Development and Application of a Rapid and Simple Method for Extracting Nucleic Acids from Microbes.

Abstract

Rapid, efficient, and easy-to-use procedures for isolating high-quality genomic DNA or RNA of pathogenic bacteria or viruses prior to gene amplification procedures are essential for the molecular identification of biowarfare agents. In this study, we developed a rapid and simple nucleic acid extraction method for bacteria and its spore. The method is based on cell disruption using zirconia/silica beads followed by precipitation of nucleic acids with isopropanol by using a personal microcentrifuge. In spectrometric and agarose gel electrophoretic analysis, we confirmed that bacterial genomic DNA was efficiently purified from gram-negative, gram-positive bacteria and spores. We evaluated this extraction method for the application for real-time PCR-based biological detection system, R.A.P.I.D.TM. The detection sensitivities of the R.A.P.I.D.TM system to the samples prepared from Bacillus anthracis vegetative cells and spores by our extraction method were 100-fold higher than those by using the IT1-2-3TM extraction kit, which is recommended by the instruction of R.A.P.I.D.TM. In the loop-mediated isothermal amplification (LAMP) detection, which we previously developed, B. anthracis DNA in the extracts of 2×102 cfu/mL of spore suspensions could be detected. In addition, B. anthracis in 0.5% (w/v) white powder (e.g. sugar) suspension was also detected by R.A.P.I.D.TM and LAMP. The DNA extraction method we developed here is highly efficient and applicable for nucleic acid amplification techniques. In addition, this method was able to be performed with simple devices. Therefore our method was considered to be suitable for the on-site use for detection of microbiological agents.