Medical Device Link.

Originally Published IVD Technology July 2004

Molecular Diagnostics

Figure 1. Whole blood is collected directly into a Liat tube (a, b). After the tube is capped, the analyzer scans the bar code (c). The tube is inserted into the analyzer (d). The analyzer then conducts all the nucleic acid testing automatically and reports the results in real time (e) (click to enlarge).

Molecular diagnostics is one of the fastest-growing segments in the IVD industry and has the potential to revolutionize biological sample testing. Bringing such sensitive tests to hospital laboratories, patients’ point-of-care (POC), production floors, and emergency first responders presents a considerable challenge because of the complex steps that are required to set up and perform molecular assays. To address this need, technology platforms have been developed which enable ordinary healthcare workers and first responders to perform such sophisticated tests in various settings.

Current Commercial Nucleic Acid Test Systems

Many challenges must be overcome when conducting nucleic acid tests (NATs) both in centralized laboratories and out in the field. Large laboratories use automated or semiautomated robotic systems for high-volume NATs, such as for detecting human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Even though they are automated, such high-throughput systems still require certain manual preparations, including sample and reagent loading, and waste removal. Moreover, the NATs offered on these automated systems account for only a small percentage of the testing menu that clinical labs need. For smaller laboratories, the cost of an automated system is often difficult to justify, considering that NATs are usually outsourced or conducted by hand. 

Another challenge is that because of the specialized skills required to conduct many NATs, only highly trained technologists can perform such long and complicated testing processes. Cross-contamination is also a problem when conducting NATs since they employ amplification technologies such as polymerase chain reaction (PCR). Clinical laboratories often use separate rooms for reagent preparation, sample preparation, amplification, and postamplification analysis. For these reasons, NATs are considered high-complexity tests under the Clinical Laboratory Improvement Amendments (CLIA). To date, no NAT system has qualified for CLIA-waived status, largely because of the difficulties in automating sample preparation and reagent handling. 

Figure 2. This figure shows the individual steps of a nucleic acid test using a Liat tube inserted into a Liat molecular analyzer. The flexible Liat tube (light blue) contains a sample (red), reagents (white), and waste (magenta). Actuators (light brown) and clamps (white) compress the tube segments in a coordinated manner, while blocks (yellow) are heated (red) to incubate the sample. Magnets (black squares) and photometers (PM) (blue) are also shown (click to enlarge).

Performing field-use or near-patient NATs involves even more challenges, especially since they will inevitably be conducted by less-experienced users in nonlab environments. The following systems have recently been developed for deployment of NATs in the field. 

The GeneXpert system by Cepheid (Sunnyvale, CA) appears to be the first PCR-based instrument that integrates sample preparation, amplification, and detection. The system ultrasonically disrupts up to 5 ml of sample, breaking cell membranes or spore coats, and extracts DNA into a microfluidic channel containing immobilized DNA probes that bind bacterial DNA as the cellular debris flows over.¹ The bound DNA is later released from its attachment site and washed off for PCR amplification.²
 
Other commercialized real-time PCR devices intended for field use include the Ruggedized Advanced Pathogen Identification Device by Idaho Technology Inc. (Salt Lake City), the Hand-Held Advanced Nucleic Acid Analyzer by Lawrence Livermore National Laboratory (Livermore, CA), and the Bio-Seeq detector by Smiths Detection (Pine Brook, NJ). However, these devices do not have automated sample-preparation and reagent-handling functions.

Because of the complex nature of sample preparation and handling, the U.S. Department of Health and Human Services has recommended against first responders using handheld assays for evaluating unknown powders suspected of being anthrax or other biological agents.³ Various studies have discussed the development hurdles that must be overcome to adapt molecular diagnostic technologies to field use.^{4, 5}

The Liat Molecular Analyzer 

Figure 3. Rapid two-step polymerase chain reaction (PCR) with a 95°C incubation for 3 seconds, a liquid transfer time of 2.5 seconds, and an incubation at 60°C ranging from 13 to 33 seconds. The fluorescence intensity is plotted as a function of time. Amplifications were performed using 103 copies of Bacillus cereus DNA (click to enlarge).

In an effort to address the requirements of a fully automated NAT system, IQuum Inc. (Allston, MA) has developed a system that is based on its proprietary lab-in-a-tube (Liat) technology platform. Consisting of the Liat analyzer and disposable Liat tubes, the Liat system automates all NAT processes, including reagent preparation, target enrichment, inhibitor removal, nucleic acid extraction, amplification, and detection in one portable device.

In this system, the testing process has been refined to three steps: collecting a raw biological sample, such as whole blood or cells, in a collection device; placing the sample into a Liat tube and capping it; and inserting the tube into a Liat analyzer (see Figure 1). The analyzer automatically executes all the required assay steps, including sample preparation and real-time PCR, and reports the test results on the analyzer’s touch screen. No further operator intervention or interpretation is required. 

Designed to be similar in size to conventional test tubes for easy handling, the Liat tube uses a flexible tube as the sample vessel and contains all assay reagents prepacked in tube segments that are separated by peelable seals. A bar code is attached to the tube frame so that the analyzer can identify the test menu and select an appropriate program for conducting the test. Another bar code sticker, such as for patient identification, can be attached to the tube frame to track the sample. After a test is completed, any biohazard waste remains enclosed in the tube for disposal. 

The Liat analyzer is small in size (approximately 3.7 ¥ 6.5 ¥ 6 in.), is light (about 4 lbs.) and can accommodate one tube at a time. An internal optical system provides four independent excitation and emission-detection channels for fluorescence signal monitoring. This allows for internal controls and multiplex detection of specific sequence variants in each test. The analyzer is also equipped with microprocessors for automation control, a color touch screen for input and result display, and a variety of connectivity ports. In addition, the analyzer can be powered internally, by an external battery pack, or by standard ac power.

Figure 4. Multiplex PCR detection of E. coli encoding Shiga-like toxins (Stx). Minor groove binding probes for the Stx1 and Stx2 genes were used to detect 103 copies of E. coli DNA isolated from strains with Stx1 only (a) and both Stx genes (b) (click to enlarge).

The Liat system is designed as a truly closed system. Once a sample is introduced into the Liat tube, it remains closed for all test processes. This reduces biohazard and cross-contamination risks, and helps to preserve sample integrity and prevent false-positive results. The Liat technology uses liquid flow and mixing to accelerate reactions. For example, a 30-cycle PCR of 50 µl of sample can be accomplished within 12 minutes. Alternatively, the system is capable of accommodating all of the isothermal nucleic acid amplification methods that use fluorescent probe reporters. This rapid speed allows a genetic test from whole blood to be conducted in 30 minutes, and a test for infectious agents in plasma in less than 1 hour.

Lab-in-a-Tube Technology 

The core technologies of the Liat platform are the flexible plastic sample tube, the modular sample processor, and the sample processing methods. 

Tube Features and Self-Contained Reagents. Many features of the Liat tubes have been developed to automate sample processing. One of the tube’s features is a peelable seal that is formed by a thermal weld of the plastic tube and is used to form tube segments for reagent storage and handling. By applying pressure to the tube segments adjacent to each seal, the seal can burst open to release reagents. 

By using peelable seals, unit-dose reagents and internal controls can be held separately in a series of tube segments in the order they are used for an assay. Buffers and organic solvents are kept in a liquid format, while biological reagents such as enzymes, oligonucleotides, and antibodies are kept in a dry format for stability and long shelf life. Since reactive solutions in the tube are reconstituted just in time for use in an assay, reaction specificity can be improved without resorting to chemically modified enzymes.

In a clinical testing lab, highly trained technicians reconstitute reagents in cleanrooms prior to test initiation. In contrast, with the Liat system, the molecular analyzer automatically performs such tasks with prepackaged reagents. 

Sample Processor and Sample Manipulation Methods. In a Liat analyzer, multiple sample-processor modules are aligned with the Liat tube (see Figure 2). Each module consists of an actuator and a clamp, whose positions can be controlled to manipulate a test sample within a tube. Each module also contains a temperature-control element that can be used to heat, cool, or incubate the sample. A retractable magnet is attached to one of the modules for manipulating magnetic beads, and a four-channel photometer is connected to another module for detection. 

Figure 5. Detection of viral particles in human plasma. Three 40-µl plasma samples spiked with approximately 103 Epstein-Barr virus (EBV) particles were added to a Liat tube containing prepacked reagents. An input control consisting of purified EBV DNA and a negative control consisting of human plasma without EBV (dashed line) are included for comparison (click to enlarge).

When a tube is loaded in the analyzer, the actuators and clamps compress the tube sequentially to move the reagents and controls from one segment to another (see Figure 2A). Similarly, by synchronizing the motion of the actuators and clamps, various sample processes can be conducted within a tube. Such processes include adjusting a liquid’s volume in a segment; releasing a reagent to the adjacent segment; mixing reagents and samples; agitating and incubating a reaction mixture at a given temperature; and washing and removing waste from a segment. Waste is moved toward a waste chamber in the cap while the purified sample moves further down the tube. By doing so, the processed sample does not contact surfaces that have been contaminated by previous steps. 

Sample-Preparation Options. The Liat technology can use a variety of magnetic-bead-based sample-processing strategies. Selecting this sample-preparation option depends on the target and the sample matrix containing the target. 

General nucleic acid extraction is based on the lysis and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate (GuSCN), and the nucleic acid– binding properties of silica particles in the presence of GuSCN.⁶

A small volume of blood or a cheek scraping can be used as starting material for genomic DNA extraction. For infectious agents, this procedure uses a mild detergent and proteinase K treatment of plasma samples to expose viral nucleic acid.

The released nucleic acids are subsequently bound to silica-coated magnetic beads in the presence of GuSCN and isopropanol. After magnetic capture, the beads are washed twice to remove unbound sample residue. Subsequently, the nucleic acids are eluted from the silica beads in a low-salt basic buffer, yielding template nucleic acid for downstream analysis. This method can be used for genotyping and detecting infectious agents in plasma. 

Another sample-processing method uses target-specific antibodies that are conjugated to magnetic beads to capture specific infectious agents from blood or environmental samples. This method controls the antibody binding conditions and temperatures in Liat tubes to facilitate efficient capture. Purified organisms can subsequently undergo nucleic acid extraction, followed by real-time PCR analysis for agent identification. 

Table I. Genotyping results for the detecton of HFE C282Y mutation (click to enlarge).

Rapid Closed-Tube PCR Detection. The Liat system’s fluid-handling and temperature-control capabilities can perform rapid PCR by moving a reaction mixture between different temperature zones. A two-temperature cycling process is used to perform closed-tube PCR (ct-PCR). Since fluid transfer replaces block temperature ramping, the two-temperature cycling protocols with a single liquid-transfer event per cycle have a speed advantage over conventional thermocyclers. For example, the duration of the device’s ct-PCR amplification reactions is one-fifth that of commercial Peltier-based thermocyclers. 

With the Liat system, the anneal or extension incubation times can be reduced significantly to accelerate PCR (see Figure 3). Target nucleic acids are detected when the exponential increase in fluorescence crosses a threshold during the PCR reaction at a cycle number. For example, the program with the 13-second-long extend or anneal incubation is capable of detecting 103 copies in 12.4 minutes. This performance compares favorably with air-cooled thermal cyclers, such as the SmartCycler by Cepheid.

The Liat analyzer can also detect as few as 10 copies of a nucleic acid. The device has a dynamic range of more than six logs using either TaqMan probes or molecular beacons. The cycle number values observed on the analyzer are comparable to those of commercial thermal cyclers. In addition, the Liat system can perform reverse transcriptase PCR for gene expression detection or RNA virus testing, as well as melting-curve analysis for sequence confirmation. 

Diagnostic Applications 

Multiplex Detection. E. coli encoding shiga-like toxins (Stx) can produce severe gastrointestinal symptoms. Tests for the presence of E. coli are part of the food industry’s hazard analysis and critical control points. The U.S. Food Safety Inspection Service mandates an estimated 1.45 million tests per year.⁷

For the Liat system, IQuum developed TaqMan minor groove binding (MGB) probes to detect the Stx1 and Stx2 genes in a single multiplex reaction. The dynamic range of the Stx1 probe and primers was validated with a dilution series. Results were obtained from a multiplex PCR reaction for detecting both Stx1 and Stx2, as well as DNA from two strains of E. coli O157, one containing only the Stx1 gene and the other containing both genes (see Figure 4).
 
Genotyping. DNA sequence variants contribute to human phenotypes and human diseases. To illustrate the Liat system’s capability for genotyping, IQuum has developed assays for the HFE C282Y locus, a single nucleotide polymorphism (SNP) associated with hemochromatosis. 

Genotyping assays performed on the Liat analyzer can use 10 µl of human blood or a cheek scrape as input samples. The reagents used for genomic DNA extraction include sample dilution buffer, proteinase K, and a chaotropic salt lysis buffer to release nucleic acids. Effective lysis can be achieved in 5 minutes at 50°C. 

Figure 6. Detection of anthrax spores on surfaces. Three swab samples from a 5 ¥ 5-cm solid surface containing 4000 anthrax spores in 1 cm2 were tested on the Liat system. The negative control comprised a swab sample collected from a clean surface without spores, and a positive control comprised boiled spores added directly to the tube (click to enlarge).

The released DNA was bound to magnetic silica beads in the presence of 40% isopropanol. The beads and bound DNA were washed, and the DNA was eluted from the surface of the beads by heating at 95°C for 2 minutes. A fixed volume of eluted DNA was mixed with primers and two TaqMan MGB probes, which can distinguish the two alleles of the HFE C282Y mutation. The temperature-cycling program for this assay consisted of a 95°C incubation for 3 seconds and a 60°C incubation for 15 seconds, with a 2.5-second liquid transfer time. The experiment yielded genotyping results of nine patient blood samples, four blood samples from wild-type donors, and a heterozygous DNA control (see Table I). This study demonstrated that the analyzer was able to determine accurately the genotype of all nine patient blood samples. 

Detection of Infectious Agents. Infectious-disease detection currently accounts for about 78% of the molecular diagnostics market.⁸ This major share of the market is dominated by tests for detecting HIV, HCV, and cytomegalovirus (CMV). Using HIV quantitation as a predictor of patient outcomes during antiretroviral therapy has created a new market segment.⁹

For the Liat system, IQuum has developed protocols that integrate sample preparation and sequence detection using plasma or serum spiked in viral particles that are grown in culture as an input sample. For these experiments, Epstein-Barr virus (EBV) was used as an infectious-agent model. Three examples of the data obtained have identical inputs of approximately103 copies of EBV spiked into 40 µl of human plasma (see Figure 5). 

The yield of viral DNA recovered from plasma by the Liat analyzer is comparable to the control extraction, suggesting that this protocol provides a quantitative measurement of viral titer in plasma. The reagents used were similar to those used in the genotyping assay. Eluted DNA was mixed with primers and TaqMan-MGB probes that are specific for the EBV genome, and with dry PCR beads containing Taq polymerase, dNTPs, buffer, and MgC2. Temperature cycling conditions were identical to those used for the genotyping assay. EBV was detected in all three samples and the positive control, while the negative control remained negative. 

Shuqi Chen, PhD, is president and chief executive officer, George Selecman is the director of engineering, and Bertrand Lemieux, PhD, is the principal investigator of new technology at IQuum Inc. (Allston, MA). They can be reached at shuqi@iquum.com,  george.selecman@iquum.com,  and bertrand@iquum.com,  respectively.

Anthrax Spore Detection. The Liat technology can also be used to detect biological warfare agents in environmental samples, such as anthrax. Bacillus anthracis, the causative agent of anthrax, is a formidable biological weapon because of the stability of its spores, the ease of culture and production, and its ability to be aerosolized. Inhalation anthrax is potentially fatal, with an estimated mortality rate of 75%, even with antibiotic therapy. 

Tests for detecting B. anthracis using PCR should implement combinations of probes that can distinguish bona fide anthrax from harmless soil bacteria and veterinary vaccine strains. For example, the Aum Shinrikyo cult once released a harmless vaccine strain of B. anthracis that is used to immunize livestock in Japan.¹⁰ This vaccine strain lacks one of the plasmids present in lethal strains of B. anthracis.

IQuum has developed sample-preparation methods that can detect B. anthracis spores in environmental samples (e.g., soil) that contain PCR inhibitors. Examples of assays show that the Liat system is capable of detecting spores spread on a dry plastic surface (see Figure 6).

In this example, 105 spores were spread on a 25 cm2 surface at the bottom of a petri dish, and the entire area was wiped with a moist swab. The collected spores were transferred to a collection buffer and enriched using antispore antibodies that were coupled to magnetic beads. These reagents can be stored in a Liat tube in dry form for several months before the assay.

The wash buffers were stored in three separate compartments: one compartment contained elution buffer, and the other two contained dry PCR reagents. The binding of spores to the magnetic beads was done at room temperature for 30 minutes, and three washes were used to remove PCR inhibitors. Spore DNA was eluted by heating at 98°C for 10 minutes, and the beads were magnetically immobilized during the transfer of the eluate to the dry PCR reagents. Temperature-cycling conditions were identical to those used for the genotyping assay. 

Conclusion 

The Liat technology platform can be adapted to a variety of assay formats and sample matrices, including whole blood, plasma, urine, and swab samples. Many established genotyping, infectious-disease, and bioweapon assays can also be adapted to the Liat platform. In addition, the technology is scalable, enabling the extension of the system from its current single-tube configuration to high-throughput multitube configurations without altering the instrument’s scientific basis. 

References

1. DP Chandler et al., “Continuous Spore Disruption Using Radially Focused, High-Frequency Ultrasound,” Analytical Chemistry 73 (2001): 3784–3789. 

2. MT Taylor et al., “Lysing Bacterial Spores by Sonication through a Flexible Interface in a Microfluidic System,” Analytical Chemistry 73 (2001): 492–496. 

3. C Bunch, “Bioterrorism Corner: Biodetection Devices,” Washington State Department of Health Elaborations 8, no. 9 (2003): 2–4. 

4. JA Higgins et al., “A Handheld Real-Time Thermal Cycler for Bacterial Pathogen Detection,” Biosensors and Bioelectronics (2003): 1115–1123. 

5. PA Emanuel et al., “Detection of Francisella Tularensis within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler,” Journal of Clinical Microbiology 41 (2003): 689–693. 

6. R Boom et al., “Rapid Purification of Hepatitis B Virus DNA from Serum,” Journal of Clinical Microbiology 29 (1991): 1804–1811. 
7. A Reder, “Taking IVD Test Technology Beyond Human Clinical Diagnostics,” IVD Technology 7, no. 5 (2001): 35–44.

8. S Halasey and R Park, “Making Sense of the IVD Global Market,” IVD Technology 8, no. 7 (2002): 29–36.

9. JW Mellors et al., “Quantitation of HIV-1 RNA in Plasma Predicts Outcome after Seroconversion,” Annals of Internal Medicine 122 (1995): 573–579. 

10. P Keim et al., “Molecular Investigation of the Aum Shinrikyo Anthrax Release in Kameido, Japan,” Journal of Clinical Microbiology 39 (2001): 4566–4567.   

Copyright ©2004 IVD Technology