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

Inami, Hisao; Tsuge, Kouichiro; Matsuzawa, Mitsuhiro; Sasaki, Y.; Togashi, Shigenori; Komano, Asuka; Seto, Yasuo

doi:10.1016/j.bios.2009.04.025

Cited by 26 publications

(21 citation statements)

References 35 publications

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“…a few hundred copies of template DNA [186]. Practical work in the area of diagnostics was done on the amplification of 16S rDNA [189] or semi-automated bacterial spore detection systems for bio-warfare agents (e.g., Bacillus anthracis) including an aerosol sampler [190]. The detection of 10 4 spores was achieved within 2 h employing microfluidics.…”

Section: Various Other Methodsmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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Section: Various Other Methodsmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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show abstract

“…These particle and matrix associated approaches are more suitable for miniaturization than the traditional chloroform-phenol extraction [6]. Various chip gadgets have been developed for preparation of DNA from bacteria [7], [8], [9], [10], [11]. Silica beads [7], [8] with superparamagnetic iron oxide cores [9] are also very common for on-chip application.…”

Section: Introductionmentioning

confidence: 99%

“…They are portable and can be used for fast analysis at the point-of-need [12]. The number of microfluidic devices which are actually used for preparation of DNA from environmental samples is still very low and their suitability was demonstrated only for specific kinds of samples like swabs [13] or aerosols [8]. A microfluidic method for preparation of DNA from Gram-negative or Gram-positive bacteria in different matrices is a prerequisite for PCR and could be an innovative tool for diagnostic purposes.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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The detection of bacterial pathogens from complex sample matrices by PCR requires efficient DNA extraction. In this study, a protocol for extraction and purification of DNA from swabs, air, and water samples using a microfluidic chip system was established. The optimized protocol includes a combination of thermal, chemical and enzymatic lysis followed by chip-based DNA purification using magnetic particles. The procedure was tested using Gram-positive Bacillus thuringiensis Berliner var. kurstaki as a model organism for Bacillus anthracis and the attenuated live vaccine strain of Francisella tularensis subsp. holarctica as Gram-negative bacterium. The detection limits corresponded to 103 genome equivalents per milliliter (GE/ml) for surface water samples spiked with F. tularensis and 102 GE/ml for B. thuringiensis. In air, 10 GE of F. tularensis per 10 L and 1 GE of B. thuringiensis per 10 L were detectable. For swab samples obtained from artificially contaminated surfaces the detection limits were 4 × 103 GE/cm2 for F. tularensis and 4 × 102 GE/cm2 for B. thuringiensis. Suitability of the chip-assisted procedure for DNA preparation of real samples was demonstrated using livestock samples. The presence of thermophilic Campylobacter spp. DNA could be confirmed in air samples collected on pig and broiler farms.

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“…In suspected cases of chemical and biological terrorism, samples are first analyzed on-site and are then brought to a laboratory for detailed analysis [4]. Our laboratories have long assessed on-site detection devices and other commercially available equipment [5][6][7][8][9][10][11][12]; we have also developed new on-site detection methods for biological warfare agents [13,14].…”

Section: Introductionmentioning

confidence: 99%

Detection of saxitoxin in counterterrorism using a commercial lateral flow immunoassay kit

et al. 2010

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The neurotoxin saxitoxin (STX) is registered in the list of the Chemical Weapons Convention. In preparation against potential terrorism by STX use, we investigated the performance of a commercially available rapid test kit for paralytic shellfish poisoning (PSP), which is essentially a lateral flow immunoassay kit. Pink lines in the test and control zones appeared after 35 min and were observed by the naked eye and were recorded by a digital scanner. The competitive displacement of gold-labeled antitoxin analog antibody by STX in the test zone was quantitatively shown using the ratio of the intensity of the test zone line to that of control zone line. As the STX concentration increased, the intensity of the pink line in the test zone on the strip decreased. The limit of detection was defined as the STX concentration that gave a ratio half that of the STX blank test, and was calculated to be about 12 ng/ml. Various matrix components, such as wheat flour and the decontamination chemical hypochlorite, were examined for their effects on false positive and false negative results in the determination of STX by the PSP Rapid Test system.

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

Cited by 26 publications

References 35 publications

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

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

Detection of saxitoxin in counterterrorism using a commercial lateral flow immunoassay kit

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