Barbara Erwin

Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities.

Rapid isothermal nucleic acid amplification technologies can enable diagnosis of human pathogens and genetic variations in a simple, inexpensive, user-friendly format. The isothermal exponential amplification reaction (EXPAR) efficiently amplifies short oligonucleotides called triggers in less than 10 min by means of thermostable polymerase and nicking endonuclease activities. We recently demonstrated that this reaction can be coupled with upstream generation of trigger oligonucleotides from a genomic target sequence, and with downstream visual detection using DNA-functionalized gold nanospheres. The utility of EXPAR in clinical diagnostics is, however, limited by a nonspecific background amplification phenomenon, which is further investigated in this report. We found that nonspecific background amplification includes an early phase and a late phase. Observations related to late phase background amplification are in general agreement with literature reports of ab initio DNA synthesis. Early phase background amplification, which limits the sensitivity of EXPAR, differs however from previous reports of nonspecific DNA synthesis. It is observable in the presence of single-stranded oligonucleotides following the EXPAR template design rules and generates the trigger sequence expected for the EXPAR template present in the reaction. It appears to require interaction between the DNA polymerase and the single-stranded EXPAR template. Early phase background amplification can be suppressed or eliminated by physically separating the template and polymerase until the final reaction temperature has been reached, thereby enabling detection of attomolar starting trigger concentrations.

Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device

ABSTRACT Molecular detection of microorganisms requires microbial cell disruption to release nucleic acids. Sensitive detection of thick-walled microorganisms such as Bacillus spores and Mycobacterium cells typically necessitates mechanical disruption through bead beating or sonication, using benchtop instruments that require line power. Miniaturized, low-power, battery-operated devices are needed to facilitate mechanical pathogen disruption for nucleic acid testing at the point of care and in field settings. We assessed the lysis efficiency of a very small disposable bead blender called OmniLyse relative to the industry standard benchtop Biospec Mini-BeadBeater. The OmniLyse weighs approximately 3 g, at a size of approximately 1.1 cm3 without the battery pack. Both instruments were used to mechanically lyse Bacillus subtilis spores and Mycobacterium bovis BCG cells. The relative lysis efficiency was assessed through real-time PCR. Cycle threshold (CT ) values obtained at all microbial cell concentrations were similar between the two devices, indicating that the lysis efficiencies of the OmniLyse and the BioSpec Mini-BeadBeater were comparable. As an internal control, genomic DNA from a different organism was spiked at a constant concentration into each sample upstream of lysis. The CT values for PCR amplification of lysed samples using primers specific to this internal control were comparable between the two devices, indicating negligible PCR inhibition or other secondary effects. Overall, the OmniLyse device was found to effectively lyse tough-walled organisms in a very small, disposable, battery-operated format, which is expected to facilitate sensitive point-of-care nucleic acid testing.

Continuous-Flow, Rapid Lysis Devices for Biodefense Nucleic Acid Diagnostic Systems

Two mechanical lysis devices have been developed as compact, robust components to provide rapid sample preparation for nucleic acid diagnostic systems. One such component, known as the Micro Bead-Beater™ (μBB™, BBTM, Claremont BioSolutions, Upland, CA), is a compact device that is capable of ultrarapid lysis (>90% lysis in 30 s) of micro volumes (<80 μL) ofBacillus spores in a continuous-flow format or in a disposable single-tube format. The μBB is also capable of processing much larger volumes of solutions containing spores or vegetative cells using a continuous-flow mode. A second mechanical lysis device designed as a disposable component is the microfluidic bead blender, which uses a small electric motor to spin vanes within the bead-laden solution. DNA quantification results using dsDNA-binding fluorescence dyes and real-time PCR are presented, comparing the lysis of Bacillus subtilis spores using the μBB™ with other well-known lysis techniques. Nanoscale imaging results obtained using scanning electron microscopy and transmission electron microscopy on B. subtilis spores lyzed using the μBB™ are also presented