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Biosens Bioelectron ; 24(11): 3299-305, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19450964

RESUMO

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.


Assuntos
Aerossóis/análise , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Técnicas Biossensoriais/instrumentação , DNA Bacteriano/análise , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Microbiologia do Ar , Algoritmos , DNA Bacteriano/genética , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Técnicas de Amplificação de Ácido Nucleico/métodos , Esporos Bacterianos/genética , Esporos Bacterianos/isolamento & purificação
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