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Jun 15, 2008 (Vol. 28, No. 12)

Overcoming Complications in Multiplexing

Push for Combination Tests Stem from the Need for Speed and Portability

  • As a new generation of biodetection assays come of age, we increasingly see implementation of multiple, simultaneous assays. In some cases, this multiplexity is modest and pragmatic—just a few assays at a time for very specific purposes. In others, ambitious assays identify a hundred pathogens simultaneously. It’s clear that the field is moving in the direction of multiplex assays, but why?

    Whether you are using an antibody-based technology, a real-time PCR assay, or something else altogether, combining tests for a number of different pathogens is pretty much guaranteed to cause problems with cross reactions and contamination. To get the best sensitivity and specificity, single-analyte assays make the most sense. The push for multiplexity, then, comes from needs that cannot be met with traditional single-analyte detection, such as speed, portability, and the ability to characterize a community of organisms.

    The specific requirements of medical, environmental, food safety, and biodefense applications invite a diversity of methods and targets for detection. The Knowledge Foundation’s “Biodetection Technologies 2008” conference to be held later this month invited a number of scientists to present multiplexed detection technologies. From this diverse menu of offerings, certain commonalities can be found. Notably, each displays singular ingenuity in overcoming the complications of multiplex detection.

  • Using Beads

    The key challenges of pathogen detection in environmental samples are purification and concentration. The sample starts out with a large volume and a high level of contamination from particulate materials as well as nontarget organisms. The sample-preparation stage needs to be very robust. At the Pacific Northwest National Laboratory, Cindy Bruckner-Lea, Ph.D., uses the BEADS (biodetection enabling analyte delivery system) platform to detect biological contaminants in water with remarkable sensitivity.

    Microbeads concentrate organisms into a smaller volume of solvent. At the next stage, cells are lysed to release DNA, and then these cell contents are ushered into a multiplex detection system such as a microarray or a bead-based array. “If you don’t clean up your sample,” says Dr. Bruckner-Lea, “you’re not going to efficiently amplify and detect. You need to remove inhibitors that can impact the detection and reliability and give false positives or false negatives.”

    In one application of BEADS, Dr. Bruckner-Lea and her colleagues attempted to detect a wide range of pathogens from a water sample. They designed the chemistry on the microbeads to allow electrostatic capture of the organisms. The group of target organisms included E. coli, Salmonella, crytosporidium, and viral simulants.

    In a 1 L water sample, they recovered 95% of the contaminants or approximately 25 to 100 organisms per liter. The system is also capable of trace detection from a small sample, according to Dr. Bruckner-Lea. “We are able to reproducibly detect 10 cells in one milliliter of sample by doing purification on magnetic beads, then lysis and PCR to amplify the DNA, and then microarray detection,” she reports.

    One of the particular challenges of this type of study is simultaneously detecting a wide range of organisms. A promising approach for multiplexed detection is to use one universal PCR reaction rather than conducting many amplification reactions using sets of specific primers for each organism of interest, explains Dr. Bruckner-Lea. DNA target sequences on a microarray are then used to detect the target organisms while excluding organisms that are not of interest.

    Fluid force discrimination (FFD) is a newer technology that can improve bead-based purification and separation. Seahawk Biosystems (a Tetracore subsidiary; www.tetracore.com) is using FFD to develop a handheld biothreat detector. The principle is that paramagnetic beads capture antibodies, which bind to antigens in the sample. Those are then captured onto a microarray by another capture antibody bound to the surface. Force (FFD) exerted by a laminar flow of liquid across the surface of the array washes away nonspecifically bound targets.

    The technology is still in development. Tetracore has an experimental benchtop unit, and a prototype handheld unit is in the design phase. “We expect to have a working prototype within about six months,” reports Gary Long, Ph.D., vp and senior scientist.

    Robert Tebbs, Ph.D., has been working on food pathogen detection assays for Applied Biosystems (www.appliedbiosystems.com) based on RT-PCR. The assay Dr. Tebbs is developing uses a fluorescent probe that binds to the template between the two primers. The probe contains a quencher so that it does not fluoresce until it is cleaved during PCR. This gives a quantitative readout on the reaction.

  • In Real Time

    “It gets complicated when you’re doing multiplexing,” comments Dr. Tebbs. “If it’s only one reaction, it’s only a couple of primers. When you start adding other reactions, you’ve got more possibility of interference. The primers can interact. The probes can interact. The effort is trying to mitigate those interaction effects and balance out the different reactions.”

    In the food pathogen detection business, there are specific regulatory requirements for which pathogens should be detected and which should not. One common problem is the detection of “nearest neighbor” organisms that are not actually target organisms themselves. For example, Vibrio mimicus looks a lot like cholera (hence, the name mimicus). There is zero tolerance for cholera in the food supply, but the presence of V. mimicus could cause false positives for cholera.

    The payoff for all this trouble is an assay of surpassing utility. “One of the big advantages of doing these multiplexes is that you get to detect and assay many different organisms at once. It takes less time to do your work.

    “Using classical methods, if you look at each one separately,” Dr. Tebbs points out, “it takes two or three days to determine if an organism is present.” In the food business this is often two or three days too long, and many people may become ill before the pathogen is found.

    Another interesting application of PCR to multiplex biodetection is the use of nested multiplex PCR. In this strategy, PCR is carried out in two stages. The first stage uses universal amplification, like the BEADS assay previously mentioned. The PCR products are then diluted and moved into an array of reaction chambers for a second stage of PCR using specific primers targeted to a much shorter sequence.

    Thus, each final reaction product has been created using four primers instead of the usual two, with the second set “nested” inside the first set. This circumvents the problem of washout by the dominant species, so that minority species can be detected.

    One big problem with nested multiplex PCR, however, is contamination of the amplicon. Idaho Technology (www.idahotech.com) has a solution to this problem. The reaction is carried out in a completely sealed chemical circuit board. It actually resembles a glorified piece of bubble wrap, but in this case each bubble is a chamber that contains a stage of the assay, beginning with sample prep all the way through a 120-plex second-stage PCR.

    The instrument that shepherds the samples through these plastic chambers is called the FilmArray™Instrument. The technology came from government field testing but it’s being developed at Idaho for medical testing. Multiplex pathogen detection will, for the first time, allow physicians to diagnose complex, multiple infections right at the bedside (in about 55 minutes), according to Idaho. These types of infections are much more prevalent than once thought, and if they can be detected in the early stages, it may be possible to prevent complications.

    The FilmArray is able to detect pathogens at concentrations ranging from 10,000 cfu/mL down to 5 cfu/mL in the same sample from up to 120 samples simultaneously, reports the company. The system includes a software analysis package developed in-house at Idaho Technology.

  • Putting It All Together

    Some biodetection applications demand even more than sensitivity, specificity, rapidity, or an elegant technology. There is great interest in the development of sensors that will sample indoor air passively and then run toxin or pathogen detection assays when triggered by positive results of the air sampling tests. ICx Technologies (www.icxt.com) is providing all this and more in the RapidPlex™ system.

    RapidPlex is being designed to operate as a triggered sensor for detecting bacteria, viruses, and toxins in an aerosol sample in under 15 minutes, reports Michael Meyer, senior director of laboratories at ICx Technologies’ biosystems division. “Most of our emphasis has been on reducing assay time to get it down to 10 or 15 minutes. We’ve designed the RapidPlex to get assays to run quickly without sacrificing sensitivity and specificity of the instrument.”

    The assay will detect 15 to 20 threat targets at a time by running two assays in parallel. One is an antibody-based assay, and the second is a nucleic acid-based assay for detection of DNA and RNA targets. The antibody assay is primarily for toxins but also provides detection of bacterial and viral threat targets. It uses a microbead fluorescence detection system. The nucleic acid assay is designed to detect bacteria and viruses and it operates under a rapid multiplex PCR assay with the same fluorescent readout as the antibody assay.

    To prevent false positives, RapidPlex uses three PCR reactions in parallel with three different 20- to 30-plex sets of primers. “To get that high confidence and specificity,” explains Meyer, “we go after multiple independent sequences for each threat target. Without independent biomarkers, it’s easier to be susceptible to a false positive. The really key thing about our whole system is the integration of the whole process from sample prep to detection in a single end-to-end process that takes less than 15 minutes.

    It’s clear that multiplexed pathogen detection is not as simple as squirting the sample into 96 wells and popping it into a plate reader. Each assay needs to be customized not only for its industry application but also to the specific panel of pathogens that are targeted by the assay.

    Needs will also vary based on whether the system is intended to be portable and handheld or to occupy a place of honor in a corner of a laboratory. Elegant solutions, however, are being put forward that will make it blessedly unnecessary to culture samples for days and days just to find out whether the water you just drank, the food you just ate, or the air you just breathed really was safe.

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