Asuka Komano

Semi-automated bacterial spore detection system with micro-fluidic chips for aerosol collection, spore treatment and ICAN DNA detection.

A semi-automated bacterial spore detection system (BSDS) was developed to detect biological threat agents (e.g., Bacillus anthracis) on-site. The system comprised an aerosol sampler, micro-fluidic chip-A (for spore germination and cell lysis), micro-fluidic chip-B (for extraction and detection of genomic DNA) and an analyzer. An aerosol with bacterial spores was first collected in the collection chamber of chip-A with a velocity of 300 l/min, and the chip-A was taken off from the aerosol sampler and loaded into the analyzer. Reagents packaged in the chip-A were sequentially applied into the chamber. The genomic DNA extract from spore lyzate was manually transferred from chip-A to chip-B and loaded into the analyzer. Genomic DNA in chip-B was first trapped on a glass bead column, washed with various reagents, and eluted to the detection chamber by sequential auto-dispensing. Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) with fluorescent measurement was adopted to amplify and detect target DNA. Bacillus subtilis was the stimulant of biological warfare agent in this experiment. Pretreatment conditions were optimized by examining bacterial target DNA recovery in the respective steps (aerosol collection, spore germination, cell lysis, and DNA extraction), by an off-chip experiment using a real-time polymerase chain reaction quantification method. Without the germination step, B. subtilis spores did not demonstrate amplification of target DNA. The detection of 10(4) spores was achieved within 2h throughout the micro-fluidic process.

Detection of proteinous toxins using the Bio-Threat Alert system, part 3: effects of heat pretreatment and interfering substances

We previously reported that the Guardian Bio-Threat Alert (BTA) system could detect (detection limit: about 0.1 μg/ml) staphylococcal enterotoxin B (SEB), botulinum toxins (BTX) A and B, and ricin, with no interference by white-powdered materials or colored matrices. In this study, the capability of the BTA system was further assessed. With 10 min of preheating at 60°C, all toxins could be detected, but with preheating at 80°C, BTX A and B and ricin became undetectable. About 20% SEB could be detected after heating at 80°C, but this detection ability was completely removed after heating at 100°C. The effects of chemicals usually used for decontamination, such as sodium hypochlorite, hydrogen peroxide, formaldehyde, and sodium nitrite, on the detectability of SEB, BTX A, or ricin in the BTA system were also tested. The concentrations giving 50% line intensity for SEB, BTX A, and ricin were 3.1, 11, and 15 μM for sodium hypochlorite and 88, 210, and 60 mM for formaldehyde, respectively. The addition of hydrogen peroxide or sodium nitrite did not decrease the detectability even when used at high concentrations.

Decontamination of nerve agents by immobilized organophosphorus hydrolase

Organophosphorus hydrolase (OPH; EC 3.1.8.1) is known to be capable of hydrolyzing a variety of organophosphorus compounds, such as sarin and paraoxon. We have developed a nerve agent decontamination method using OPH. The gene that encodes OPH was cloned from the bacterial strain Sphingobium fuliginis ATCC 27551, and several OPH gene fusion plasmids were constructed. Escherichia coli was utilized as the expression host for the resulting plasmids. The activities of the recombinant OPH enzymes expressed (KGU0060, KGU0092, and KGU0094) were determined by measuring paraoxon hydrolysis activities. The recombinant OPH enzymes that lacked the signal peptide regions (KGU0092 and KGU0094) were remarkably activated by zinc ion; while the OPH enzyme that contained the signal peptide region (KGU0060) was activated by both zinc and cobalt ions, although the specific activity of this enzyme was much lower than that of KGU0092 or KGU0094. The pH profile demonstrated that the OPH enzymes effectively hydrolyzed the substrate under alkaline conditions. We applied the recombinant OPH to the degradation of nerve agents; it hydrolyzed sarin, tabun, cyclohexyl sarin, and soman with high activities, but not O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX). The enzyme was immobilized to a nickel-chelating sepharose resin. Similarly, all nerve agents could be hydrolyzed by the immobilized enzyme except VX. Our results suggest the possibility for developing a powerful and ecological nerve agent detoxication system using OPH.

Detection of proteinous toxins using the Bio-Threat Alert system, part 4. Differences in detectability according to manufactural lots and according to toxin subtypes

In a series of experiments, we have tested the usefulness and limitations of the Guardian Bio-Threat Alert (BTA) system for detection of staphylococcal enterotoxin B (SEB), botulinum toxins (BTXs) A and B, and ricin. In this report, the BTA system has been further evaluated for toxin subtypes and the detection ability of manufactural lots of the BTA strips. The SEB strips failed to detect staphylococcal enterotoxin A, C, and D; the BTX strips generally failed to detect BTXs C, D, E, and F, but one lot showed positive results for BTXs C and D with very low sample values. Differences were observed in sample values at 1 μg/ml for all main toxins according to the different manufactural strip lots: 3.9-fold difference for SEB, 6.3-fold difference for BTX A, 10.9-fold difference for BTX B, and 6.4-fold difference for ricin. The ricin strips showed high cross reactivity toward RCA120. The BioWarfare Agent Detection Devices system showed much lower sensitivity than the BTA system for BTX and ricin (detection limit: about 10 μg/ml).