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Jun 1, 2012 (Vol. 32, No. 11)

Real-Time Sample Prep on the Horizon

  • Hospital Labs

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    FilmArray RP is Idaho Technology’s first clinical diagnostic test designed to run on the company’s Film Array system.

    Idaho Technology’s solution is the single-sample FilmArray. Its first commercialized product, Respiratory Panel, has been approved by the FDA to test for viral pathogens from a nasal pharyngeal swab. The system is reportedly simple: all the reagents for sample processing and amplification are contained in a single-use, disposable “pouch”.

    The first step is to hydrate the freeze-dried reagents in the pouch by injection of buffer under vacuum. The patient sample is then washed off the swab and resuspended in lysis buffer. Approximately 250 µL of that sample is then loaded at the other end of the pouch under vacuum.

    The barcoded FilmArray pouch is processed by placing it into the FilmArray benchtop instrument. Any microbial material in the sample is then subjected to a bead-beating mechanical lysis using ceramic beads that can effectively bust open spores or other difficult to lyse pathogens. Nucleic acids are scavenged from the lysis mix by magnetic beads for subsequent processing.

    “Respiratory pathogens, in particular viruses, are notoriously difficult to culture,” said Stephanie Thatcher, director of systems integration. “Given the effectiveness of the sample-prep step and sensitivity of the rtPCR and detection PCR reactions in the FilmArray, a culture step is not necessary. We are able to process the material routinely collected in the hospital when patients come in with a respiratory infection. Processed in the hospital lab, the pathogens are identified in less than 60 minutes so that the proper course of therapy can be initiated.”

    The FilmArray system is easy to use, explained Thatcher. First, the FilmArray extracts and purifies all nucleic acids from the sample. Next, the FilmArray performs a nested multiplex PCR in a single large volume multiplexed reaction. Then individual singleplex second-stage PCR reactions detect the product from the first-stage PCR in each chamber of the array.

    Using endpoint melting curve analysis with double-stranded binding dyes, which pathogens are present in the patient sample can be determined. The system is optimized for low-level detection of pathogens from clinical samples. A control organism is provided in the sample buffer, which serves to provide confidence that the sample signal, whether positive or negative, is valid.

  • Enzyme-Based Technology

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    ZyGEM’s family of products, derived from its collection of microorganisms from extreme environments, enables nucleic acid extraction from diverse sample types.

    ZyGEM has taken a different approach to nucleic acid sample prep. Rather than using solid-phase extraction (SPE), ZyGEM offers an enzyme-based technology to strip the sample down to nucleic acid. While the technology is amenable to use on liquid handlers in the research lab, James Landers, Ph.D., was brought into ZyGEM as CSO to adapt the technology to microfluidics.

    As professor of chemistry and mechanical engineering and assistant professor of pathology at the University of Virgina, Dr. Landers has abandoned SPE for ZyGEM’s enzymatic approach because of its simplicity and elegance, he said. With the adoption of microfluidics, the extraction process has been shrunk to sub-microliter volumes, which saves on enzyme and other reagent costs. In addition, the reaction kinetics are sped up; the entire process has been reduced to 35 minutes.

    The reaction is controlled by temperature. The EA1 proteinase, a highly active metallo-endo-proteinase isolated from a thermophile in a volcanic vent, is inactive at room temperature. Peak activity occurs at 75°C where the enzyme works as a protease. Raising the reaction temperature to 94°C irreversibly kills enzyme activity.

    Regarding the macro-to-micro interface, ZyGEM deals with real world samples; a buccal swab or blood droplet is collected in 100 µL volume. Of that starting material, only 1 µL is needed for downstream processing; the rest of the material can be stored for reanalysis, if necessary. Front-end concentration of the sample has not been determined to be necessary. From a buccal swab, ZyGEM routinely liberates purified DNA in the range of 6–12 ng/µL, which is in excess of the 4 ng/µL that is needed for PCR, Dr. Landers explained.

    The ZyGEM approach has a narrow scope; the sample-prep method provides material that is “PCR-ready”, but is not limited to microfluidic devices. Kits are also available for use in microtiter plates on liquid handlers, where the enzyme approach is reportedly a dramatic improvement over SPE protocols as the number of sample-handling steps is reduced, avoiding loss of sample from transfer steps.

    “It is important to remember that the ZyGEM process doesn’t help with finding a needle in a haystack. DNA that is liberated from the reaction comes from all sources in that sample. You must depend on the use of proper PCR primers to amplify up rare sequences in the mix or resort to SPE-based selection of target from the mix,” explained Dr. Landers.

    “But with its simplicity and elegance, PoC applications are not far off. For example, where the challenge is to differentiate between viral and bacterial Encephalitis meningitis when the patient presents with similar symptoms, given the rapid kinetics of the sample prep of Cerebral Spinal Fluid, and subsequent PCR, you could determine the right course of treatment within the first two hours of hospitalization,” Dr. Landers claimed.

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