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RESEARCH PROFILE A PCR-based anthrax detector After the September 11, 2001, attacks on the World Trade Center and the Pentagon, an unknown bioterrorist sent letters laced with Bacillus anthracis (anthrax) spores through the U.S. mail. Five people died and 13 others fell ill from exposure to the pathogen. But exposure to the spores might have been drastically reduced if an “anthrax detector”, similar to a smoke detector, had been installed in the victims’ buildings. An anthrax/biothreat detector is exactly what Bill Colston at Lawrence Livermore National Laboratory and colleagues at Microfluidic Systems, Inc., and Global FIA, Inc., are developing. An integral piece of the detection system is the reusable and continuous flow-through (F-T) PCR system that they describe in an Analytical Chemistry paper published online May 30 (DOI: 10.1021/ac034062u). The real-time, autonomous PCR system can detect fewer than three copies of a B. anthracis genome within minutes, whereas conventional PCR can take up to 2 h. Although a few other groups have reported rapid F-T PCR systems, co-author Philip Belgrader at Microfluidic Systems explains that those systems have not been optimized for the low pathogen concentrations present in real environmental samples. Setting up an autonomous PCRbased detector requires a different way of thinking, says Belgrader. Current PCR-based detection systems used in the field have disposable, one-time-use components, but he explains that this is not practical for an inexpensive continuous monitoring system. Instead, Belgrader envisions an instrument that performs thousands of reactions unattended; a cartridge containing reagents to replenish the F-T PCR system would be replaced only weekly or monthly. Indeed, Colston says that this is the unique aspect of the F-T PCR system. “The remarkable part about this is that you can get rid of all billion copies [produced by PCR amplification] every
time you make a Fluid in Light source measurement,” he Filter says, which eliminates carry-over contamination. Silicon heater B. anthracis gePhoto detectors Light nomic DNA and a Filters PCR “master mix” containing DNA Cool air polymerase, a comintake mercial fluorescent probe, primers, and Flow-through other reagents were tubing kept in separate vials. The master Turning mirrors Fluid mix and B. anthracis out DNA were pumped toward the F-T PCR Schematic of the F-T PCR system. reaction chamber, is comparable to the efficiency obtained and the reaction mix zone was positioned at an optical window in the ther- with conventional PCR. Blanks were run immediately after three of the B. mal chamber, allowing fluorescence deanthracis samples, and no signal was detection of a positive signal. Once the tected with any of them, suggesting that PCR cycles finished, the reagents and all the PCR products were eliminated products were pumped to a waste comby the washing procedure. partment. Belgrader says that instead of The detector is flexible and can be sending the amplicons to waste, they easily adapted to monitor different could be routed to other instruments, pathogens as the need arises. In fact, such as a mass spectrometer, flow cyBelgrader says that the detector could tometer, or capillary electrophoresis debe modified to sense the virus that causvice, for further analysis. After each es severe acute respiratory syndrome, PCR run, the chamber and tubing were known as SARS, or any other virus or flushed with a sodium hypochlorite sobacterium. “Once the sequence inforlution and water. mation is available about the microbe of The researchers prepared diluted samples of B. anthracis DNA containing interest, the PCR primers and probes just have to be made, and you can put a calculated average of 0.3–30,000 that into the system,” he says. copies of the genome. The PCR cycle Both Microfluidic Systems and Globnumber at which each particular sample al FIA plan to market the device and its reached a set fluorescence threshold components. Within the next year or value was reproducible to within 0.5 year and a half, Colston thinks the decycles, except for those containing
