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

https://doi.org/10.1016/j.bios.2009.04.025Get rights and content

Abstract

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 104 spores was achieved within 2 h throughout the micro-fluidic process.

Introduction

The sarin gas attack on the Tokyo subway in 1995 (Seto et al., 2000) and the anthrax attack using USA postal system in 2001 (Inglesby et al., 2002, Matsumoto, 2003) were acts of chemical and biological terrorism. Biological warfare agents (BWAs) (Centers for Disease Control and Prevention, www.bt.cdc.gov/agent/agentlist-category.asp) do not cause disease immediately after dispersion. They therefore have been detected and identified by surveillance and diagnosis of infected patients (Franz et al., 1997), or laboratory analysis of specimens. Immunological (Andreotti et al., 2003, Peruski and Peruski, 2003), molecular biological (Ivnitski et al., 2003) and microbiological (Stanier et al., 1976, Schuch et al., 2002) methods have been developed, and new technologies (e.g., DNA microarray (Edelstein et al., 2000, Song et al., 2006), mass spectrometry (Fenselau and Demirev, 2001, Warscheid et al., 2003, Fergenson et al., 2004)) have recently been introduced. The BioWatch program was subsequently implemented in the USA: air samples were continuously taken from public places and collected aerosol samples transferred to a specific laboratory to detect BWAs (Shea and Lister, 2003). Considering the time-consuming analysis and diagnosis currently required by laboratories, rapid on-site detection undertaken by first responders or a stand-alone automated detection system would minimize the scale of disasters, contributing to early diagnosis and medical treatment before the outbreak of infectious diseases. Adaptability in the field, rapid-alarm capability, adequate sensitivity, ease of automated operation, and adequate selectivity are required to detect on-site BWAs. New on-site detection technologies have been proposed and reviewed (Paddle, 1996, Ivnitski et al., 1999, Casagrande, 2000, Walt and Franz, 2000, Deisingh and Thompson, 2002, Deisingh and Thompson, 2004). Various types of on-site equipment have been used by the military worldwide; some have been introduced for civil defense and implemented by first responders (Fitch et al., 2003). Of these, lateral flow immunoassay test-strips are usually employed in the field (Iura et al., 2004). Field-portable real-time polymerase chain reaction (RT-PCR) instruments are used by civil defense and the military (Higgins et al., 2003a, Higgins et al., 2003b), but require specialized skill for the very complex procedure of preparing samples and extracting DNA.

Micro-electromechanical system (MEMS) technology provides automated down-sized laboratory-like analysis in the field (Cheng et al., 1998). It adopts the detection mechanisms of DNA hybridization (Kelly and Woolley, 2005) or immunological molecular recognition (Rowe-Taitt et al., 2000). The Mechanical Engineering Laboratory of Hitachi Limited developed MEMS technology using a silicon elastomer (polydimethylsiloxane (PDMS)) chip. The latter uses hard glass or silicon chips, is inexpensive to manufacture, and is compatible with biological reagents (Boone et al., 2002). Our research group has developed a semi-automated bacterial spore detection system (BSDS) that continuously collects aerosols, treats spores, and extracts and detects DNA with disposable PDMS chips. The target selected was the spore form of the anthrax-simulated bacteria Bacillus subtilis. Conventional on-site automated equipment can detect genetic DNA from vegetative bacterial cells because the cell membrane can be readily destroyed using biochemical protease or detergents. Analyzing the DNA of spore forms has not been possible because of the difficulty of extracting DNA from spores with rigid cell membranes. Detection of spore forms should be considered because anthrax spores were employed in the attacks using the USA postal system (Inglesby et al., 2002). Rupturing rigid spore coats by mechanical force (e.g., ultrasonication) is necessary. Considering the hardware limitation for treating samples mechanically in the field, biochemical pretreatment for spore lysis has been adopted using germination whereby spores are transformed to vegetative cells in a “nutrient broth” (Stopa et al., 1999). In our previous contribution, we utilized germination to increase the sensitivity of detecting B. subtilis spore forms with adenosine triphosphate detection using bioluminescence (Fujinami et al., 2004). Liu et al. (2007) successfully detected anthrax spores using a germination process in electrical measurement of polar and ionic chemicals released from spores in a micro-fluidic biochip system. We therefore adopted germination to treat spores in the BSDS.

Section snippets

Chemicals

Lysozyme from chicken egg albumin (biochemical reagent grade), guanidium hydrochloride (guanidium HCl), glycerol, and agarose were purchased from Wako Pure Chemicals (Osaka, Japan). Proteinase-K, ϕX174 Hae III digest and ICAN kits were obtained from Takara Bio Incorporated (Ohtsu, Japan). Glass beads (DNA analytical glass particles, 325 mesh) were purchased from Wako Pure Chemicals. All other chemicals were of analytical reagent grade. The water purification system was a Milli-Q gradient A-10

Results and discussion

Analytical conditions were optimized for aerosol collection, germination, cell lysis, and DNA extraction of B. subtilis spores. This was followed by off-chip study of fluorescence using RT-PCR quantification.

Conclusion

We developed a semi-automated bacterial spore detection system (BSDS) with micro-fluidic chips for aerosol collection, spore treatment, and ICAN DNA detection to detect B. subtilis spores. 104 spores were detected within 2 h throughout the micro-fluidic process. Therefore, our BSDS can raise alerts about airborne bacterial spore contamination considerably more sensitive and easier than other on-site air-collection systems that involve tedious and specialized methods of culturing and examining

Acknowledgements

This work was partly undertaken under a research program called the “Effective Promotion of Joint Research with Industries, Academia, and Government” sponsored by Special Coordination Funds for Promoting Science and Technology supported by the Ministry of Education, Culture, Sports, Science and Technology.

References (39)

  • G.P. Anderson et al.

    Biosens. Bioelectron.

    (2000)
  • R.L. Edelstein et al.

    Biosens. Biolectron.

    (2000)
  • E.W. Henningson et al.

    J. Aerosol Sci.

    (1994)
  • J.A. Higgins et al.

    Biosens. Bioelectron.

    (2003)
  • D. Ivnitski et al.

    Biosens. Bioelectron.

    (1999)
  • B.M. Paddle

    Biosens. Bioelectron.

    (1996)
  • C.A. Rowe-Taitt et al.

    Biosens. Bioelectron.

    (2000)
  • P.E. Andreotti et al.

    BioTechnique

    (2003)
  • T.D. Boone et al.

    Anal. Chem.

    (2002)
  • R. Casagrande

    Sci. Am. 2000

    (2000)
  • J. Cheng et al.

    Nat. Biotechnol.

    (1998)
  • A.K. Deisingh et al.

    Analyst

    (2002)
  • A.K. Deisingh et al.

    Can. J. Microbiol.

    (2004)
  • C. Fenselau et al.

    Mass Spectrom. Rev.

    (2001)
  • D.P. Fergenson et al.

    Anal. Chem.

    (2004)
  • J.P. Fitch et al.

    Science

    (2003)
  • D.R. Franz et al.

    J. Am. Med. Assoc.

    (1997)
  • Y. Fujinami et al.

    J. Health Sci.

    (2004)
  • J.A. Higgins et al.

    Appl. Environ. Microbiol.

    (2003)
  • Cited by (24)

    • Analysis of bioaerosols

      2023, Aeromicrobiology
    • Recent progress in nanomaterial-based sensing of airborne viral and bacterial pathogens

      2021, Environment International
      Citation Excerpt :

      Once all are put together, these steps take more than 2 h to limit its application to real-time monitoring. However, incorporation of semi-automation and microfluidics in PCR-based technologies has been an option to reduce the total analysis time (e.g., up to 70 min) (Inami et al., 2009; Jiang et al., 2014). Till now, PCR-based approaches have also been employed to detect various airborne viruses, e.g., influenza (Huynh et al., 2008; Pyankov et al., 2007), vaccinia (Agranovski et al., 2006), porcine circovirus (Verreault et al., 2010), rhinovirus (Huynh et al., 2008; Myatt et al., 2003), parainfluenza (Huynh et al., 2008), H3N2 viruses (Lednicky and Loeb 2013), rhinoviruses (Myatt et al., 2003).

    • Evaluation of a microfluidic chip system for preparation of bacterial DNA from swabs, air, and surface water samples

      2016, Biologicals
      Citation Excerpt :

      Various chip gadgets have been developed for preparation of DNA from bacteria [7–11]. Silica beads [7,8] with superparamagnetic iron oxide cores [9] are also very common for on-chip application. Microfluidic lab-on-a-chip devices need only small volumes of reagents and samples.

    • Perspective: Microfluidic applications in microbiology

      2010, Journal of Microbiological Methods
    View all citing articles on Scopus
    View full text