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 cm 3 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 (C T ) 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 C T 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, batteryoperated format, which is expected to facilitate sensitive point-of-care nucleic acid testing.

Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

“…Spores were harvested from B. anthracis Sterne strain 34F2, B.cereus 13061, B. mycoides, B. subtilis, and B. thuringiensis Kurstaki 4D9 as described in Walper et al [15]. Purified spores were mechanically sheared using 1.0 mm glass beads, a BioSpec Products Mini-Bead Beater, and a protocol modified from that described by Vandeventer et al [22]. Protein concentrations obtained for several of the spore samples, particularly B. mycoides and B. subtilis, were significantly lower (10-fold) than those obtained from the other spore samples.…”

Polymeric-Based In Vitro Diagnostic Devices

“…Functional beads, when incorporated with other actuation techniques, are effective and worthwhile tools for cell lysis studies. A disposable bead-based blender can be used for thick-walled bacillus spores and mycobacterium cell lysis for molecular diagnostics [205]. Similarly, a laser can be used to irradiate magnetic particles for rapid lysis aimed at the extraction of DNA from Escherichia coli and the hepatitis B virus (Fig.…”

Isolation and Detection of Carcinogenic Nucleic Acid Adducts

“…Mechanical methods are used for samples such as animal tissues that require great force to disrupt. 19 …”