IQuum (www.iquum.com) developed the Liat Analyzer for rapid, fully automated biological sample testing in a closed system. In March, the company received a Phase II contract from the U.S. Department of Homeland Security Advanced Research Project Agency (HSARPA) for the development of its bioaerosol monitoring systemthe Liat Bioagent Autonomous Networked Detector (BAND)for monitoring outdoor urban areas for bacteria, viruses, and toxins. The company also received a Phase I contract from HSARPA to develop a Food Assay Sensor System for biodefense applications.
A drawback of conventional amplification-based nucleic acid testing is the potential for sample contamination when the sample tubes are opened during processing, Dr. Chen says. The company’s lab-in-a-tube platform is fully self-contained and disposable. The processing tube is only opened at the beginning to load the sample.
According to Dr. Chen, an additional challenge is assay automation. The Liat system utilizes the lab-in-a-tube technology to allow for automation of the entire assay, including sample preparation, target enrichment, inhibitor removal, nucleic acid extraction, PCR amplification, and real-time detection. The system processes the sample through a series of segmented sections in the Liat Tube, with each segment containing the necessary reagent for an assay step.
Peristaltic pumps move the reaction mixture from one segment of the tube to the next, breaking and reclamping the seals that divide the segments. For PCR, one segment is set at the denaturing temperature and another is set at the annealing-extension temperature, and the reaction mixture is alternatively cycled between these two segments.
"Since only the reaction mixture is flow-cycled between the temperatures, the Liat Analyzer can rapidly perform nucleic acid tests," Dr. Chen adds. The system can complete 30 cycles on a 50-µL reaction volume in seven minutes. This allows an entire genetic test from whole blood to be completed in about 30 minutes, and detection of an infectious agent in plasma takes about 60 minutes.
Dr. Chen’s vision is for nonspecialized laboratory personnel or individuals in a field setting to be able to perform these tests with minimal training. He expects the company to release its first product for research use only by the end of this year and to receive approval for a clinical application in 2007.
IQuum has launched a clinical trial for detection of the infectious agent that causes leishmaniasis and is developing assays for Chlamydia sepsis, cytomegalovirus, and Epstein Barr virus, as well as for biodefense targets, such as anthrax and E. coli. SNP detection for genotyping applications is also in development.
IQuum’s BAND system for environmental monitoring uses the same lab-in-a-tube assay technology as the Liat Analyzer but requires different instrumentation to enable collection of large-volume air samples.
Fractal Systems (www.fractalsystems.com) is developing a PCR-free molecular detection system for biodefense diagnostic applications. The system utilizes an ultrathin conductive polymer film that when exposed to an oxidizing or reducing agent in the environment, changes its conductivity. Measurement of the change in conductivity correlates with the amount of material present in the environment. In this way, the electrochemical response corresponds to the concentration of the analyte, which can be quantified.
The nanocomposites comprised of the conductive polymers are reuseable, as the change in conductivity is reversible. In the presence of the target analyte the redox reaction oxidizes the polymer backbone. Removal of the analyte causes the coated electrode to reverse to its original state.
DNA acts as an oxidizing agent. By exploiting this characteristic to embed a single-stranded oligonucleotide into the polymer backbone, the subsequent recognition and hybridization of a specific, complementary DNA segment will increase the volume of the polymer, displacing the conjugated backbones and altering its conductivity. In addition, hybridization will diminish the interaction between the single-stranded oligo and the polymer backbone, without actually separating the oligo from the polymer. This, too, leads to a change in conductivity.
The company’s first project, funded by an SBIR grant from the Department of Defense’s Threat Reduction Agency, focused on developing highly specific viral sensors. Its second project, funded by the U.S. Army, uses aptamers to detect toxins.
BioHelix (www.biohelix.com) is developing a portable detection system for use in the field to identify biothreat organisms, under a cooperative agreement with the Department of Homeland Security. The company’s isothermal, helicase-dependent amplification (HDA) technology for amplifying and analyzing DNA maintains a constant reaction temperature and eliminates the need for thermocycling equipment. BioHelix plans to commercialize HDA (linked to a downstream detector) as a rapid screening platform for use in the point-of-care molecular diagnostics market. The company’s first assays in development target infectious diseases such as herpes. HDA relies on a DNA helicase that unwinds the two strands of a DNA helix, creating single-stranded templates for primer hybridization and subsequent extension by a DNA polymerase.
Huimin Kong, Ph.D., president and CEO, cites HDA’s low cost and ease of use as two important advantages of the technology compared to traditional PCR approaches. Also, HDA can be used to amplify either DNA or RNA and %is more robust than strand-displacement methods,% says Dr. Kong. In late April, the company entered into an exclusive licensing agreement with Harvard University to commercialize a rapid, primase-based whole-genome amplification technology.
GE Healthcare (www.gehealthcare.com) introduced the illustra Hot Start Master Mix to amplify target DNA sequences that the company says provides an increased level of PCR specificity compared to conventional techniques. The Hot Start Master Mix achieves hot start activation by sequestering primer until the template DNA is denatured. It then releases the PCR primers, and the cycle of amplificationprimer:template annealing, extension, and denaturationthen proceeds using traditional thermal cycling protocols.
Unlike hot start PCR systems that rely on chemically modified or antibody-based methods, HotStart Master Mix does not interfere with the thermostable polymerase, resulting in no loss of enzyme efficiency. By effectively inhibiting primer:dimer formation without compromising enzyme function, Hot Start Master Mix maximizes target amplification specificity and efficiency for better overall PCR results.