Threat Agent Detection Impetus For Handheld Nucleic Acid Analyzer Development

Successful performance of PCR obviously depends on the ability of the thermal cycling platform to maintain accurate gradations of temperature in the manifold or block containing the reaction tubes. The heating and cooling apparatus necessary to provide these temperature changes places constraints on the size and weight of the thermal cycler. ''First-generation'' thermal cyclers, such as the Perkin Elmer 480 model instrument, while reasonably light in weight and small in footprint, were not designed with field use in mind. However, during operations in the Persian Gulf in 1990-1991, field laboratories for detection of threat agents were deployed and operated by both the U.S. Army and the U.S. Navy. Polymerase chain reaction on conventional thermal cyclers, with agarose gel electrophoresis used to confirm amplification products, was used on a regular basis by these laboratories with a high degree of success, despite the trying conditions associated with performing molecular assays in a desert environment. By the mid-1990s the U.S. Army adopted PCR-based assays as a critical component of the field-deployable 520th Theatre Area Medical Laboratory (TAML), which was designed to provide rapid threat agent detection in battlefield conditions.

Just as Western defense and public health personnel were coming to grips with the disclosure of the size and scope of the Iraqi bioweapons program, it was revealed that the Soviet Union had a clandestinely operated sizeable, well-funded program of its own. Among the pathogens selected for weaponization were smallpox, plague, tularemia, and anthrax. The likelihood that U.S. and NATO troops may encounter such agents heightened the importance of on-site diagnostic techniques and, consequently, research in this area was prioritized by the Department of Defense, primarily under the auspices of the Defense Advanced Research Projects Agency (DARPA).

Research on portable nucleic acid-based detection devices was considerably aided by the discovery in 1991 that the 5'-3' exonuclease activity of Taq polymerase, the enzyme mediating PCR, could be harnessed to provide real-time monitoring of the reaction via the hybridization of a fluorogenic oligonucleotide probe.[1] In 1995 researchers at Genentech, Inc., and Applied Biosystems reported the first use of a commercial probe expressly designed for real-time PCR, the ''TaqMan®'' probe.[2] With this assay system, fluorescence generated by the probe could be detected by the appropriate instrumentation and the user provided with a graphical depiction of the accumulation of the PCR amplicons over the length of the reaction. The TaqMan assay was notable for being conducted in the same reaction tube from start to finish; there was no need for postreaction manipulations. Probe design and synthesis was relatively straightforward and a TaqMan-based protocol for the detection of the food-borne bacterium Listeria monocytogenes was published in 1995.[3] These results gave impetus to the design and evaluation of TaqMan probes against a variety of bacterial and viral threat agents, such as orthopoxviruses.[4]

Food Bacteria Analyzer

Fig. 1 The Smiths Detection BioSeeq™ instrument weighs 6.5 lb (2.9 kg) and measures 12 x 8 x 2 in. (30.4 x 20.3 x 5.0 cm) and can run for up to 1 hr on the power provided by eight alkaline ''C'' batteries. There are six independently programmable reaction modules and data are displayed to the user via a small inset screen; the instrument can also be linked to a laptop computer for more involved assay programming and data display. (Photo courtesy of P. Emanuel, U.S. Army, Edgewood, MD.) (View this art in color at www.dekker.com.)

Fig. 1 The Smiths Detection BioSeeq™ instrument weighs 6.5 lb (2.9 kg) and measures 12 x 8 x 2 in. (30.4 x 20.3 x 5.0 cm) and can run for up to 1 hr on the power provided by eight alkaline ''C'' batteries. There are six independently programmable reaction modules and data are displayed to the user via a small inset screen; the instrument can also be linked to a laptop computer for more involved assay programming and data display. (Photo courtesy of P. Emanuel, U.S. Army, Edgewood, MD.) (View this art in color at www.dekker.com.)

The implementation of fluorogenic probes freed the user from the need to conduct electrophoresis to determine whether or not a PCR assay was successful, and had obvious implications for reducing dependence on a laboratory infrastructure. However, the need for power sources and optics capable of monitoring the fluorescence produced by a real-time PCR assay was a major impediment to the design of portable thermal cyclers. For example, the first commercial platform dedicated to real-time PCR, the Perkin Elmer/Applied Biosystems 7700 model Prism® instrument, appeared in 1996-1997, but was too heavy (140 kg) for field use. U.S. Department of Defense-sponsored research sought to address this issue, and in 1997 researchers at the Lawrence Livermore National Laboratory (Livermore, CA) succeeded in fabricating a battery-powered, suitcase-sized real-time thermal cycler capable of using TaqMan probes: the MATCI, or Miniature Analytical Thermal Cycler Instrument.1-5-1 Central to the operation of the device was its use of silicon ''thermalcycler'' units to mediate the heating and cooling of the associated 25-p.l volume plastic reaction tube; this allowed for a one cycle per minute cycling speed. The MATCI provided sufficient temperature fidelity to allow for differentiation between sequences, with a high degree of homogeneity (such as poxvirus hemagglutinin gene), on the basis of a single nucleotide substitution.1-6-1

The MATCI could only accommodate one reaction tube at a time; the next iteration of the technology, the Advanced Nucleic Acid Analyzer (ANAA), which appeared in 1998, could perform 10 reactions simultaneously, and detection of as few as 500 cells of Erwinia was accomplished after only 7 min of thermal cycling.[7] The ANAA was a suitcase-sized instrument and researchers at the Lawrence Livermore National Laboratory were interested in advancing the miniaturization of the system to a handheld format; this led to the fabrication of the Handheld Advanced Nucleic Acid Analyzer (HANAA), arguably the world's first handheld, real-time thermal cycler instrument. The HANAA could perform four reactions simultaneously, with the use of two light emitting diodes (at 490 and 525 nm, with a combined emission of over 1 mW of power) allowing for monitoring of two dyes (e.g., FAM in one channel and JOE in the other).[8] The HANAA weighed less than 1 kg, and several sequential reactions could be performed, powered off a 12 V/3.5 A battery pack.[9] Reactions of up to 25 ml in volume were conducted in customized plastic tubes, and an improved silicon/platinum thermalcycler unit (22 x 6.6 x 2 mm) permitted ''traditional'' cycling parameters (e.g., 95°C for 0 sec, 55°C for 10 sec, and 72°C for 15 sec for 40 cycles) to be conducted in less than 20 min; strongly positive samples could generate positive results by 13 min of assay time.[10]

Prototype HANAAs were distributed to a variety of testers and feedback was favorable enough to result in licensing of the technology to Smiths Detection, Edge-wood, MD; the result was production of the BioSeeq™ handheld real-time thermal cycler, available for purchase in 2003 (Figs. 1 and 2). The BioSeeq can accommodate up to six independently programmable reactions, each with a volume of 25 mL. In their evaluation of the BioSeeq during an 8-month beta testing phase, Emanuel et al.[11]

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Fig. 2 Demonstration of the use of the BioSeeq™ instrument in the field. (Photo courtesy of P. Emanuel, U.S. Army, Edgewood, MD.) (View this art in color at www.dekker.com.)
Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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