by Catherine Shaffer
When a patient is waiting to be diagnosed with a life threatening disease, every minute counts. Conventional tests for viral infections, such as Herpes Simplex virus and enterovirus, require a three- or four-day delay while the sample is being cultured for the virus. During this time, the patient's clinical condition often deteriorates, forcing the doctor to try risky empirical therapy while waiting for results. A number of biotechnology companies have introduced new tools and technologies for pathogen detection for the clinical and research markets.
TrimGen has developed a new type of diagnostic for viruses based on real-time PCR. Before beginning work on specific viruses, TrimGen researchers spent an entire year familiarizing themselves with every real-time PCR system on the market, gaining an in-depth understanding of the abilities and limitations of each system. Focusing in on just a couple of systems, the ABI 7500, ABI 7900, and Roche LightCycler, TrimGen scientists optimized the PCR ingredients and conditions to create a proprietary technology they call enhanced real-time PCR, or eQ-PCR™. Diagnostics based on eQ-PCR™ are capable of detecting as few as one or two copies of virus in a sample, with a run time of an hour and a half.
Says Howard Doong, PhD: "The biggest advantage is that it's very sensitive. If there are only one or two copies of viruses in your spinal fluid, most platforms cannot detect it. You have to wait until the virus grows. When you detect a large amount, the patient is very sick already ... our sensitivity is a major plus for our kits."
TrimGen products include tests for Herpes Simplex virus (HSV), enterovirus (ENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and hepatitis B virus (HBV). Tests are targeted at both research and clinical markets. Currently, only the combo kit for HSV and ENV detection has been validated for the clinical market, but work is underway on the other tests. The TrimGen pipeline includes new tests developed in collaboration with Canadian company Spartan Biosciences for a real-time PCR-based diagnostic test that runs in under thirty minutes, for a true bedside clinical application.
The combination of a real-time PCR-based pathogen detection test with a microarray format assay and detection capabilities opens up the possibility of the simultaneous detection of multiple pathogens. The OpenArray™ is a custom product from Biotrove Inc. that can be designed to detect a particular pathogen strain or species in panel format. It has been used in a variety of applications. Michigan State University's Environmental Engineering and Microbial Ecology departments are using OpenArray™ to study pathogens in the water supply. Array-based pathogen detection systems can also be used to study clonality, as in a recent collaboration between BioTrove and the Centers for Disease Control and Prevention (CDC) to identify bacterial strains of E. coli, salmonella, botulism, and other organisms in tainted food.
OpenArray technology is optimized for panelized assays grouped according to the sample tested, and for fast results. Says Kevin Munnelly, vice president and general manager for Biotrove, "The system allows for rapid turn-around times, and provides 'surge capacity' in the case of outbreaks." Currently, BioTrove's products are exclusively custom applications based on genetic sequences provided by users. Future plans for Biotrove include the development of ready-to-use panels of assays for standard groups of pathogens.
The ability to detect multiple pathogens can be as important in clinical applications as it is in environmental or food safety studies. Idaho Technology's Film Array system is targeted toward a clinical diagnostics market, where Todd Ritter, chief corporate development officer, estimates that 35 percent of clinical infection samples will contain multiple pathogen strains. During ordinary pathogen detection tests, the dominant strain tends to swamp the test, obscuring evidence of other pathogen strains. The ability to test for many pathogens at one time will allow doctors to choose more appropriate treatments, earlier.
The Film Array is an automated sample preparation device that can screen one sample for up to 120 different genetic targets. The completely automated assay takes 52 to 55 minutes to run, with highly multiplexed real-time PCR detection. The technology is based in a novel "chemical circuit board" technology, which is essentially a series of plastic pouches. The concept was originally developed for remote field testing applications, and reinvented by Idaho for pathogen detection. The entire assay is contained in something resembling a high-tech Ziploc® bag with divided pockets for solutions. The sample gets "squished" through two stages of PCR, and then into a tiny microarray plate contained right inside the pouch for detection. A dilution step after the first stage PCR prevents the dominant bacterial strain from washing out minor strains, and the completely enclosed system avoids problems with contamination of the lab area by the amplicon. Says Todd Ritter: "Our system has a lot of linearity—a great span for picking up minority species. Usually the dominant species overwhelms everything and consumes all the chemicals inside ... we're able to dilute out dominant species, and keep less dominant species in the reaction."
All of the Idaho Technology instruments and components have been developed in-house, including the software analysis suite. However, if you want to put together your own real-time PCR/microarray pathogen detection system, you don't have to hire an army of software engineers to design your data analysis package. Premier Biosoft offers a program called Allele ID for designing qPCR diagnostic assays and microarrays for rapid detection of pathogens. For example, AlleleID allows researchers to detect a species or taxa from a complex mix of flora. It can distinguish a particular strain of E. Coli, an enteric bacteria, from contaminated water even when the water contains strains that are as high as 99% homologous to the strain of interest. Premier Biosoft developed Allele ID in response to customer demand. Says Kay Brown, President and CEO, "Our users know what they want to do, but they don't know what is possible, so we did one better. We gave them the design capability for not only species specific detection, but added taxa identification functionality, cross-species design capabilities for phylogenic studies and a new capability called Minimal Set." Minimal Set designs the fewest number of oligos required for detection, lowering assay costs.
There is no question that new assays based on real-time PCR are quickly replacing older-generation pathogen detection methods, with faster, more powerful, multiplex pathogen detection. Combined with robust bioinformatics solutions, these systems have impressive potential for clinical and research applications.
