out for testing at a reference laboratory. A simple, instrumentation-free molecular assay would enable hospitals to eliminate this testing problem and potentially reduce the incidence of life-threatening pseudomembranous colitis.

Using Lateral-Flow Detection

One possible solution to the challenge of creating an economical, simple-to-use molecular test is the adoption of nucleic acid lateral flow as a detection format, rather than the more complicated fluorescence detection.¹⁴ Lateral flow is a format commonly used in the point-of-care diagnostic industry for immunotesting. It can provide a simple, cost-effective, and easily interpretable format for molecular testing as well, particularly when coupled with an isothermal amplification method.

Unlike PCR, which, even when used with lateral-flow detection, requires the use of a thermocycler for the amplification reaction, HDA can be performed in a water bath or in a heat block, making it unnecessary to purchase thermocycling equipment. Nor does HDA require a brief denaturation step at 95°C to initiate the assay, as many of the other isothermal methods do. Therefore, a single water bath or heat block is the only instrumentation necessary to perform the assay.

The familiarity of the lateral-flow immunoassay format to clinical labs means that staff will not need additional training to operate a new or complicated piece of equipment. BioHelix’s IsoAmp On Demand molecular analyzer enables clinical laboratories to perform rapid molecular diagnostic tests using a simple disposable nucleic acid detection device in combination with an isothermal target nucleic acid amplification platform.

The HDA assay can be taken from a water bath and placed directly inside a plastic housing that holds the closed tube and the lateral-flow strip. This allows the tube contents to be transferred to the strip in a completely enclosed format, thus preventing release of amplicons after the amplification reaction.

Figure 4. (click to enlarge) Schematic diagram of asymmetric HDA. Amplification results in the generation of multiple copies of single-stranded amplicon for probe detection.

BioHelix is developing an HDA-based assay for its molecular analyzer that targets the mecA gene from S. aureus, an indicator of methicillin resistance. As depicted in Figure 1, HDA utilizes two sequence-specific primers to amplify a targeted sequence isothermally at 65°C. In order to most effectively couple this method with lateral-flow detection, BioHelix developed an asymmetric HDA reaction method whereby a larger quantity of one labeled primer than of the other is provided in the amplification reaction (see Figure 4). This results in the generation of single-stranded amplicons that can anneal to sequence-specific DNA probes present during the reaction.

Figure 5. (click to enlarge) Detection of MRSA by asymmetric HDA was carried out with a pair of mecA gene-specific primers and genomic DNA in the presence of a biotin-labeled probe in a total volume of 50 µl. After HDA, 10 µl was applied to 2% agarose gel (upper panels). Another 10 µl of the products were applied to lateral-flow strips following incubation with a gold-conjugated antibiotin antibody (lower panels). A visible red line indicates a positive signal (*). (a) Determination of the limit of detection (LOD) of the HDA-based MRSA assay with genomic DNA from the Mu50 MRSA strain. (b)Testing the specificity of HDA-based MRSA assay in the presence of non-MRSA S. aureus (SA) cells.

A positive signal is generated by incubating the HDA reaction with a gold-conjugated antibody to the labeled probe and applying this to a lateral-flow test strip that contains an antibody to the biotinylated primer. This ensures that detection is specific for the amplified product.

The limits of detection for the HDA MRSA assay by gel electrophoresis and by lateral-flow detection can be illustrated (see Figure 5). A positive signal corresponding to the correct product can be detected in samples containing from as many as 5 × 10⁵ copies of MRSA genomic DNA down to approximately 5 copies of MRSA genomic DNA, demonstrating the high degree of sensitivity of this method. The right-hand portion of the figure shows the ability of the HDA MRSA assay to detect specifically the presence of 100 MRSA cells in samples that also contain 106 nonresistant SA cells. Discrimination of MRSA from nonresistant SA in clinical samples is critical, as MRSA is often found concomitantly with nonresistant SA in infected individuals. In the absence of any input MRSA (lane 11 in the figure), a primer-dimer artifact is generated. However, this is not detected on the lateral-flow test strip.

Conclusion

Generally, isothermal amplification methods such as those described in this article can offer a great deal of application utility and choice for both clinical diagnostics and biodefense. They lay especially strong claim to consideration in applications in which performance, equipment requirements, and cost are important factors in the selection of a molecular diagnostics platform.

 

 

 

Huimin Kong is the president and CEO of BioHelix Corporation (Beverly, MA). He can be reached at kong@biohelix.com.	Bertrand Lemieux is senior director of technology development at BioHelix and he can be reached at lemieux@biohelix.com.	Tamara Ranalli is manager for business development and quality system at BioHelix and she can be reached at ranalli@biohelix.com.

 

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