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1.
Persistence of Bacillus atrophaeus subsp. globigii spores on corroded iron coupons in drinking water was studied using a biofilm annular reactor. Spores were inoculated at 10(6) CFU/ml in the dechlorinated reactor bulk water. The dechlorination allowed for observation of the effects of hydraulic shear and biofilm sloughing on persistence. Approximately 50% of the spores initially adhered to the corroded iron surface were not detected after 1 month. Addition of a stable 10 mg/liter free chlorine residual after 1 month led to a 2-log(10) reduction of adhered B. atrophaeus subsp. globigii, but levels on the coupons quickly stabilized thereafter. Increasing the free chlorine concentration to 25 or 70 mg/liter had no additional effect on inactivation. B. atrophaeus subsp. globigii spores injected in the presence of a typical distribution system chlorine residual (approximately 0.75 mg/liter) resulted in a steady reduction of adhered B. atrophaeus subsp. globigii over 1 month, but levels on the coupons eventually stabilized. Adding elevated chlorine levels (10, 25, and 70 mg/liter) after 1 month had no effect on the rate of inactivation. Decontamination with elevated free chlorine levels immediately after spore injection resulted in a 3-log(10) reduction within 2 weeks, but the rate of inactivation leveled off afterward. This indicates that free chlorine did not reach portions of the corroded iron surface where B. atrophaeus subsp. globigii spores had adhered. B. atrophaeus subsp. globigii spores are capable of persisting for an extended time in the presence of high levels of free chlorine.  相似文献   

2.
Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R(2) > 0.98) over a 7-log-unit dynamic range down to 10(1) B. atrophaeus cells or spores. Quantification of S. marcescens (R(2) > 0.98) was linear over a 6-log-unit dynamic range down to 10(2) S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.  相似文献   

3.
Bacterial spores dried on aluminium strips are used in microbiological validation of packaging and processing systems. Vortex agitation and sonication in Butterfield's buffer, 70% ethanol or 0·1% Tween 80 were evaluated for ease of recovery of bacillus spores dried on aluminium strips to compare the concentration of dried spores to dilutions used to inoculate such strips. The highest recovery for Bacillus subtilis var. globigii spores was observed with sonication in 70% ethanol with average recovery close to the initial inoculum. The highest recovery for B. stearothermophilus spores was with sonication in Butterfield's buffer, averaging 0·8 log less recovery than the initial inoculum. Bacillus subtilis var. globigii spores were recovered from strips in greater numbers than B. stearothermophilus spores for all treatment medium combinations. Scanning electron microscopy revealed unrecovered spores adhering to strips after treatment. Recovery of B. subtilis var. globigii spores decreased with time over the 4 week storage period.  相似文献   

4.
An instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system has been developed for the rapid and specific identification of biological warfare (BW) agents. The system has been designed to assay for up to eight agents simultaneously and provides an indication of the absence or presence of a threat within 15 min. Parameters affecting the mixing of the reagents within the instrument's fluidic lines were investigated and optimized. Measurements of blank samples and samples containing Bacillus subtilis spores in the concentration range of 10(4) to 10(6) cfu/ml indicate the limit of detection (LOD) is 3 x 10(3) cfu/ml for B. subtilus. Although the LOD is higher than that of several technologies currently under development, this instrument offers an immediate interim approach for addressing the need to rapidly detect biological warfare agents in the field.  相似文献   

5.
Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 microg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25 degrees C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.  相似文献   

6.
Unique patterns of biomarkers were reproducibly characterized by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry and were used to distinguish Bacillus species members from one another. Discrimination at the strain level was demonstrated for Bacillus cereus spores. Lipophilic biomarkers were invariant in Bacillus globigii spores produced in three different media and in B. globigii spores stored for more than 30 years. The sensitivity was less than 5,000 cells deposited for analysis. Protein biomarkers were also characterized by MALDI analysis by using spores treated briefly with corona plasma discharge. Protein biomarkers were readily desorbed following this treatment. The effect of corona plasma discharge on the spores was examined.  相似文献   

7.
Spores of Bacillus subtilis SA22 harvested after 22 d incubation on nutrient agar at 30°C were more resistant to 0–04% peracetic acid at 20°C than spores harvested following 2 d incubation. Similarly, spores of B. subtilis globigii B17, harvested after 7 d incubation on a sporulation agar were up to 10 times less resistant to 0.04% peracetic acid at 20°C than spores harvested after 35 d incubation. An increase in resistance to heating at 100°C and to exposure to 17.7% hydrogen peroxide at 20°C occurred as the age of B. subtilis SA22 spores prior to harvesting increased, whereas differences in resistance were not observed with spores of B. subtilis globigii B17.  相似文献   

8.
The feasibility of utilizing vapor-phase hydrogen peroxide (VPHP) as a surface decontaminant and sterilant was evaluated in a centrifuge application. The prototype VPHP decontamination system, retrofitted into a Beckman L8-M ultracentrifuge, was designed to vaporize a 30% (wt/wt) solution of aqueous hydrogen peroxide continuously injecting and withdrawing VPHP in a deep-vacuum flow-through system. VPHP cycles of 4, 8, 16, and 32 min were examined for cidal activity against spores of Bacillus subtilis subsp. globigii and Bacillus stearothermophilus. Spore inocula (approximately 10(6)/coupon) were dried onto 0.5-in. (1.27-cm)-square stainless-steel coupons, and coupons were suspended in the centrifuge chamber, the space between the refrigeration can and the barrier ring (inner gap), and the space between the barrier ring and the vacuum ring (outer gap). At a chamber temperature of 4 degrees C, B. subtilis subsp. globigii spores were inactivated within 8 min, while inactivation of spores located in the outer gap at 27 degrees C required 32 min. The elevated temperature and high surface area/volume ratios in the outer gap may serve to decompose the gas more rapidly, thus reducing cidal efficacy. Of the two test spores, B. stearothermophilus was more resistant to VPHP. Nonetheless, VPHP was shown to possess significant sporicidal capability. For practical decontamination applications of the type described, VPHP shows promise as an effective and safer alternative to currently used ethylene oxide or formaldehyde vapors.  相似文献   

9.
Short peptides are capable of tight and specific binding to physiological or fortuitous receptors on the surface of cells. These peptides can be used to tag or capture target cells in an assortment of detector platforms. As part of an effort to identify small-molecule ligands for advanced detectors for spores of Bacillus anthracis, the causative agent of anthrax, we are screening (or biopanning) commercial phage display peptide libraries for peptides that bind tightly and selectively to spores of several Bacillus species. In addition to B. anthracis, these species include B. cereus, B. subtilis, and B. globigii. This review summarizes the methods used in our studies, the results from the biopanning experiments, and the characterization of the spore-binding peptides identified to date. Briefly, several unique families of peptides, with consensus sequences< or = seven-amino-acids long, were identified that exhibit preferential binding to spores (but not vegetative cells) of either one or only a few Bacillus species. At least one peptide family binds well to spores of multiple strains of B. anthracis, while binding poorly or not at all to spores of phylogenetically similar species. This review also discusses other points of interest regarding the use of peptide ligands for spore detection and for the detection of other types of cells.  相似文献   

10.
Highly selective detection of Bacillus anthrax spores has attracted worldwide attention because Bacillus anthrax spores not only are harmful to the health of human beings and animals, but also can be used as biological warfare agents. Here, we report a simple platform by mixing EuCl3·6H2O and sodium polyacrylate in aqueous solution and further investigate its luminescence response towards Bacillus anthrax biomarker dipicolinic acid (DPA) as a turn‐on luminescence probe. Importantly, our probe has good sensitivity, lower detection limit, excellent selectivity as well as great anti‐interference ability due to the great luminescence enhancement of Eu3+. Moreover, a test paper is constructed to realize the purpose of portable detection. These results indicate that our probe is an excellent candidate for sensing DPA.  相似文献   

11.
Vapor-phase hydrogen peroxide as a surface decontaminant and sterilant   总被引:2,自引:0,他引:2  
The feasibility of utilizing vapor-phase hydrogen peroxide (VPHP) as a surface decontaminant and sterilant was evaluated in a centrifuge application. The prototype VPHP decontamination system, retrofitted into a Beckman L8-M ultracentrifuge, was designed to vaporize a 30% (wt/wt) solution of aqueous hydrogen peroxide continuously injecting and withdrawing VPHP in a deep-vacuum flow-through system. VPHP cycles of 4, 8, 16, and 32 min were examined for cidal activity against spores of Bacillus subtilis subsp. globigii and Bacillus stearothermophilus. Spore inocula (approximately 10(6)/coupon) were dried onto 0.5-in. (1.27-cm)-square stainless-steel coupons, and coupons were suspended in the centrifuge chamber, the space between the refrigeration can and the barrier ring (inner gap), and the space between the barrier ring and the vacuum ring (outer gap). At a chamber temperature of 4 degrees C, B. subtilis subsp. globigii spores were inactivated within 8 min, while inactivation of spores located in the outer gap at 27 degrees C required 32 min. The elevated temperature and high surface area/volume ratios in the outer gap may serve to decompose the gas more rapidly, thus reducing cidal efficacy. Of the two test spores, B. stearothermophilus was more resistant to VPHP. Nonetheless, VPHP was shown to possess significant sporicidal capability. For practical decontamination applications of the type described, VPHP shows promise as an effective and safer alternative to currently used ethylene oxide or formaldehyde vapors.  相似文献   

12.
Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.  相似文献   

13.
New biochip technology for label-free detection of pathogens and their toxins   总被引:10,自引:0,他引:10  
microSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label-free transduction. The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface. Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip. SERS, like other whole-organism fingerprinting techniques, is very specific. Our initial studies have shown that the Gram-positive Listeria and Gram-negative Legionella bacteria, Bacillus spores and Cryptosporidium oocysts can often be identified at the subspecies/strain level on the basis of SERS fingerprints collected from single organisms. Therefore, pathogens can be individually identified by microSERS, even when organisms that cross-react with the capture biomolecules are present in a sample. Moreover, the SERS fingerprint reflects the physiological state of a bacterial cell, e.g., when pathogenic Listeria and Legionella were cultured under conditions known to affect virulence, their SERS fingerprints changed significantly. Similarly, nonviable (e.g., heat- or UV-killed) microorganisms could be differentiated from their viable counterparts by SERS fingerprinting. Finally, microSERS is also capable of the sensitive and highly specific detection of toxins. Toxins that comprised as little as 0.02% by weight of the biomolecule-toxin complex produced strong, unique fingerprints when spectra collected from the complexes were subtracted from the spectra of the uncomplexed biomolecules. For example, aflatoxins B(1) and G(1) could be detected and individually identified when biochips bearing pixels of antibody or enzyme capture biomolecules were incubated in samples containing one or both aflatoxins, and the spectra were then collected for 20 s from an area of the biomolecule pixel approximately 1 microm in diameter. In the future, we plan to investigate the use of hyperspectral imaging Raman microscopy for collecting fingerprints from all the pixels on the biochip, individually yet simultaneously, to enable the rapid detection of diverse pathogens and their toxins in a sample, using a single biochip.  相似文献   

14.
Bacillus subtilis-Bacillus globigii hybrids were made by integration of the B. globigii aromatic region (aroB to aroE) as an intergenote in the B. subtillis chromosome. Transformation of the heterologous intergenote by B. subtillis DNA (or vice versa) occurred at about 10% of the frequency of homologous transformation by hybrid donors into the same region. Heterologous intergenote crosses were unusually sensitive to shear fragmentations of donor DNA to sizes less than 30 X 10(6) to 40 X 10(6) daltons. In all cases, the entire intergenote was transferred en bloc. Homologous transformation of intergenote markers by B. globigii DNA was not unusually shear sensitive, and linkage was normal for markers in the intergenote. A model is proposed in which efficient heterologous intergenote transformation occurs by recognition and base pairing of homologous DNA sequences of both flanks of the intergenote.  相似文献   

15.
Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.  相似文献   

16.
AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.  相似文献   

17.
Point-of-care testing (POCT) of infectious bacterial agents offers substantial benefits for disease diagnosis, mainly by shortening the time required to obtain results and by making the test available bedside or at remote care centers. Immunochromatographic lateral flow biosensors offer a low cost, highly sensitive platform for POCT. In this article, we describe the fabrication and testing of a multiplex immuno-disc sensor for the specific detection of Pseudomonas aeruginosa and Staphylococcus aureus. Antibody conjugated gold nanoparticles were used as the signaling agents. The detection range of the bacteria lies within 500-5000 CFU/ml. The advantage of the immuno-disc sensor is that it does not require any preprocessing of biological sample and is capable of whole cell bacterial detection. We also describe the design and fabrication of a compact portable device which converts the color intensity of the gold nanoparticles that accumulate at the test region into a quantitative voltage reading proportional to the bacterial concentration in the sample. The combination of the immuno-disc and the portable color reader provides a rapid, sensitive, low cost, and quantitative tool for the detection of a panel of infectious agents present in the patient sample.  相似文献   

18.
An optical waveguide array biosensor suitable for rapid detection of multiple bio-hazardous agents is presented. SpectroSens? optical microchip sensors contain multiple spatially-separated waveguide channels with integral high-precision Bragg gratings sensitive to changes in refractive-index; selective surface-functionalisation of discrete sensing channels with different antibodies as bio-recognition elements enables selective multi-analyte biological detection. Interactions between target antigens in the test sample and respective surface-immobilised antibodies result in localised changes in refractive-index; the biosensor response manifests as increases in wavelength of light reflected from specific sensing channels. Multiplexed, label-free detection of 8 different biological agents, encompassing bacterial spores, vegetative cells, viruses and proteinaceous toxins has been demonstrated in real-time. Selective detection of Bacillus atrophaeus (BG) spores, Escherichia coli cells, MS2 viruses and ovalbumin (OVA) protein (simulant bio-hazardous agents) was first demonstrated as proof-of-concept; subsequently, detection of Bacillus anthracis (BA) spores (UM23CL2 strain), Franciscella tularensis (FT) cells (live vaccine strain), Vaccinia viruses (heat-killed) and ricin toxin (bio-hazardous agents) was proven. Two optical microchip sensors, each comprising 8 sensing channels were packaged into a single disposable cartridge allowing simultaneous 16-channel data acquisition. The specific antibody deposition sequence used in this study enabled detection of either 4 simulants or 4 bio-hazardous agents using a single consumable. The final device, a culmination of the multidisciplinary convergence of the fields of biology, chemistry, optoelectronics and microfluidics, is man-portable and inherently robust. The performance characteristics of the SpectroSens? technology platform highlight its potential for exploitation as a ‘detect to warn/treat’ biodetector in security and defence operations.  相似文献   

19.
Three strains of Bacillus: Bacillus atrophaeus (formally Bacillus globigii), Bacillus thuringiensis, and Bacillus cereus were tested for their intrinsic magnetic susceptibility. All three strains when sporulated demonstrated significant magnetic susceptibility using an instrument referred to as Cell Tracking Velocimetry. Energy dispersive spectroscopy also confirmed the presence of paramagnetic elements, Fe and Mn, in the spore form of the bacteria. It was demonstrated that this magnetic susceptibility is sufficient to separate and deposit these spores on glass slides in a magnetic deposition system. These results indicate the potential to separate spores with intrinsic magnetic susceptibility directly out of water or air samples.  相似文献   

20.
生物光学传感器是一种对生物物质敏感并将其浓度转换为光信号,再由光电器件转换成电信号进行检测的仪器。由于随着微加工技术和纳米技术的进步,生物光学传感器将不断的微型化,各种便携式生物光学传感器的出现使得人们在家中进行疾病诊断、在市场上直接检测食品及在野外快速检测环境污染成为可能。便携式生物光学传感器一般由光源、光学通路和光电元件三部分组成。传感器结构中各个组件的优化处理将有利于检测设备在实际运用中的便利性和在复杂环境中的适用性,同时也有利于提高生物检测的灵敏度。主要从激励光源的选择、生物光学检测的原理、用于传感光源分析的半导体光敏元件3方面,描述近年来常见的便携式生物光学传感器的研究进展。未来生物检测器件将趋于成本低廉、便携快捷、智能高效等特点,基于生物光学反应特性的研究和传感器结构制备的优化将使得便携式生物光学传感器在未来传感检测应用中具有巨大的商业价值和广泛的实用价值。  相似文献   

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