The events of Fall 2001 have underscored the need for rapid reliable identification of potential biological warfare agents. Any means of genetic identification requires that the organism's DNA be obtained and liberated from cellular compounds that may inhibit analytical processes such as the Polymerase Chain Reaction (PCR). Current methods commonly employed for sample preparation remain labor intensive and costly or rely on wet chemistry which requires the transportation of solvents, generates unwanted waste and, most importantly, introduces the risk of losing organisms present in low numbers. Furthermore, different microorganisms require different protocols in order to disrupt the outer covering to release DNA. For certain microorganisms, these protocols may include repetitive freezing/thawing cycles, grinding, pulverizing, or crushing which are certainly not compatible with field deployed troops.
An Innovative Solution: the PlasmaGen APR-510-S
Atmospheric Glow Technologies has developed a device for rapid, one-step, reagent-free preparation of BW samples for PCR in seconds using the patented One Atmosphere Uniform Glow Discharge Plasma (OAUGDP®). The PlasmaGen APR-510-S provides for the release of PCR-ready DNA from all microorganisms in 90 seconds utilizing OAUGDP®. Recent independent results have verified the release of amplifiable DNA from a mixed population of microorganisms in this timeframe with no additional steps. The PlasmaGen APR-510-S simplifies sample preparation by eliminating both the need for boiling, freezing, or enzymatically digesting cell walls as well as any chemical-based precipitation of proteins that inhibit PCR.
Simple Operation
The PlasmaGen APR-510-S requires only ambient air and 120 VAC power to liberate the DNA from microorganisms collected manually or with separate sampling systems. Matrices that can be sampled include air, water, organic substances, and any surface using swabs. Following a 90 second PlasmaGen APR-510-S exposure, the nucleic acid on the filter is ready for amplification with no additional preparatory steps. To date, the vast majority of samples treated in the PlasmaGen APR-510-S have used this protocol.
The PlasmaGen APR-510-S uses a parallel set of dielectrically-coated electrodes to create a uniform plasma which disrupts the integrity of the outer barrier of microorganisms in order to release DNA for further analysis.
Comparison with Standard Protocols
In validating the performance of the PlasmaGen APR-510-S, a comparison was made to more customary DNA preparation methods. As demonstrated in Figure 1, samples of Staphylococcus aureus (rt) and Pseudomonas aeruginosa as representative Gram positive and negative bacteria, respectively, were treated according to a standard phenol/chloroform protocol (top half of gel) or for 30 seconds in the PlasmaGen APR-510-S (bottom half of gel). Equivalent or superior amplification product was generated by the PlasmaGen APR-510-S in a one-step process without the need for reagents or separate precipitation steps.
Figure 1. Comparison of APR-510-S (bottom) withstandard protocol (top). Lanes 1-5 represent five
replicates.
Broad Spectrum Applicability
In order to be accepted as a tool for the preparation of unknown biological samples for identification, any method must verify that it is applicable to all microorganisms. To that end, AGT has performed DNA preparation assays with the PlasmaGen APR-510-S for a wide range of microorganisms with positive results for all tested to date. Figure 2 provides details on this effort. The PlasmaGen APR-510-S has proven to be a means of liberating DNA from vegetative bacteria as well as bacterial endospores, yeast, fungi, and viruses.
Figure 2. Applicability of APR-510-S. (A) Saccharomyces cerevisiae exposedfor 30 seconds. (B) Aspergillus niger hyphae exposed for 30 seconds. (C) T2
bacteriophage exposed for 135 seconds. For all: T indicates Tris buffer;
H indicates water; TE indicates Tris-EDTA.
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