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Sample records for rapid bacteria detection

  1. Rapid methods for detection of bacteria

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Andersen, B.Ø.; Miller, M.

    2006-01-01

    Traditional methods for detection of bacteria in drinking water e.g. Heterotrophic Plate Counts (HPC) or Most Probable Number (MNP) take 48-72 hours to give the result. New rapid methods for detection of bacteria are needed to protect the consumers against contaminations. Two rapid methods...

  2. ETV Tech Brief: Rapid Fungi and Bacteria Detection Technologies

    Science.gov (United States)

    Technical brief that summarizes the results for Mycometer, Inc. Mycometer®-test and Bactiquant®-test, which are rapid detection technologies for fungi and bacteria. The brief summarizes the results of the verification report and statement.

  3. Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection

    Science.gov (United States)

    Kang, Dong-Ku; Ali, M. Monsur; Zhang, Kaixiang; Huang, Susan S.; Peterson, Ellena; Digman, Michelle A.; Gratton, Enrico; Zhao, Weian

    2014-01-01

    Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed ‘Integrated Comprehensive Droplet Digital Detection’ (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime. PMID:25391809

  4. Upconversion nanoparticles based FRET aptasensor for rapid and ultrasenstive bacteria detection.

    Science.gov (United States)

    Jin, Birui; Wang, Shurui; Lin, Min; Jin, Ying; Zhang, Shujing; Cui, Xingye; Gong, Yan; Li, Ang; Xu, Feng; Lu, Tian Jian

    2017-04-15

    Pathogenic bacteria cause serious harm to human health, which calls for the development of advanced detection methods. Herein, we developed a novel detection platform based on fluorescence resonance energy transfer (FRET) for rapid, ultrasensitive and specific bacteria detection, where gold nanoparticles (AuNPs, acceptor) were conjugated with aptamers while upconversion nanoparticles (UCNPs, donor) were functionalized with corresponding complementary DNA (cDNA). The spectral overlap between UCNPs fluorescence emission and AuNPs absorption enables the occurrence of FRET when hybridizing the targeted aptamer and cDNA, causing upconversion fluorescence quenching. In the presence of target bacteria, the aptamers preferentially bind to bacteria forming a three-dimensional structure and thereby dissociate UCNPs-cDNA from AuNPs-aptamers, resulting in the recovery of upconversion fluorescence. Using the UCNPs based FRET aptasensor, we successfully detected Escherichia coli ATCC 8739 (as a model analyte) with a detection range of 5-106cfu/mL and detection limit of 3cfu/mL. The aptasensor was further used to detect E. coli in real food and water samples (e.g., tap/pond water, milk) within 20min. The novel UCNPs based FRET aptasensor could be used to detect a broad range of targets from whole cells to metal ions by using different aptamer sequences, holding great potential in environmental monitoring, medical diagnostics and food safety analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Rapid, sensitive detection of bacteria in platelet samples with Fountain Flow Cytometry.

    Science.gov (United States)

    Johnson, Paul; Moriwaki, Mika; Johnson, Joseph

    2017-11-01

    There is a current need to develop a technique for bacterial screening of platelet donations that is more rapid, sensitive, and economical than alternatives. The objective of this research was to perform a pilot test of the viability of Fountain Flow Cytometry (FFC), for the rapid and sensitive detection of bacteria in platelet donations. Platelet samples were inoculated with serial dilutions of five selected bacterial strains. Samples were then centrifuged, reconstituted in buffer, and stained with a live/dead bacterial stain cocktail. The resulting aqueous sample was measured by FFC, in which the sample passed as a stream in front of an LED, which excited the fluorescent labels. Fluorescence was detected with a digital camera as the sample flowed toward it. Fountain Flow Cytometry enumeration yielded results that were linear with bacterial concentration, having an R2 of ≥0.98 with a detection efficiency of 92%±3%. Measurements of uninoculated samples showed a false-positive detection rate at ~400 colony forming units (CFU)/mL. Detection of bacterial concentrations in platelets above this threshold can be made in ~15 minutes, including sample preparation time. This pilot study supports the efficacy of FFC for the rapid and sensitive screening of platelet donations for bacteria. © 2017 Wiley Periodicals, Inc.

  6. Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria.

    Science.gov (United States)

    Liu, Hai-Bin; Zang, Yu-Xuan; Du, Xin-Jun; Li, Ping; Wang, Shuo

    2017-09-01

    The efficient and timely detection of pathogens is a major concern worldwide. The aim of this study was to establish a rapid detection method for Salmonella bacteria in food samples to facilitate timely treatment. Widely used detection methods currently include culture-based methods and PCR-based methods. The former are time consuming, requiring 2 to 3 d, whereas the latter have higher accuracy but are typically complicated, requiring expertise and expensive instruments. In this study, a sensitive and rapid approach for the visual and point-of-use detection of Salmonella bacteria based on recombinase polymerase amplification (RPA) and a lateral-flow (LF) nucleic acid strip was established. We designed a pair of primers according to the invA gene of Salmonella bacteria: one was modified with digoxin, and the other was modified with biotin. In the presence of the biotin- and digoxin-modified primers and target DNA, the RPA produced a substantial amount of duplex DNA attached to biotin and digoxin. The products were detected using LF strips through immunoreaction: anti-digoxin antibodies on the gold nanoparticles, digoxin on the duplex, streptavidin on the LF test line, and biotin on the duplex. The developed RPA-LF assay allowed detection of Salmonella genomic DNA in less than 20 min with simple water bath equipment or portable thermal equipment. In addition, the RPA-LF assay was highly sensitive, with a detection limit as low as 20 fg of target DNA or 1.05 × 101 cfu of bacteria in pure culture, and highly specific, exhibiting no cross-reaction with Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Enterobacter aerogenes, or Campylobacter jejuni. Importantly, Salmonella could be detected in milk and chicken breast at concentrations as low as 1.05 × 100 cfu/mL or 1.05 × 100 cfu/g after enrichment for 2 h and in eggs at 1.05 × 100 cfu/g after enrichment for 4 h. Furthermore, RPA was more sensitive than PCR, which requires a thermal cycling

  7. Real-time PCR for rapidly detecting aniline-degrading bacteria in activated sludge.

    Science.gov (United States)

    Kayashima, Takakazu; Suzuki, Hisako; Maeda, Toshinari; Ogawa, Hiroaki I

    2013-05-01

    We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Decomposable quantum-dots/DNA nanosphere for rapid and ultrasensitive detection of extracellular respiring bacteria.

    Science.gov (United States)

    Wen, Junlin; Zhou, Shungui; Yu, Zhen; Chen, Junhua; Yang, Guiqin; Tang, Jia

    2018-02-15

    Extracellular respiring bacteria (ERB) are a group of bacteria capable of transferring electrons to extracellular acceptors and have important application in environmental remediation. In this study, a decomposable quantum-dots (QDs)/DNA nanosphere probe was developed for rapid and ultrasensitive detection of ERB. The QDs/DNA nanosphere was self-assembled from QDs-streptavidin conjugate (QDs-SA) and Y-shaped DNA nanostructure that is constructed based on toehold-mediated strand displacement. It can release numerous fluorescent QDs-SA in immunomagnetic separation (IMS)-based immunoassay via simple biotin displacement, which remarkably amplifies the signal of antigen-antibody recognizing event. This QDs/DNA-nanosphere-based IMS-fluorescent immunoassay is ultrasensitive for model ERB Shewanella oneidensis, showing a wide detection range between 1.0 cfu/mL and 1.0 × 108 cfu/mL with a low detection limit of 1.37 cfu/mL. Moreover, the proposed IMS-fluorescent immunoassay exhibits high specificity, acceptable reproducibility and stability. Furthermore, the proposed method shows acceptable recovery (92.4-101.4%) for detection of S. oneidensis spiked in river water samples. The proposed IMS-fluorescent immunoassay advances an intelligent strategy for rapid and ultrasensitive quantitation of low-abundance analyte and thus holds promising potential in food, medical and environmental applications. Copyright © 2017. Published by Elsevier B.V.

  9. Rapid Detection of Pathogenic Bacteria from Fresh Produce by Filtration and Surface-Enhanced Raman Spectroscopy

    Science.gov (United States)

    Wu, Xiaomeng; Han, Caiqin; Chen, Jing; Huang, Yao-Wen; Zhao, Yiping

    2016-04-01

    The detection of Salmonella Poona from cantaloupe cubes and E. coli O157:H7 from lettuce has been explored by using a filtration method and surface-enhanced Raman spectroscopy (SERS) based on vancomycin-functionalized silver nanorod array substrates. It is found that with a two-step filtration process, the limit of detection (LOD) of Salmonella Poona from cantaloupe cubes can be as low as 100 CFU/mL in less than 4 h, whereas the chlorophyll in the lettuce causes severe SERS spectral interference. To improve the LOD of lettuce, a three-step filtration method with a hydrophobic filter is proposed. The hydrophobic filter can effectively eliminate the interferences from chlorophyll and achieve a LOD of 1000 CFU/mL detection of E. coli O157:H7 from lettuce samples within 5 h. With the low LODs and rapid detection time, the SERS biosensing platform has demonstrated its potential as a rapid, simple, and inexpensive means for pathogenic bacteria detection from fresh produce.

  10. Exploration of Simple Analytical Approaches for Rapid Detection of Pathogenic Bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Salma [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    Many of the current methods for pathogenic bacterial detection require long sample-preparation and analysis time, as well as complex instrumentation. This dissertation explores simple analytical approaches (e.g., flow cytometry and diffuse reflectance spectroscopy) that may be applied towards ideal requirements of a microbial detection system, through method and instrumentation development, and by the creation and characterization of immunosensing platforms. This dissertation is organized into six sections. In the general Introduction section a literature review on several of the key aspects of this work is presented. First, different approaches for detection of pathogenic bacteria will be reviewed, with a comparison of the relative strengths and weaknesses of each approach, A general overview regarding diffuse reflectance spectroscopy is then presented. Next, the structure and function of self-assembled monolayers (SAMs) formed from organosulfur molecules at gold and micrometer and sub-micrometer patterning of biomolecules using SAMs will be discussed. This section is followed by four research chapters, presented as separate manuscripts. Chapter 1 describes the efforts and challenges towards the creation of imunosensing platforms that exploit the flexibility and structural stability of SAMs of thiols at gold. 1H, 1H, 2H, 2H-perfluorodecyl-1-thiol SAM (PFDT) and dithio-bis(succinimidyl propionate)-(DSP)-derived SAMs were used to construct the platform. Chapter 2 describes the characterization of the PFDT- and DSP-derived SAMs, and the architectures formed when it is coupled to antibodies as well as target bacteria. These studies used infrared reflection spectroscopy (IRS), X-ray photoelectron spectroscopy (XPS), and electrochemical quartz crystal microbalance (EQCM), Chapter 3 presents a new sensitive, and portable diffuse reflection based technique for the rapid identification and quantification of pathogenic bacteria. Chapter 4 reports research efforts in the

  11. Gold Nanorod-based Photo-PCR System for One-Step, Rapid Detection of Bacteria.

    Science.gov (United States)

    Kim, Jinjoo; Kim, Hansol; Park, Ji Ho; Jon, Sangyong

    2017-01-01

    The polymerase chain reaction (PCR) has been an essential tool for diagnosis of infectious diseases, but conventional PCR still has some limitations with respect to applications to point-of-care (POC) diagnostic systems that require rapid detection and miniaturization. Here we report a light-based PCR method, termed as photo-PCR, which enables rapid detection of bacteria in a single step. In the photo-PCR system, poly(enthylene glycol)-modified gold nanorods (PEG-GNRs), used as a heat generator, are added into the PCR mixture, which is subsequently periodically irradiated with a 808-nm laser to create thermal cycling. Photo-PCR was able to significantly reduce overall thermal cycling time by integrating bacterial cell lysis and DNA amplification into a single step. Furthermore, when combined with KAPA2G fast polymerase and cooling system, the entire process of bacterial genomic DNA extraction and amplification was further shortened, highlighting the potential of photo-PCR for use in a portable, POC diagnostic system.

  12. Rapid detection and typing of live bacteria from human joint fluid samples by utilizing an integrated microfluidic system.

    Science.gov (United States)

    Chang, Wen-Hsin; Wang, Chih-Hung; Lin, Chih-Lin; Wu, Jiunn-Jong; Lee, Mel S; Lee, Gwo-Bin

    2015-04-15

    Periprosthetic joint infection (PJI) is one of the most dreading complications that hinder the merits of an arthroplasty. A prerequisite for treatment of the above procedure is rapid detection of live bacteria to prevent its recurrence and proper choice of antibiotics. Conventional culture methods are time-consuming and associated with a high false negative rate. Amplification of bacterial genetic materials requires a tedious process but is associated with a high false positive rate. An integrated microfluidic system capable of molecular diagnosis for detecting live bacteria was reported in our previous work. However, the system could not provide detailed information about infectious bacteria for the subsequent antibiotic choices. Furthermore, it took at least 55min to finish the entire process. In this work, a microfluidic platform using ethidium monoazide (EMA) which can only penetrate into dead bacteria is presented for live bacteria detection and typing within a short period of time (30min for the detection of live bacteria and another 40min for the typing of bacteria strains). We tested the proposed system by using human joint fluid samples and found its limit of detection for bacterial detection equal to 10(2)CFU (colony formation unit) for live bacteria detection with gold nanoparticle probes and 10(2)-10(4)CFU for typing bacteria by an on-chip polymerase chain reaction. The whole procedure of the integrated microfluidic system is automated with little human intervention. Moreover, this is the first time that sequential live bacteria detection and typing are demonstrated on the same microfluidic platform. Based on the promising results, the proposed system may become in the near future an auxiliary tool for immediate medical decision and choice of antibiotics in routine arthroplasties or PJI's. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Rapid and high-throughput detection of highly pathogenic bacteria by Ibis PLEX-ID technology.

    Directory of Open Access Journals (Sweden)

    Daniela Jacob

    Full Text Available In this manuscript, we describe the identification of highly pathogenic bacteria using an assay coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS run on an Ibis PLEX-ID high-throughput platform. The biothreat cluster assay identifies most of the potential bioterrorism-relevant microorganisms including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Burkholderia mallei and pseudomallei, Brucella species, and Coxiella burnetii. DNA from 45 different reference materials with different formulations and different concentrations were chosen and sent to a service screening laboratory that uses the PCR/ESI-MS platform to provide a microbial identification service. The standard reference materials were produced out of a repository built up in the framework of the EU funded project "Establishment of Quality Assurances for Detection of Highly Pathogenic Bacteria of Potential Bioterrorism Risk" (EQADeBa. All samples were correctly identified at least to the genus level.

  14. Self-Assembled Biosensors on a Solid Interface for Rapid Detection and Growth Monitoring of Bacteria

    CERN Document Server

    Kinnunen, Paivo; Craig, Elizabeth; Brahmasandra, Sundu; McNaughton, Brandon H

    2012-01-01

    Developing rapid methods for pathogen detection and growth monitoring at low cell and analyte concentrations is an important goal, which numerous technologies are working towards solving. Rapid biosensors have already made a dramatic impact on improving patient outcomes and with continued development, these technologies may also help limit the emergence of antimicrobial resistance and reduce the ever expanding risk of foodborne illnesses. One technology that is being developed with these goals in mind is asynchronous magnetic bead rotation (AMBR) biosensors. Self-assembled AMBR biosensors have been demonstrated at water/air and water/oil interfaces, and here, for the first time, we report on self-assembled AMBR biosensors used at a solid interface. The solid interface configuration was used to measure the growth of Escherichia coli with two distinct phenomena at low cell concentrations: firstly, the AMBR rotational period decreased and secondly, the rotational period increased after several division times. Ta...

  15. Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

    Science.gov (United States)

    Hoefel, Daniel; Monis, Paul T.; Grooby, Warwick L.; Andrews, Stuart; Saint, Christopher P.

    2005-01-01

    Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 105 bacteria ml−1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 103 active AOB ml−1 were detected in the membrane-intact fraction, and 1.40 × 104 active AOB ml−1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml−1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an

  16. Enzyme characteristics of beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria and their interference in rapid methods for detection of waterborne coliforms and Escherichia coli.

    Science.gov (United States)

    Tryland, I; Fiksdal, L

    1998-03-01

    Bacteria which were beta-D-galactosidase and beta-D-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the beta-D-galactosidase-positive nontarget bacteria but none of the beta-D-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5 degrees C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria. The induced and noninduced beta-D-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced beta-D-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean beta-D-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.

  17. Comparison of two rapid biochemical tests and four chromogenic selective media for detection of carbapenemase-producing Gram-negative bacteria.

    Science.gov (United States)

    Hinić, Vladimira; Amrein, Ivo; Stammler, Sabrina; Heckendorn, Judith; Meinel, Dominik; Frei, Reno; Egli, Adrian

    2017-04-01

    We evaluated RAPIDEC® CARBA NP, Neo-Rapid CARB, chromID® CARBA SMART (CARB/OXA), Brilliance™ CRE/ESBL, ChromArt CRE and BBL™ CHROMagar™ CPE for the detection of carbapenemase-producing bacteria. The analytical sensitivity of RAPIDEC® CARBA NP was better than that of Neo-Rapid CARB. A combination of carbapenemase and ESBL screening plates could be advantageous. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Rapid and Sensitive Detection of Bacteria Response to Antibiotics Using Nanoporous Membrane and Graphene Quantum Dot (GQDs-Based Electrochemical Biosensors

    Directory of Open Access Journals (Sweden)

    Weiwei Ye

    2017-05-01

    Full Text Available The wide abuse of antibiotics has accelerated bacterial multiresistance, which means there is a need to develop tools for rapid detection and characterization of bacterial response to antibiotics in the management of infections. In the study, an electrochemical biosensor based on nanoporous alumina membrane and graphene quantum dots (GQDs was developed for bacterial response to antibiotics detection. Anti-Salmonella antibody was conjugated with amino-modified GQDs by glutaraldehyde and immobilized on silanized nanoporous alumina membranes for Salmonella bacteria capture. The impedance signals across nanoporous membranes could monitor the capture of bacteria on nanoporous membranes as well as bacterial response to antibiotics. This nanoporous membrane and GQD-based electrochemical biosensor achieved rapid detection of bacterial response to antibiotics within 30 min, and the detection limit could reach the pM level. It was capable of investigating the response of bacteria exposed to antibiotics much more rapidly and conveniently than traditional tools. The capability of studying the dynamic effects of antibiotics on bacteria has potential applications in the field of monitoring disease therapy, detecting comprehensive food safety hazards and even life in hostile environment.

  19. Rapidly evolving genes in pathogens: methods for detecting positive selection and examples among fungi, bacteria, viruses and protists.

    Science.gov (United States)

    Aguileta, Gabriela; Refrégier, Guislaine; Yockteng, Roxana; Fournier, Elisabeth; Giraud, Tatiana

    2009-07-01

    The ongoing coevolutionary struggle between hosts and pathogens, with hosts evolving to escape pathogen infection and pathogens evolving to escape host defences, can generate an 'arms race', i.e., the occurrence of recurrent selective sweeps that each favours a novel resistance or virulence allele that goes to fixation. Host-pathogen coevolution can alternatively lead to a 'trench warfare', i.e., balancing selection, maintaining certain alleles at loci involved in host-pathogen recognition over long time scales. Recently, technological and methodological progress has enabled detection of footprints of selection directly on genes, which can provide useful insights into the processes of coevolution. This knowledge can also have practical applications, for instance development of vaccines or drugs. Here we review the methods for detecting genes under positive selection using divergence data (i.e., the ratio of nonsynonymous to synonymous substitution rates, d(N)/d(S)). We also review methods for detecting selection using polymorphisms, such as methods based on F(ST) measures, frequency spectrum, linkage disequilibrium and haplotype structure. In the second part, we review examples where targets of selection have been identified in pathogens using these tests. Genes under positive selection in pathogens have mostly been sought among viruses, bacteria and protists, because of their paramount importance for human health. Another focus is on fungal pathogens owing to their agronomic importance. We finally discuss promising directions in pathogen studies, such as detecting selection in non-coding regions.

  20. Multicenter evaluation of the Verigene Gram-negative blood culture nucleic acid test for rapid detection of bacteria and resistance determinants in positive blood cultures.

    Science.gov (United States)

    Uno, Naoki; Suzuki, Hiromichi; Yamakawa, Hiromi; Yamada, Maiko; Yaguchi, Yuji; Notake, Shigeyuki; Tamai, Kiyoko; Yanagisawa, Hideji; Misawa, Shigeki; Yanagihara, Katsunori

    2015-12-01

    The Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN) is a microarray-based assay that enables rapid detection of 9 common Gram-negative bacteria and 6 resistance determinants directly from positive blood cultures. We compared the performance of BC-GN with currently used automated systems, testing 141 clinical blood cultures and 205 spiked blood cultures. For identification of BC-GN target organisms in clinical and spiked blood cultures, the BC-GN assay showed 98.5% (130/132) and 98.9% (182/184) concordance, respectively. Of 140 resistance genes positively detected in clinical and spiked blood cultures with the BC-GN test, 139 (99.3%) were confirmed by PCR, and the detection results were consistent with the resistance phenotypes observed. The BC-GN assay, thus, can potentially improve care for sepsis patients by enabling timely detection and targeted antimicrobial therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Detection of the coliform bacteria Escherichia coli and Salmonella sp. in water by a sensitive and rapid immunomagnetic electrochemiluminescence (ECL) technique

    Science.gov (United States)

    Yu, H.; Bruno, J.

    1995-10-01

    Hemorrhagic Escherichia coli O157:H7 and other fecal coliform bacteria, such as species of Salmonella, could pose a serious health threat in contaminated water resources. Traditional bacterial culture methods and ELISA based assays for identification of fecal coliforms are relatively slow and ambiguous. Polymerase chain reaction of extracted DNA from such bacteria and immunomagnetic separation (IMS) methods appear promising for this application. Although PCR can be a definitive identification technique, it is relatively time consuming when compared to IMS. In this work, the IMS technique has been coupled with an electrochemiluminescence (ECL) technology to separate specific bacteria from their media and quantitatively detect the bacteria within one hour. The sensitivity of the IMS-ECL assay for E.coli O157 strain and Salmonella sp. is as low as 10 - 100 cells/mL in water samples. In addition, IMS was accomplished in dense washings of food and environmental samples followed by ECL assay. These results suggest strongly use of the IMS-ECL methodology for rapid and facile screening of various bacterial contaminations in water resources or other environmental samples for the low level presence of pathogenic coliforms.

  2. Rapid detection of Gram-negative bacteria and their drug resistance genes from positive blood cultures using an automated microarray assay.

    Science.gov (United States)

    Han, Eunhee; Park, Dong-Jin; Kim, Yukyoung; Yu, Jin Kyung; Park, Kang Gyun; Park, Yeon-Joon

    2015-03-01

    We evaluated the performance of the Verigene Gram-negative blood culture (BC-GN) assay (CE-IVD version) for identification of Gram-negative (GN) bacteria and detection of resistance genes. A total of 163 GN organisms (72 characterized strains and 91 clinical isolates from 86 patients) were tested; among the clinical isolates, 86 (94.5%) isolates were included in the BC-GN panel. For identification, the agreement was 98.6% (146/148, 95% confidence interval [CI], 92.1-100) and 70% (7/10, 95% CI, 53.5-100) for monomicrobial and polymicrobial cultures, respectively. Of the 48 resistance genes harbored by 43 characterized strains, all were correctly detected. Of the 19 clinical isolates harboring resistance genes, 1 CTX-M-producing Escherichia coli isolated in polymicrobial culture was not detected. Overall, BC-GN assay provides acceptable accuracy for rapid identification of Gram-negative bacteria and detection of resistance genes, compared with routine laboratory methods despite that it has limitations in the number of genus/species and resistance gene included in the panel and it shows lower sensitivity in polymicrobial cultures. Copyright © 2015. Published by Elsevier Inc.

  3. Bioreporter bacteria for landmine detection

    Energy Technology Data Exchange (ETDEWEB)

    Burlage, R.S. [Oak Ridge National Lab., TN (United States); Youngblood, T. [Frisby Technologies, Aiken, SC (United States); Lamothe, D. [American Technologies, Inc., Huntsville, AL (United States). Ordnance/Explosives Environmental Services Div.

    1998-04-01

    Landmines (and other UXO) gradually leak explosive chemicals into the soil at significant concentrations. Bacteria, which have adapted to scavenge low concentrations of nutrients, can detect these explosive chemicals. Uptake of these chemicals results in the triggering of specific bacterial genes. The authors have created genetically recombinant bioreporter bacteria that detect small concentrations of energetic chemicals. These bacteria are genetically engineered to produce a bioluminescent signal when they contact specific explosives. A gene for a brightly fluorescent compound can be substituted for increased sensitivity. By finding the fluorescent bacteria, you find the landmine. Detection might be accomplished using stand-off illumination of the minefield and GPS technology, which would result in greatly reduced risk to the deminers. Bioreporter technology has been proven at the laboratory scale, and will be tested under field conditions in the near future. They have created a bacterial strain that detects sub-micromolar concentrations of o- and p-nitrotoluene. Related bacterial strains were produced using standard laboratory protocols, and bioreporters of dinitrotoluene and trinitrotoluene were produced, screening for activity with the explosive compounds. Response time is dependent on the growth rate of the bacteria. Although frill signal production may require several hours, the bacteria can be applied over vast areas and scanned quickly, producing an equivalent detection speed that is very fast. This technology may be applicable to other needs, such as locating buried explosives at military and ordnance/explosive manufacturing facilities.

  4. Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures.

    Directory of Open Access Journals (Sweden)

    Masayoshi Tojo

    Full Text Available We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA, an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods, Citrobacter spp. (7/7, Escherichia coli (87/87, Klebsiella oxytoca (13/13, and Proteus spp. (11/11; Enterobacter spp. (29/30; Klebsiella pneumoniae (62/72; Pseudomonas aeruginosa (124/125; and Serratia marcescens (18/21; respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes, including 54 bla(CTX-M, 119 bla(IMP, 8 bla(KPC, 16 bla(NDM, 24 bla(OXA-23, 1 bla(OXA-24/40, 1 bla(OXA-48, 4 bla(OXA-58, and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

  5. Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures.

    Science.gov (United States)

    Tojo, Masayoshi; Fujita, Takahiro; Ainoda, Yusuke; Nagamatsu, Maki; Hayakawa, Kayoko; Mezaki, Kazuhisa; Sakurai, Aki; Masui, Yoshinori; Yazaki, Hirohisa; Takahashi, Hiroshi; Miyoshi-Akiyama, Tohru; Totsuka, Kyoichi; Kirikae, Teruo; Ohmagari, Norio

    2014-01-01

    We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 bla(CTX-M), 119 bla(IMP), 8 bla(KPC), 16 bla(NDM), 24 bla(OXA-23), 1 bla(OXA-24/40), 1 bla(OXA-48), 4 bla(OXA-58), and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

  6. Rapid Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Perlin

    2005-08-14

    Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleic acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development

  7. Development of a multiplexed microsphere PCR for rapid, culture-free detection and Gram-typing of bacteria in human blood samples.

    Science.gov (United States)

    Liang, Fang; Browne, Daniel J; Gray, Megan J; Gartlan, Kate H; Smith, David D; Barnard, Ross; Hill, Geoff R; Corrie, Simon Robert; Markey, Kate

    2018-01-19

    Blood stream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected blood stream infection remain centered around blood culture (highly variable timing, hours to days), and empiric use of broad-spectrum antibiotics is often employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g. combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives), however current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5-hour time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labelled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labelled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

  8. Rapid Detection of Cellular Response to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan R

    2005-01-01

    Our program objective is to develop simple and rapid methods for detecting at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  9. Rapid Detection of Cellular Responses to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan

    2004-01-01

    Our program objective is to develop simple and rapid methods for detecting, at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  10. Rapid Detection of Cellular Responses to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan

    2003-01-01

    Our program objective is to develop simple and rapid methods for detecting, at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  11. Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; PReal-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

  12. Bacteriophage-based nanoprobes for rapid bacteria separation

    Science.gov (United States)

    Chen, Juhong; Duncan, Bradley; Wang, Ziyuan; Wang, Li-Sheng; Rotello, Vincent M.; Nugen, Sam R.

    2015-10-01

    The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes.The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying

  13. A Basic Study to Assess the Potential Usefulness of Resonance Raman Spectroscopy as a Means of Rapidly Detecting and Identifying Bacteria and other Microorganisms.

    Science.gov (United States)

    1985-02-15

    Inc., Metuchen, N.J., we were able to laser illuminate and count two types of bacteria under a microscope . Because it was possible to see the laser...difficult at best for typical organisms. The fluorecence "backgroxun will obliterate most resonance Raman spectra since the resonance Raman spectra tend to

  14. Molecular probe technology detects bacteria without culture

    Directory of Open Access Journals (Sweden)

    Hyman Richard W

    2012-03-01

    Full Text Available Abstract Background Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples. Results While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together. Conclusions Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent.

  15. Rapid separation of very low concentrations of bacteria from blood.

    Science.gov (United States)

    Buchanan, Clara M; Wood, Ryan L; Hoj, Taalin R; Alizadeh, Mahsa; Bledsoe, Colin G; Wood, Madison E; McClellan, Daniel S; Blanco, Rae; Hickey, Caroline L; Ravsten, Tanner V; Husseini, Ghaleb A; Robison, Richard A; Pitt, William G

    2017-08-01

    A rapid and accurate diagnosis of the species and antibiotic resistance of bacteria in septic blood is vital to increase survival rates of patients with bloodstream infections, particularly those with carbapenem-resistant enterobacteriaceae (CRE) infections. The extremely low levels in blood (1 to 100CFU/ml) make rapid diagnosis difficult. In this study, very low concentrations of bacteria (6 to 200CFU/ml) were separated from 7ml of whole blood using rapid sedimentation in a spinning hollow disk that separated plasma from red and white cells, leaving most of the bacteria suspended in the plasma. Following less than a minute of spinning, the disk was slowed, the plasma was recovered, and the bacteria were isolated by vacuum filtration. The filters were grown on nutrient plates to determine the number of bacteria recovered from the blood. Experiments were done without red blood cell (RBC) lysis and with RBC lysis in the recovered plasma. While there was scatter in the data from blood with low bacterial concentrations, the mean average recovery was 69%. The gender of the blood donor made no statistical difference in bacterial recovery. These results show that this rapid technique recovers a significant amount of bacteria from blood containing clinically relevant low levels of bacteria, producing the bacteria in minutes. These bacteria could subsequently be identified by molecular techniques to quickly identify the infectious organism and its resistance profile, thus greatly reducing the time needed to correctly diagnose and treat a blood infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    Directory of Open Access Journals (Sweden)

    Sandra P van Tongeren

    Full Text Available For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

  17. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    Science.gov (United States)

    van Tongeren, Sandra P; Roest, Hendrik I J; Degener, John E; Harmsen, Hermie J M

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

  18. Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

    Science.gov (United States)

    McFeters, G. A.; Pyle, B. H.; Lisle, J. T.; Broadaway, S. C.

    1999-01-01

    Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between

  19. Rapid Separation of Bacteria from Blood—Review and Outlook

    Science.gov (United States)

    Alizadeh, Mahsa; Husseini, Ghaleb A.; McClellan, Daniel S.; Buchanan, Clara M.; Bledsoe, Colin G.; Robison, Richard A.; Blanco, Rae; Roeder, Beverly L.; Melville, Madison; Hunter, Alex K.

    2017-01-01

    The high morbidity and mortality rate of bloodstream infections involving antibiotic-resistant bacteria necessitate a rapid identification of the infectious organism and its resistance profile. Traditional methods based on culturing the blood typically require at least 24 h, and genetic amplification by PCR in the presence of blood components has been problematic. The rapid separation of bacteria from blood would facilitate their genetic identification by PCR or other methods so that the proper antibiotic regimen can quickly be selected for the septic patient. Microfluidic systems that separate bacteria from whole blood have been developed, but these are designed to process only microliter quantities of whole blood or only highly diluted blood. However, symptoms of clinical blood infections can be manifest with bacterial burdens perhaps as low as 10 CFU/mL, and thus milliliter quantities of blood must be processed to collect enough bacteria for reliable genetic analysis. This review considers the advantages and shortcomings of various methods to separate bacteria from blood, with emphasis on techniques that can be done in less than 10 min on milliliter-quantities of whole blood. These techniques include filtration, screening, centrifugation, sedimentation, hydrodynamic focusing, chemical capture on surfaces or beads, field-flow fractionation, and dielectrophoresis. Techniques with the most promise include screening, sedimentation, and magnetic bead capture, as they allow large quantities of blood to be processed quickly. Some microfluidic techniques can be scaled up. PMID:27160415

  20. A new ultrasonic signal amplification method for detection of bacteria

    Science.gov (United States)

    Kant Shukla, Shiva; Resa López, Pablo; Sierra Sánchez, Carlos; Urréjola, José; Segura, Luis Elvira

    2012-10-01

    A new method is presented that increases the sensitivity of ultrasound-based techniques for detection of bacteria. The technique was developed for the detection of catalase-positive microorganisms. It uses a bubble trapping medium containing hydrogen peroxide that is mixed with the sample for microbiological evaluation. The enzyme catalase is present in catalase-positive bacteria, which induces a rapid hydrolysis of hydrogen peroxide, forming bubbles which remain in the medium. This reaction results in the amplification of the mechanical changes that the microorganisms produce in the medium. The effect can be detected by means of ultrasonic wave amplitude continuous measurement since the bubbles increase the ultrasonic attenuation significantly. It is shown that microorganism concentrations of the order of 105 cells ml-1 can be detected using this method. This allows an improvement of three orders of magnitude in the ultrasonic detection threshold of microorganisms in conventional culture media, and is competitive with modern rapid microbiological methods. It can also be used for the characterization of the enzymatic activity.

  1. Using Fluorescent Viruses for Detecting Bacteria in Water

    Science.gov (United States)

    Tabacco, Mary Beth; Qian, Xiaohua; Russo, Jaimie A.

    2009-01-01

    A method of detecting water-borne pathogenic bacteria is based partly on established molecular-recognition and fluorescent-labeling concepts, according to which bacteria of a species of interest are labeled with fluorescent reporter molecules and the bacteria can then be detected by fluorescence spectroscopy. The novelty of the present method lies in the use of bacteriophages (viruses that infect bacteria) to deliver the fluorescent reporter molecules to the bacteria of the species of interest.

  2. Detection of phenols using engineered bacteria

    Science.gov (United States)

    Wise, Arlene A.; Kuske, Cheryl R.; Terwilliger, Thomas C.

    2007-12-04

    Detection of phenols using engineered bacteria. A biosensor can be created by placing a reporter gene under control of an inducible promoter. The reporter gene produces a signal when a cognate transcriptional activator senses the inducing chemical. Creation of bacterial biosensors is currently restricted by limited knowledge of the genetic systems of bacteria that catabolize xenobiotics. By using mutagenic PCR to change the chemical specificity of the Pseudomonas species CF600 DmpR protein, the potential for engineering novel biosensors for detection of phenols has been demonstrated. DmpR, a well-characterized transcriptional activator of the P. CF600's dmp operon mediates growth on simple phenols. Transcription from Po, the promoter heading the dmp operon, is activated when the sensor domain of DmpR interacts with phenol and mono-substituted phenols. By altering the sensor domain of the DmpR, a group of DmpR derivatives that activate transcription of a Po-lacZ fusion in response to eight of the EPA's eleven priority pollutant phenols has been created. The assays and the sensor domain mutations that alter the chemical specificity of DmpR is described.

  3. Nanomaterial-enabled Rapid Detection of Water Contaminants.

    Science.gov (United States)

    Mao, Shun; Chang, Jingbo; Zhou, Guihua; Chen, Junhong

    2015-10-28

    Water contaminants, e.g., inorganic chemicals and microorganisms, are critical metrics for water quality monitoring and have significant impacts on human health and plants/organisms living in water. The scope and focus of this review is nanomaterial-based optical, electronic, and electrochemical sensors for rapid detection of water contaminants, e.g., heavy metals, anions, and bacteria. These contaminants are commonly found in different water systems. The importance of water quality monitoring and control demands significant advancement in the detection of contaminants in water because current sensing technologies for water contaminants have limitations. The advantages of nanomaterial-based sensing technologies are highlighted and recent progress on nanomaterial-based sensors for rapid water contaminant detection is discussed. An outlook for future research into this rapidly growing field is also provided. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Rapid detection of threshold VEPs.

    Science.gov (United States)

    Mackay, Alison M; Bradnam, Michael S; Hamilton, Ruth

    2003-06-01

    To determine whether a one-dimensional (1D) Laplacian analysis detects steady-state visual evoked potentials (ssVEPs) faster than the standard O(z)-F(z) montage and to establish the optimum position of Laplacian reference electrodes. Twenty-two normal adults were shown reversing checks ranging from 1.5' to 60'. Three electrode montages were investigated: O(z)-F(z), LO-F(z) and a 1D Laplacian analysis of 3 occipital electrodes (2O(z)-(RO+LO)). RO and LO were placed symmetrically and horizontally about O(z). Five different locations for RO and LO were investigated. Recordings were analysed in the frequency domain and the presence (and detection time, DT) or absence of a ssVEP defined statistically. Effects of individual, reference electrode site and check size on DT and phase differences between recording montages were investigated. Laplacian analysis detected ssVEPs to small (3') checks faster than O(z)-F(z), by 12.3 and 4.1s on average with Laplacian reference electrodes at 15 and 20% of half-head circumference, respectively. The optimum position of reference electrodes was governed by the instantaneous spatial spread of the response and the noise coherence between midline and lateral electrodes. A 1D Laplacian analysis can reduce the time to statistical detection of ssVEPs compared to the traditional O(z)-F(z) recording for stimuli near the normal acuity threshold of adults. This in turn could be used to minimise the length of a VEP acuity assessment.

  5. Development of omics methodologies for the detection and indetification of pathogenic and spoilage bacteria in seafood

    OpenAIRE

    Böhme, Karola

    2012-01-01

    The project bases on the development of analytical methods for the rapid detection and identification of the main pathogenic and food-borne spoilage microorganisms, present in fish products and products of the aquaculture. Traditionally the methods to identify bacteria were time consuming and tediously. However the application of molecular methods by genomic and proteomic analysis offer an attractive alternative for the rapid and simple identification and characterization of bacteria. The mai...

  6. Optical detection of asymmetric bacteria utilizing electro orientation

    OpenAIRE

    Choi, Jae-Woo; Pu, Allen; Psaltis, Demetri

    2006-01-01

    We propose a bacterial detection scheme which uses no biochemical markers and can be applied in a Point-of-Care setting. The detection scheme aligns asymmetric bacteria with an electric field and detects the optical scattering.

  7. Isolation, amplification and characterization of foodborne pathogen disease bacteria gene for rapid kit test development

    Science.gov (United States)

    Nurjayadi, M.; Santoso, I.; Kartika, I. R.; Kurniadewi, F.; Saamia, V.; Sofihan, W.; Nurkhasanah, D.

    2017-07-01

    There is a lot of public concern over food safety. Food-safety cases recently, including many food poisoning cases in both the developed and developing countries, considered to be the national security threats which involved police investigation. Quick and accurate detection methods are needed to handle the food poisoning cases with a big number of sufferers at the same time. Therefore, the research is aimed to develop a specific, sensitive, and rapid result molecular detection tool for foodborne pathogen bacteria. We, thus, propose genomic level approach with Polymerase Chain Reaction. The research has successfully produced a specific primer to perform amplification to fim-C S. typhi, E. coli, and pef Salmonella typhimurium genes. The electrophoresis result shows that amplification products are 95 base pairs, 121 base pairs, and 139 base pairs; and all three genes are in accordance with the size of the in silico to third genes bacteria. In conclusion, the research has been successfully designed a specific detection tool to three foodborne pathogen bacteria genes. Further stages test and the uses of Real-time PCR in the detection are still in the trial process for better detection method.

  8. Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity

    Science.gov (United States)

    Ning, Xinghai; Lee, Seungjun; Wang, Zhirui; Kim, Dongin; Stubblefield, Bryan; Gilbert, Eric; Murthy, Niren

    2011-08-01

    The diagnosis of bacterial infections remains a major challenge in medicine. Although numerous contrast agents have been developed to image bacteria, their clinical impact has been minimal because they are unable to detect small numbers of bacteria in vivo, and cannot distinguish infections from other pathologies such as cancer and inflammation. Here, we present a family of contrast agents, termed maltodextrin-based imaging probes (MDPs), which can detect bacteria in vivo with a sensitivity two orders of magnitude higher than previously reported, and can detect bacteria using a bacteria-specific mechanism that is independent of host response and secondary pathologies. MDPs are composed of a fluorescent dye conjugated to maltohexaose, and are rapidly internalized through the bacteria-specific maltodextrin transport pathway, endowing the MDPs with a unique combination of high sensitivity and specificity for bacteria. Here, we show that MDPs selectively accumulate within bacteria at millimolar concentrations, and are a thousand-fold more specific for bacteria than mammalian cells. Furthermore, we demonstrate that MDPs can image as few as 105 colony-forming units in vivo and can discriminate between active bacteria and inflammation induced by either lipopolysaccharides or metabolically inactive bacteria.

  9. A simple and rapid method for optical visualization and quantification of bacteria on textiles

    Science.gov (United States)

    Stiefel, Philipp; Schneider, Jana; Amberg, Caroline; Maniura-Weber, Katharina; Ren, Qun

    2016-01-01

    To prevent bacterial contamination on textiles and the associated undesired effects different biocidal coatings have been investigated and applied. However, due to health and environmental concerns anti-adhesive coatings preventing the binding of bacteria would be favored. To develop such anti-adhesive coatings simple assays for reliable and fast screening are beneficial. Here an easy-to-handle, robust and rapid assay to assess bacteria on textiles utilizing a tetrazolium salt was reported. The assay allowed direct eye visualization of the color change of the textiles containing bacteria, facilitating fast screening. Quantification of the adhered bacteria could be done by generating standard curves which correlate the staining intensity to cell numbers. An additional advantage of the described assay is that with the same detection method anti-adhesive and biocidal effects can be investigated. The method was applied to different coatings, using Pseudomonas aeruginosa and Staphylococcus aureus as model organisms. The detection limit was found to be between 2.5 * 106 and 9.4 * 108 for P. aeruginosa and between 1 * 106 and 3.3 * 108 for S. aureus. The anti-adhesive coating PLUMA was demonstrated to reduce bacterial adhesion without killing them, whereas the biocidal coating TH22-27 caused a clear reduction in the number of viable cells. PMID:28004762

  10. Method of Detecting Coliform Bacteria and Escherichia Coli Bacteria from Reflected Light

    Science.gov (United States)

    Vincent, Robert (Inventor)

    2013-01-01

    The present invention relates to a method of detecting coliform bacteria in water from reflected light and a method of detecting Eschericha Coli bacteria in water from reflected light, and also includes devices for the measurement, calculation and transmission of data relating to that method.

  11. Rapid diagnosis of tuberculosis. Detection of drug resistance mechanisms.

    Science.gov (United States)

    Viñuelas-Bayón, Jesús; Vitoria, María Asunción; Samper, Sofía

    2017-10-01

    Tuberculosis is still a serious public health problem, with 10.8 million new cases and 1.8 million deaths worldwide in 2015. The diversity among members of the Mycobacterium tuberculosis complex, the causal agent of tuberculosis, is conducive to the design of different methods for rapid diagnosis. Mutations in the genes involved in resistance mechanisms enable the bacteria to elude the treatment. We have reviewed the methods for the rapid diagnosis of M. tuberculosis complex and the detection of susceptibility to drugs, both of which are necessary to prevent the onset of new resistance and to establish early, appropriate treatment. Copyright © 2017 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  12. Indigenous people's detection of rapid ecological change.

    Science.gov (United States)

    Aswani, Shankar; Lauer, Matthew

    2014-06-01

    When sudden catastrophic events occur, it becomes critical for coastal communities to detect and respond to environmental transformations because failure to do so may undermine overall ecosystem resilience and threaten people's livelihoods. We therefore asked how capable of detecting rapid ecological change following massive environmental disruptions local, indigenous people are. We assessed the direction and periodicity of experimental learning of people in the Western Solomon Islands after a tsunami in 2007. We compared the results of marine science surveys with local ecological knowledge of the benthos across 3 affected villages and 3 periods before and after the tsunami. We sought to determine how people recognize biophysical changes in the environment before and after catastrophic events such as earthquakes and tsunamis and whether people have the ability to detect ecological changes over short time scales or need longer time scales to recognize changes. Indigenous people were able to detect changes in the benthos over time. Detection levels differed between marine science surveys and local ecological knowledge sources over time, but overall patterns of statistically significant detection of change were evident for various habitats. Our findings have implications for marine conservation, coastal management policies, and disaster-relief efforts because when people are able to detect ecological changes, this, in turn, affects how they exploit and manage their marine resources. © 2014 Society for Conservation Biology.

  13. Method of Detecting Coliform Bacteria from Reflected Light

    Science.gov (United States)

    Vincent, Robert K. (Inventor)

    2014-01-01

    The present invention relates to a method of detecting coliform bacteria in water from reflected light, and also includes devices for the measurement, calculation and transmission of data relating to that method.

  14. Species-specific detection of hydrocarbon-utilizing bacteria.

    Science.gov (United States)

    Wilson, V L; Tatford, B C; Yin, X; Rajki, S C; Walsh, M M; LaRock, P

    1999-12-01

    Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of developing rapid tests for hydrocarbon-degrading bacteria, species-specific PCR primer sets have been developed for Pseudomonas aeruginosa, Stentrophomonas (Xanthomonas) maltophilia, and Serratia marsescens. Highly variable regions of the 16S rRNA gene were used to design these primer sets. The amplification products of these primer sets have been verified and validated with hemi-nested PCR and with ligase chain reaction (LCR) techniques, and have been applied to the analyses of environmental water samples. These species-specific primer sets were also chosen to amplify in conjunction with a universal set of PCR primers chosen from highly conserved neighboring sequences in the same gene. These multiplex or competitive PCR procedures enable testing with an internal marker and/or the quantitative estimation of the relative proportion of the microbial community that any one of these species occupies. In addition, this universal PCR primer set amplified the same size amplicon from a wide spectrum of procaryotic and eucaryotic organisms and may have potential in earth biota analyses.

  15. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    Science.gov (United States)

    Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

    2015-05-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

  16. DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

    Directory of Open Access Journals (Sweden)

    Kenjiro Nagamine

    2015-01-01

    Full Text Available Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani , and Staphylococcus aureus , which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

  17. Rapid Detection of the Varicella Zoster Virus

    Science.gov (United States)

    Lewis, Michelle P.; Harding, Robert

    2011-01-01

    1.Technology Description-Researchers discovered that when the Varicella Zoster Virus (VZV) reactivates from latency in the body, the virus is consistently present in saliva before the appearance of skin lesions. A small saliva sample is mixed with a specialized reagent in a test kit. If the virus is present in the saliva sample, the mixture turns a red color. The sensitivity and specificity emanates from an antibody-antigen reaction. This technology is a rapid, non-invasive, point of-of-care testing kit for detecting the virus from a saliva sample. The device is easy to use and can be used in clinics and in remote locations to quickly detect VZV and begin treatment with antiviral drugs. 2.Market Opportunity- RST Bioscience will be the first and only company to market a rapid, same day test kit for the detection of VZV in saliva. The RST detection test kit will have several advantages over existing, competitive technology. The test kit is self contained and laboratory equipment is not required for analysis of the sample. Only a single saliva sample is required to be taken instead of blood or cerebral spinal fluid. The test kit is portable, sterile and disposable after use. RST detection test kits require no electrical power or expensive storage equipment and can be used in remote locations. 3.Market Analysis- According to the CDC, it is estimated that 1 million cases of shingles occur each year in the U.S. with more than half over the age of sixty. There is a high demand for rapid diagnostics by the public. The point-of-care testing (POCT) market is growing faster than other segments of in vitro diagnostics. According to a July 2007 InteLab Corporation industry report the overall market for POCT was forecast to increase from $10.3 billion in 2005 to $18.7 billion by 2011. The market value of this test kit has not been determined. 4.Competition- The VZV vaccine prevents 50% of cases and reduces neuralgia by 66%. The most popular test detects VZV-specific IgM antibody

  18. HB&L System: rapid determination of antibiotic sensitivity of bacteria isolated from blood cultures.

    Directory of Open Access Journals (Sweden)

    Simone Barocci

    2010-03-01

    Full Text Available Introduction. Blood culture is an important method to detect microbial pathogens on blood, very useful for diagnosing bacterial infections. Unfortunately, classical diagnostic protocols cannot directly identify bacteria responsible for sepsis and accordingly their antimicrobial profiles. This problem causes a delay of almost two days in the availability of a specific antimicrobial profile. Objective. Among the main causes of death, sepsis have a relevant importance. For this reason it is important both to identify pathogens and to perform an antimicrobial susceptibility test in the shortest time as possible. For this purpose, the main aim of this study is the evaluation of the performances of an antimicrobial susceptibility determination directly performed on positive blood cultures. Materials and methods. This study has been performed on 70 positive blood cultures, during the period from January to July 2009. A number of 35 blood cultures were positive for Gram negative bacteria, and 35 were positive for Gram positive bacteria. From these positive blood cultures, after a short sample preparation, it has been possible to directly determine antimicrobial susceptibility profiles by using the HB&L (formerly URO-QUICK instrument. Results. The HB&L system results showed a very good correlation with both the classical disk diffusion method and VITEK 2 automatic system.The performances between the methods carried out in this study were equivalent. Conclusions. From data reported, thanks to the rapidity and simplicity of the method used, we can assert that the direct susceptibility test available with the HB&L system, is useful for a rapid and early choice of the antibiotic treatment.

  19. Rapid genetic detection of ingested Amanita phalloides.

    Science.gov (United States)

    Gausterer, Christian; Penker, Martina; Krisai-Greilhuber, Irmgard; Stein, Christina; Stimpfl, Thomas

    2014-03-01

    Mushrooms are often poorly digested by humans. Thus, their remains (tissues, spores) may persist in the gastrointestinal tract and can be detected in feces several days after mushroom consumption. In this report, we present protocols for the rapid PCR-based detection of fungal traces in a variety of complex samples. Novel primers were designed to amplify portions of ribosomal DNA from deadly poisonous European members of the genus Amanita, namely the death cap (A. phalloides), the destroying angel (A. virosa) and the fool's mushroom (A. verna), respectively. Assay sensitivity was sufficient to discover diluted DNA traces in amounts below the genomic content of a single target mushroom cell. Specificity testing was performed with DNA extracts from a variety of mushroom species. Template amplification was exclusively observed with intended targets and it was not compromised by a vast excess of non-target DNA (i.e. DNA from human and human fecal origin, respectively). A series of experiments was conducted with prepared specimens in order to follow the course of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fried and digested mixed mushrooms. To improve assay performance with fecal samples, a rapid protocol for sample pre-processing (including water-ether sedimentation and bead beating) and a modified PCR reaction mix were applied. Thereby, it was possible to detect the presence of A. phalloides DNA in spiked feces as well as in clinical samples (vomit, stool) from two independent cases of suspected mushroom poisoning. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. pH-switchable bacteria detection using zwitterionic fluorescent polymer.

    Science.gov (United States)

    Khoerunnisa; Mazrad, Zihnil A I; In, Insik; Park, Sung Young

    2017-04-15

    A zwitterionic fluorescent polymer with high sensitivity to pH changes was constructed for the detection and imaging of both gram-positive and gram-negative pathogenic bacteria. A detection probe using the zwitterionic fluorescent polymer was synthesized with single boron dipyrromethane (BODIPY) as a hydrophobic dye and bromoethane as a cationic group for bacteria binding with conjugated poly(sulfobetaine methacrylate) (BOD/BE-PSM). The zwitterionic fluorescent polymer bound to bacteria through ionic complexes between anionic groups on the bacterial surface and cationic BOD/BE-PSM groups after 1h incubation. This finding demonstrated that the fluorescence on/off system operated via changes in the hydrophilic and hydrophobic nature of the zwitterionic fluorescent polymer, depending on the pH (6.0, 7.4, or 9.0), at a fixed 1mg/mL polymer concentration. The system showed good stability with a limit of detection of 1mg/mL. Quenching caused by interactions with the hydrophobic BODIPY dye was also observed, enabling bacteria detection, as shown by fluorescence spectroscopy and confocal microscopy images. Our results indicated that the zwitterionic fluorescent polymer could be used to detect bacteria over a wide range of pH values. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Multi-scale magnetic nanoparticle based optomagnetic bioassay for sensitive DNA and bacteria detection

    DEFF Research Database (Denmark)

    Tian, Bo; Zardán Gómez De La Torre, Teresa; Donolato, Marco

    2016-01-01

    Benefiting from their rapid readout, highly flexible devices and low-cost portable systems, optomagnetic biosensors have drawn increased attention in recent years as bioassay technologies for small molecules, biomarkers, DNA, and bacteria. Herein, an optomagnetic bioassay strategy suitable...... for point-of-care diagnostics, utilizing functionalized magnetic nanoparticles (100 nm) with Brownian relaxation behavior is optimized in order to obtain higher detection sensitivity for DNA molecules and bacteria. Presence of target DNA sequences or bacteria changes the dynamic behavior of the magnetic...... to previous work, we systematically optimize the concentration of 100 nm magnetic nanoparticles to increase the assay sensitivity and lower the limit of detection. To enable biplex detection, we perform this optimization in the presence of larger 250 nm magnetic nanoparticles that do not interact...

  2. Universal primers for rapid detection of hytrosaviruses.

    Science.gov (United States)

    Abd-Alla, Adly M M; Salem, Tamer Z; Parker, Andrew G; Wang, Yongjie; Jehle, Johannes A; Vreysen, Marc J B; Boucias, Drion

    2011-01-01

    Hytrosaviridae is a proposed virus family encompassing viruses that cause salivary gland hypertrophy (SGH) syndrome in infected insects and reduce the fertility in their dipteran insect hosts. They contain a large, double stranded DNA genome of 120-190 kbp. To date, these viruses have been detected only in adult Diptera. These include hytrosaviruses detected in various tsetse fly species (Glossina spp.), the narcissus bulb fly Merodon equestris and the house fly Musca domestica. The limited number of hytrosaviruses reported to date may be a reflection of the frequent absence of external symptoms in infected adult flies and the fact that the virus does not cause rapid mortality. Based on the complete genome sequence of Glossinia pallidipes (GpSGHV) and Musca domestica (MdSGHV) salivary gland hypertrophy viruses, a PCR based methodology was developed to detect the viruses in these species. To be able to detect hytrosaviruses in other Diptera, five degenerate primer pairs were designed and tested on GpSGHV and MdSGHV DNA using gradient PCR with annealing temperatures from 37 to 61°C. Two pairs of primers were selected from p74, two pairs from PIF-1 and one pair from ODV-e66 homologous proteins. Four primer pairs generated a virus specific PCR product on both MdSGHV and GpSGHV at all tested annealing temperatures, while the ODV-e66 based primers did not generate a virus specific product with annealing temperatures higher that 47°C. No non-specific PCR product was found when using genomic DNA of infected flies as template DNA. These results offer new sets of primers that could be used to detect hytrosaviruses in other insects. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. Nanostructured bioluminescent sensor for rapidly detecting thrombin.

    Science.gov (United States)

    Chen, Longyan; Bao, Yige; Denstedt, John; Zhang, Jin

    2016-03-15

    Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470 nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10 min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8 nM to 8 μM in both buffer and human urine spiked samples. The limit of detection is as low as 80 pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the international space station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    NARCIS (Netherlands)

    Tongeren, van S.P.; Roest, H.I.J.; Degener, J.E.; Harmsen, H.J.M.

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order

  5. Bacillus anthracis-Like Bacteria and Other B. cereus Group Members in a Microbial Community Within the International Space Station : A Challenge for Rapid and Easy Molecular Detection of Virulent B. anthracis

    NARCIS (Netherlands)

    van Tongeren, Sandra P.; Roest, Hendrik I. J.; Degener, John E.; Harmsen, Hermie J. M.

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order

  6. Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns.

    Science.gov (United States)

    Correia Carreira, S; Spencer, J; Schwarzacher, W; Seddon, A M

    2016-06-15

    In order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, with S. aureus being immobilized to a greater extent than E. coli Finally, low numbers of magnetically labeled E. coli bacteria (Gram-negative bacteria. Antimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved within 15 min. This approach could be extended to encompass the capture and concentration of specific pathogens, for example, by functionalizing magnetic nanoparticles with antibodies or small molecule probes. Copyright © 2016 Correia Carreira et al.

  7. Method Designed To Detect Alginate-Degrading Bacteria

    OpenAIRE

    Kitamikado, Manabu; Yamaguchi, Kuniko; Tseng, Chao-Huang; Okabe, Bun'Ichi

    1990-01-01

    A simple turbidimetric method was developed to detect alginate degradation. Bacteria were grown in alginate-containing media, and culture fluids were mixed with an acidic albumin solution. Failure to develop a white turbidity indicated an alginate degrader. The method showed alginate degradation by Vibrio alginolyticus ATCC 17749, in contrast to prior descriptions.

  8. Bioluminescence-based system for rapid detection of natural transformation.

    Science.gov (United States)

    Santala, Ville; Karp, Matti; Santala, Suvi

    2016-07-01

    Horizontal gene transfer plays a significant role in bacterial evolution and has major clinical importance. Thus, it is vital to understand the mechanisms and kinetics of genetic transformations. Natural transformation is the driving mechanism for horizontal gene transfer in diverse genera of bacteria. Our study introduces a simple and rapid method for the investigation of natural transformation. This highly sensitive system allows the detection of a transformation event directly from a bacterial population without any separation step or selection of cells. The system is based on the bacterial luciferase operon from Photorhabdus luminescens The studied molecular tools consist of the functional modules luxCDE and luxAB, which involve a replicative plasmid and an integrative gene cassette. A well-established host for bacterial genetic investigations, Acinetobacter baylyi ADP1, is used as the model bacterium. We show that natural transformation followed by homologous recombination or plasmid recircularization can be readily detected in both actively growing and static biofilm-like cultures, including very rare transformation events. The system allows the detection of natural transformation within 1 h of introducing sample DNA into the culture. The introduced method provides a convenient means to study the kinetics of natural transformation under variable conditions and perturbations. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Detection of beer spoilage bacteria Megasphaera and Pectinatus by polymerase chain reaction and colorimetric microplate hybridization.

    Science.gov (United States)

    Satokari, R; Juvonen, R; Mallison, K; von Wright, A; Haikara, A

    1998-12-08

    Anaerobic bacteria of the genera Megasphaera and Pectinatus cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. A polymerase chain reaction (PCR) method and a colorimetric microplate hybridization assay were developed for the rapid and specific detection of Megasphaera cerevisiae and Pectinatus spp. A biotinylated primer pair was designed for the amplification of a 403 base pair (bp) fragment of the M. cerevisiae 16S rRNA gene and a primer pair from literature was used for the amplification of an 816 bp fragment of Pectinatus 16S rRNA gene. Amplified PCR products were analyzed by the colorimetric microplate hybridization method in which a biotinylated PCR product was captured by streptavidin and hybridized with a digoxigenin-labelled oligonucleotide probe. In the final step an enzyme-linked antibody and a colorimetric reaction were utilized. A simple and rapid sample treatment was set up for the PCR detection of contaminants in beer. Detection of M. cerevisiae (> or = 5 x 10(3) colony forming units [cfu]/100 ml) and Pectinatus frisingensis (> or = 5 x 10(5) cfu/100 ml) in beer was successful, but the sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

  10. Enhanced Microbial Detection Capabilities by a Rapid Portable Instrument

    Science.gov (United States)

    Morris, Heather; Monaco, Lisa; Wainwright, Norm; Steele, Andrew; Damon, Michael; Schenk, Alison; Stimpson, Eric; Maule, Jake; Effinger, Michael

    2010-01-01

    We present data describing a progression of continuing technology development - from expanding the detection capabilities of the current PTS unit to re-outfitting the instrument with a protein microarray increasing the number of detectable compounds. To illustrate the adaptability of the cartridge format, on-orbit operations data from the ISS demonstrate the detection of the fungal cell wall compound beta-glucan using applicable LOCAD-PTS cartridges. LOCAD-PTS is a handheld device consisting of a spectrophotometer, an onboard pumping mechanism, and data storage capabilities. A suite of interchangeable cartridges lined with four distinct capillaries allow a hydrated sample to mix with necessary reagents in the channels before being pumped to the optical well for spectrophotometric analysis. The reagents housed in one type of cartridge trigger a reaction based on the Limulus Amebocyte Lysate (LAL) assay, which results in the release of paranitroaniline dye. The dye is measured using a 395 nm filter. The LAL assay detects the Gram-negative bacterial cell wall molecule, endotoxin or lipopolysaccharide (LPS). The more dye released, the greater the concentration of endotoxin in the sample. Sampling, quantitative analysis, and data retrieval require less than 20 minutes. This is significantly faster than standard culture-based methods, which require at least a 24 hour incubation period.Using modified cartridges, we demonstrate the detection of Gram negative bacteria with protein microarray technology. Additionally, we provide data from multiple field tests where both standard and advanced PTS technologies were used. These tests investigate the transfer of target microbial molecules from one surface to another. Collectively, these data demonstrate that the new cartridges expand the number of compounds detected by LOCAD-PTS, while maintaining the rapid, in situ analysis characteristic of the instrument. The unit provides relevant data for verifying sterile sample collection

  11. Rapid, in situ detection of Agrobacterium tumefaciens attachment to leaf tissue.

    Science.gov (United States)

    Simmons, Christopher W; Nitin, N; Vandergheynst, Jean S

    2012-01-01

    Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging-based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum-infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post-infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  12. Rapid methods: the detection of foodborne pathogens

    NARCIS (Netherlands)

    Beumer, R.R.; Hazeleger, W.C.

    2009-01-01

    Although bacteria are the first type of microorganisms that come to mind when discussing microbial food safety, they are by no means the only pathogenic foodborne microorganisms. Mycotoxin producing moulds, human enteric viruses, protozoan parasites and marine biotoxins are also of importance.

  13. Detection of Foodborne Pathogenic Bacteria using Bacteriophage Tail Spike Proteins

    Science.gov (United States)

    Poshtiban, Somayyeh

    Foodborne infections are worldwide health problem with tremendous social and financial impacts. Efforts are focused on developing accurate and reliable technologies for detection of food contaminations in early stages preferably on-site. This thesis focuses on interfacing engineering and biology by combining phage receptor binding proteins (RBPs) with engineered platforms including microresonator-based biosensors, magnetic particles and polymerase chain reaction (PCR) to develop bacterial detection sensors. We used phage RBPs as target specific bioreceptors to develop an enhanced microresonator array for bacterial detection. These resonator beams are optimized to feature a high natural frequency while offer large surface area for capture of bacteria. Theoretical analysis indicates a high mass sensitivity with a threshold for the detection of a single bacterial cell. We used phage RBPs as target specific bioreceptors, and successfully demonstrated the application of these phage RBB-immobilized arrays for specific detection of C. jejuni cells. We also developed a RBP-derivatized magnetic pre-enrichment method as an upstream sample preparation method to improve sensitivity and specificity of PCR for detection of bacterial cells in various food samples. The combination of RBP-based magnetic separation and real-time PCR allowed the detection of small number of bacteria in artificially contaminated food samples without any need for time consuming pre-enrichment step through culturing. We also looked into integration of the RBP-based magnetic separation with PCR onto a single microfluidic lab-on-a-chip to reduce the overall turnaround time.

  14. Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry

    Science.gov (United States)

    He, Shengbin; Hong, Xinyi; Huang, Tianxun; Zhang, Wenqiang; Zhou, Yingxing; Wu, Lina; Yan, Xiaomei

    2017-06-01

    A laboratory-built high-sensitivity flow cytometer (HSFCM) was employed for the rapid and accurate detection of lactic acid bacteria (LAB) and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the HSFCM. Ultra-high temperature processing milk and skim milk spiked with Lactobacillus casei were used as the model systems for the optimization of sample pretreatment and staining. The viable LAB counts measured by the HSFCM were in good agreement with those of the plate count method, and the measured ratios between the live and dead LAB matched well with the theoretical ratios. The established method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands. Moreover, the concentration and viability status of LAB in ambient yogurt, a relatively new yet popular milk product in China, are also reported.

  15. Label and label-free based surface-enhanced Raman scattering for pathogen bacteria detection: A review.

    Science.gov (United States)

    Liu, Yu; Zhou, Haibo; Hu, Ziwei; Yu, Guangxia; Yang, Danting; Zhao, Jinshun

    2017-08-15

    Rapid, accurate detection of pathogen bacteria is a highly topical research area for the sake of food safety and public health. Surface-enhanced Raman scattering (SERS) is being considered as a powerful and attractive technique for pathogen bacteria detection, due to its sensitivity, high speed, comparatively low cost, multiplexing ability and portability. This contribution aims to give a comprehensive overview of SERS as a technique for rapid detection of pathogen bacteria based on label and label-free strategies. A brief tutorial on SERS is given first of all. Then we summarize the recent trends and developments of label and label-free based SERS applied to detection of pathogen bacteria, including the relatively complete interpretation of SERS spectra. In addition, multifunctional SERS platforms for pathogen bacteria in matrix are discussed as well. Furthermore, an outlook of the work done and a perspective on the future directions of SERS as a reliable tool for real-time pathogen bacteria detection are given. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. [Detection of enteric pathogenic bacteria from surface waters by quantitative polymerase chain reaction (QPCR)].

    Science.gov (United States)

    Liu, Yong-Jun; Zhang, Chong-Miao; Wang, Xiao-Chang; Lü, Ying-Jun; Zuo, Li-Li

    2008-05-01

    A rapid quantitative polymerase chain reaction (QPCR) analysis method with universal primers was developed to detect cell densities of the enteric pathogenic bacteria from 5 surface water of Xi'an City for 4 months continuously. And the detection results by QPCR method were compared with counts of coliforms colony-forming units (CFU) determined by membrane filter (MF) analysis. The results showed that QPCR method had an estimated 94% confidence, and detection limit was 2.7 Escherichia coli cells per sample in undiluted DNA extracts. For five surface waters (N = 60), the geometric mean of pathogenic bacteria concentration determined by QPCR was 2.2-5 times of corresponding coliform CFU determined by MF analysis. Using QPCR analysis, these geometric means of pathogenic bacteria concentration ranged from 25 CCE/100 mL to 67 000 CCE/100 mL. Using MF culture analysis, coliforms ranged from 3 CFU/100 mL to 45 000 CFU/100 mL. Regression analysis showed that there was a significant positive correlation between pathogenic bacteria determined by QPCR method and coliforms determined by MF method, the correlation coefficient (r) was 0.983.

  17. Detection of sulfonamide resistant bacteria and resistance genes in soils

    Science.gov (United States)

    Wu, Nan; Zhang, Weiyu; Liu, Huifen; Wang, Xiaobo; Yang, Fan; Zeng, Ming; Chen, Pinpin; Wang, Xiao

    2017-04-01

    Manure application could accelerate the environmental dissemination of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in soils. In this study, the prevalence of sulfonamide resistant bacteria and resistance genes was investigated in agricultural soils to which organic manures had been applied in Tianjin, China. Anti-sulfonamide bacteria were found in the range of 3.29 × 104 to 1.70 × 105 CFU/g dry soil, occupying 1.5% to 2.2% of total viable counts. And sulI and sulII genes were detected in all sampling sites, with relative abundances of 5.69 × 10-5 to 6.95 × 10-4 and 4.28 × 10-4 to 1.25 × 10-3 respectively. No significant correlations between cultivable sulfonamide resistant bacteria and sul genes were found in this study. While sulI showed significant positive correlation with soil organic matter. Overall, the results highlight that soil plays an important role in resistance genes capture as the environmental reservoir.

  18. Evaluating the use of dedicated swab for rapid antigen detection ...

    African Journals Online (AJOL)

    Background: Group A streptococcus (GAS) is the most common and fearful bacterial cause in pediatric acute pharyngitis due to its serious complications. Several generations of rapid antigen detection tests (RADTs) have been developed to facilitate rapid detection of GAS pharyngitis. We assessed the value of using a ...

  19. Rapid assessment of assignments using plagiarism detection software.

    Science.gov (United States)

    Bischoff, Whitney R; Abrego, Patricia C

    2011-01-01

    Faculty members most often use plagiarism detection software to detect portions of students' written work that have been copied and/or not attributed to their authors. The rise in plagiarism has led to a parallel rise in software products designed to detect plagiarism. Some of these products are configurable for rapid assessment and teaching, as well as for plagiarism detection.

  20. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    Directory of Open Access Journals (Sweden)

    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  1. PCR detection of groundwater bacteria associated with colloidal transport

    Energy Technology Data Exchange (ETDEWEB)

    Cruz-Perez, P.; Stetzenbach, L.D.; Alvarez, A.J.

    1996-02-29

    Colloidal transport may increase the amount of contaminant material than that which could be transported by water flow alone. The role of colloids in groundwater contaminant transport is complicated and may involve many different processes, including sorption of elements onto colloidal particles, coagulation/dissolution, adsorption onto solid surfaces, filtration, and migration. Bacteria are known to concentrate minerals and influence the transport of compounds in aqueous environments and may also serve as organic colloids, thereby influencing subsurface transport of radionuclides and other contaminants. The initial phase of the project consisted of assembling a list of bacteria capable of sequestering or facilitating mineral transport. The development and optimization of the PCR amplification assay for the detection of the organisms of interest, and the examination of regional groundwaters for those organisms, are presented for subsequent research.

  2. Detection and categorization of bacteria habitats using shallow linguistic analysis.

    Science.gov (United States)

    Karadeniz, İlknur; Özgür, Arzucan

    2015-01-01

    Information regarding bacteria biotopes is important for several research areas including health sciences, microbiology, and food processing and preservation. One of the challenges for scientists in these domains is the huge amount of information buried in the text of electronic resources. Developing methods to automatically extract bacteria habitat relations from the text of these electronic resources is crucial for facilitating research in these areas. We introduce a linguistically motivated rule-based approach for recognizing and normalizing names of bacteria habitats in biomedical text by using an ontology. Our approach is based on the shallow syntactic analysis of the text that include sentence segmentation, part-of-speech (POS) tagging, partial parsing, and lemmatization. In addition, we propose two methods for identifying bacteria habitat localization relations. The underlying assumption for the first method is that discourse changes with a new paragraph. Therefore, it operates on a paragraph-basis. The second method performs a more fine-grained analysis of the text and operates on a sentence-basis. We also develop a novel anaphora resolution method for bacteria coreferences and incorporate it with the sentence-based relation extraction approach. We participated in the Bacteria Biotope (BB) Task of the BioNLP Shared Task 2013. Our system (Boun) achieved the second best performance with 68% Slot Error Rate (SER) in Sub-task 1 (Entity Detection and Categorization), and ranked third with an F-score of 27% in Sub-task 2 (Localization Event Extraction). This paper reports the system that is implemented for the shared task, including the novel methods developed and the improvements obtained after the official evaluation. The extensions include the expansion of the OntoBiotope ontology using the training set for Sub-task 1, and the novel sentence-based relation extraction method incorporated with anaphora resolution for Sub-task 2. These extensions resulted in

  3. Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring.

    Science.gov (United States)

    Salma, Mohammad; Rousseaux, Sandrine; Sequeira-Le Grand, Anabelle; Alexandre, Hervé

    2013-01-01

    In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10(6) CFU ml(-1)) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R (2) ≥ 0.97, white wine R (2) ≥ 0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine (R (2) ≥ 0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of wine and applied for microbiological quality control in wineries.

  4. Microconductometric immunosensor for label-free and sensitive detection of Gram-negative bacteria.

    Science.gov (United States)

    El Ichi, Sarra; Leon, Fanny; Vossier, Ludivine; Marchandin, Helene; Errachid, Abdelhamid; Coste, Joliette; Jaffrezic-Renault, Nicole; Fournier-Wirth, Chantal

    2014-04-15

    Blood safety is a global health goal. In developed countries, bacterial contamination of platelet concentrates is the highest infectious risk in transfusion despite the current preventive strategies. We aimed to develop a conductometric biosensor for the generic, rapid and sensitive detection of Gram-negative bacteria. Our strategy is based on immunosensors: addressable magnetic nanoparticles coupled with anti-LPS antibodies were used for the generic capture of Gram-negative bacteria. Bacterial capture was characterized by impedancemetric and microscopic measurements. The results obtained with conductometric measurements allowed real-time, sensitive detection of Escherichia coli or Serratia marcescens cultures from 1 to 10(3) CFU mL(-1). The ability of the immunosensor to detect Gram negative bacteria was also tested on clinically relevant strains. The conductometric immunosensor allowed the direct detection of 10-10(3) CFU mL(-1) of Pseudomonas aeruginosa and Acinetobacter baumannii strains that were undetectable using standard immunoblot methods. Results showed that the conductometric response was not inhibited in 1% serum. © 2013 Published by Elsevier B.V.

  5. Rapid detection of Mycobacterium avium subsp. paratuberculosis ...

    African Journals Online (AJOL)

    Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, a suspect causative agent of Crohns disease in man, is an emerging disease of international proportions affecting all ruminants. Early stage detection of Mycobacterium avium subsp. paratuberculosis infection would accelerate progress in control ...

  6. RAPID DETECTION OF MICROBIAL CONTAMINATION IN GHANA ...

    African Journals Online (AJOL)

    2014-06-01

    Jun 1, 2014 ... pathogens in herbal medicines from Ghana. Methods: We employed different DNA extraction ... kits yielded significant amounts of DNA. PCR was able to detect pathogens present in the samples directly. ..... safety of dried spices and herbs from production and retail premises in the United Kingdom. Food.

  7. The Rapid Detection of Single Bacterial Cells by Deep UV Micro Raman Spectroscopy.

    Science.gov (United States)

    1992-04-01

    developed for the purpose of rapid bacterial detection. Techniques include mass spectroscopy and its various combinations with chromatography and pyrolysis...Methods: Chromatography and Mass Spectroscopy", Plenum Press, N.Y. 1990. 6. P.J.H. Jackman in "Methods in Microbiology", Vol. 19, eds., R.R., Colwell and R...4847198 issued July 11, 1989. 5. "Ultraviolet Resonance Raman Spectra of Bacteria, Bacterial Spores, Protoplasts and Calcium Dipicolinate", R

  8. Evaluation of the Rapidec Carba NP Test Kit for Detection of Carbapenemase-Producing Gram-Negative Bacteria.

    Science.gov (United States)

    Garg, Atul; Garg, Jaya; Upadhyay, G C; Agarwal, Anurag; Bhattacharjee, Amitabha

    2015-12-01

    Recently, bioMérieux, France, introduced the Rapidec Carba NP test kit for rapid detection of carbapenemase-producing Gram-negative bacteria. This kit was evaluated in this study, and we report sensitivity, specificity, and positive and negative predictive values of 92.6%, 96.2%, 95.83%, and 92.6%, respectively. The test was easy to perform and interpret and relatively inexpensive ($5/Rs 300 per test) and provides a practical solution for early detection of carbapenemase-producing, multidrug-resistant Gram-negative bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates

    Science.gov (United States)

    Sarantopoulos, Christos; Ongchangco, Deryn; Sry, Jeremy; Cesario, Thomas

    2014-01-01

    The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance. PMID:25519429

  10. Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates.

    Science.gov (United States)

    Berger, Martina; Sarantopoulos, Christos; Ongchangco, Deryn; Sry, Jeremy; Cesario, Thomas

    2015-07-01

    The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance. © 2014 by the Society for Experimental Biology and Medicine.

  11. Reliable, rapid and simple voltammetric detection of urea nitrate explosive.

    Science.gov (United States)

    Cagan, Avi; Lu, Donglai; Cizek, Karel; La Belle, Jeff; Wang, Joseph

    2008-05-01

    A highly selective and rapid electrochemical assay of the improvised explosive urea nitrate (UN) is reported. The method involves a short ( approximately 10 s) acid-catalyzed reaction of UN with 4-nitrotoluene (NT) followed by a rapid ( approximately 2 s) square-wave voltammetric (SWV) detection of the 2,4-dinitrotoluene (DNT) product. The new protocol offers great promise for a reliable field detection of UN, with significant advantages of speed, sensitivity, portability, simplicity, and cost.

  12. Rapid method for detection of salmonella in meat

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a rapid method for the detection of Salmonella in meat as well as to a kit for performing said method. The method provides a time-to-result of less than 8 hours.......The present invention relates to a rapid method for the detection of Salmonella in meat as well as to a kit for performing said method. The method provides a time-to-result of less than 8 hours....

  13. A bacteriophage endolysin-based electrochemical impedance biosensor for the rapid detection of Listeria cells.

    Science.gov (United States)

    Tolba, Mona; Ahmed, Minhaz Uddin; Tlili, Chaker; Eichenseher, Fritz; Loessner, Martin J; Zourob, Mohammed

    2012-12-21

    The objective of this study was to develop a biosensor using the cell wall binding domain (CBD) of bacteriophage-encoded peptidoglycan hydrolases (endolysin) immobilized on a gold screen printed electrode (SPE) and subsequent electrochemical impedance spectroscopy (EIS) for a rapid and specific detection of Listeria cells. The endolysin was amine-coupled to SPEs using EDC/NHS chemistry. The CBD-based electrode was used to capture and detect the Listeria innocua serovar 6b from pure culture and 2% artificially contaminated milk. In our study, the endolysin functionalized SPEs have been characterized using X-ray photoelectron spectroscopy (XPS). The integration of endolysin-based recognition for specific bacteria and EIS can be used for direct and rapid detection of Listeria cells with high specificity against non-Listeria cells with a limit of detection of 1.1 × 10(4) and 10(5) CFU mL(-1) in pure culture and 2% milk, respectively.

  14. Sulfate reducing bacteria detection in gas pipelines; Deteccao de bacterias redutoras de sulfato em gasodutos

    Energy Technology Data Exchange (ETDEWEB)

    Lutterbach, Marcia Teresa S.; Oliveira, Ana Lucia C. de; Cavalcanti, Eduardo H. de S. [Instituto Nacional de Tecnologia (INT), Rio de Janeiro, RJ (Brazil). Div. de Corrosao e Degradacao]. E-mails: marciasl@int.gov.br; analucia@int.gov.br; eduardoh@int.gov.br

    2004-07-01

    Microbiology induced corrosion (MIC) process associated with sulfate reducing bacteria (BRS) are one of the most important matter of concern for the oil and gas industry as 77% of failures have been attributed this sort of degradation. Corrosion products found present in gas transportation pipelines, the so-called 'black-powder' problem, are also a nuisance and source of economic losses for the gas industry. According to the literature, the incidence of black-powder can be ascribed to the metabolism of BRS that can be found in the gas environment. Integrity monitoring programs of gas pipelines adopt pigging as an important tool for internal corrosion monitoring. Solid residue such as the black-powder, collected by pigging, as well as the condensed, can be seen as a very valuable samples for microbiological analyses that can be used to detect and quantify bacteria related to the incidence of MIC processes. In the present work results concerning samples collected by pigging and condensed are presented. Small populations of viable BRS have been found in the pipeline. It can be seen that the inclusion of microbiological analyses of solid and liquid residues as a complementary action in the integrity monitoring programs adopted by gas transportation industry can be very helpful on the decision making concerning preventive and corrective actions to be taken in order to maintain the CIM processes under control. (author)

  15. Freeze-dried recombinant bacteria for on-site detection of phenolic compounds by color change.

    Science.gov (United States)

    Shin, Hae Ja; Park, Hoo Hwi; Lim, Woon Ki

    2005-09-22

    We herein report the development of a recombinant bacterial biosensor for the rapid and easy detection of phenolic compounds in the field. A plasmid was designed to encode a beta-galactosidase reporter gene under the control of capR, an activator involved in phenolic compound degradation. The construct was transformed into Escherichia coli, and transformed cells were stored after being freeze-dried in the presence of sucrose. For detection of phenolic compounds, the cells were rehydrated, and used instantly, without any growth step. In the presence of 0.1 microM-10mM phenol, we observed a red color from hydrolysis of chlorophenol red beta-D-galactopyranoside (CPRG) or an indigo color from hydrolysis of X-galactopyranoside (X-gal). Other phenolic compounds could be detected by this system, including catechol, 2-methylphenol, 2-chlorophenol, 3-methylphenol, 2-nitrophenol, and 4-chlorophenol. These results suggest that this novel bacteria biosensor may be useful for easy, on-site detection of phenolic compounds without the need for unwieldy equipment or sample pretreatment. Indeed, biosensor systems involving beta-galactosidase-expressing freeze-dried recombinant bacteria could prove useful for the in situ detection of many more compounds in the future.

  16. A rapid Salmonella detection method involving thermophilic helicase-dependent amplification and a lateral flow assay.

    Science.gov (United States)

    Du, Xin-Jun; Zhou, Tian-Jiao; Li, Ping; Wang, Shuo

    2017-08-01

    Salmonella is a major foodborne pathogen that is widespread in the environment and can cause serious human and animal disease. Since conventional culture methods to detect Salmonella are time-consuming and laborious, rapid and accurate techniques to detect this pathogen are critically important for food safety and diagnosing foodborne illness. In this study, we developed a rapid, simple and portable Salmonella detection strategy that combines thermophilic helicase-dependent amplification (tHDA) with a lateral flow assay to provide a detection result based on visual signals within 90 min. Performance analyses indicated that the method had detection limits for DNA and pure cultured bacteria of 73.4-80.7 fg and 35-40 CFU, respectively. Specificity analyses showed no cross reactions with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Enterobacter aerogenes, Shigella and Campylobacter jejuni. The results for detection in real food samples showed that 1.3-1.9 CFU/g or 1.3-1.9 CFU/mL of Salmonella in contaminated chicken products and infant nutritional cereal could be detected after 2 h of enrichment. The same amount of Salmonella in contaminated milk could be detected after 4 h of enrichment. This tHDA-strip can be used for the rapid detection of Salmonella in food samples and is particularly suitable for use in areas with limited equipment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device

    Directory of Open Access Journals (Sweden)

    Shunsuke Furutani

    2016-01-01

    Full Text Available Rapid detection of food-borne pathogens is essential to public health and the food industry. Although the conventional culture method is highly sensitive, it takes at least a few days to detect food-borne pathogens. Even though polymerase chain reaction (PCR can detect food-borne pathogens in a few hours, it is more expensive and unsatisfactorily sensitive relative to the culture method. We have developed a method to rapidly detect Salmonella enterica by using a compact disc (CD-shaped device that can reduce reagent consumption in conventional PCR. The detection method, which combines culture and PCR, is more rapid than the conventional culture method and is more sensitive and cheaper than PCR. In this study, we also examined a sample preparation method that involved collecting bacterial cells from food. The bacteria collected from chicken meat spiked with S. enterica were mixed with PCR reagents, and PCR was performed on the device. At a low concentration of S. enterica, the collected S. enterica was cultured before PCR for sensitive detection. After cultivation for 4 h, S. enterica at 1.7 × 104 colony-forming units (CFUs·g−1 was detected within 8 h, which included the time needed for sample preparation and detection. Furthermore, the detection of 30 CFUs·g−1 of S. enterica was possible within 12 h including 8 h for cultivation.

  18. FT-IR microspectroscopy in rapid identification of bacteria in pure and mixed culture

    Science.gov (United States)

    Fontoura, Inglid; Belo, Ricardo; Sakane, Kumiko; Cardoso, Maria Angélica Gargione; Khouri, Sônia; Uehara, Mituo; Raniero, Leandro; Martin, Airton A.

    2010-02-01

    In recent years FT-IR microspectroscopy has been developed for microbiology analysis and applied successfully in pure cultures of microorganisms to rapidly identify strains of bacteria, yeasts and fungi. The investigation and characterization of microorganism mixed cultures is also of growing importance, especially in hospitals where it is common to poly-microbial infections. In this work, the rapid identification of bacteria in pure and mixed cultures was studied. The bacteria were obtained from the Institute Oswaldo Cruz culture collection at Brazil. Escherichia coli ATCC 10799 and Staphylococcus aureus ATCC 14456 were analyzed, 3 inoculations were examined in triplicate: Escherichia coli, Staphylococcus aureus and a mixed culture of them. The inoculations were prepared according to McFarland 0.5, incubated at 37 ° C for 6 hours, diluted in saline, placed in the CaF2 window and store for one hour at 50°C to obtain thin film. The measurement was performed by Spectrum Spotlight 400 (Perkin-Elmer) equipment in the range of 4000-900 cm-1, with 32 scans using a transmittance technique with point and image modes. The data were processed (baseline, normalization, calculation of first derivate followed by smoothing with 9 point using a Savitzky-Golay algorithm) and a cluster analysis were done by Ward's algorithm and an excellent discrimination between pure and mixed culture was obtained. Our preliminary results indicate that the FT-IR microspectroscopy associated with cluster analysis can be used to discriminate between pure and mixed culture.

  19. Office Paper Platform for Bioelectrochromic Detection of Electrochemically Active Bacteria using Tungsten Trioxide Nanoprobes

    Science.gov (United States)

    Marques, A. C.; Santos, L.; Costa, M. N.; Dantas, J. M.; Duarte, P.; Gonçalves, A.; Martins, R.; Salgueiro, C. A.; Fortunato, E.

    2015-01-01

    Electrochemically active bacteria (EAB) have the capability to transfer electrons to cell exterior, a feature that is currently explored for important applications in bioremediation and biotechnology fields. However, the number of isolated and characterized EAB species is still very limited regarding their abundance in nature. Colorimetric detection has emerged recently as an attractive mean for fast identification and characterization of analytes based on the use of electrochromic materials. In this work, WO3 nanoparticles were synthesized by microwave assisted hydrothermal synthesis and used to impregnate non-treated regular office paper substrates. This allowed the production of a paper-based colorimetric sensor able to detect EAB in a simple, rapid, reliable, inexpensive and eco-friendly method. The developed platform was then tested with Geobacter sulfurreducens, as a proof of concept. G. sulfurreducens cells were detected at latent phase with an RGB ratio of 1.10 ± 0.04, and a response time of two hours. PMID:25891213

  20. Rapid prototyping and parametric optimization of plastic acoustofluidic devices for blood-bacteria separation.

    Science.gov (United States)

    Silva, R; Dow, P; Dubay, R; Lissandrello, C; Holder, J; Densmore, D; Fiering, J

    2017-09-01

    Acoustic manipulation has emerged as a versatile method for microfluidic separation and concentration of particles and cells. Most recent demonstrations of the technology use piezoelectric actuators to excite resonant modes in silicon or glass microchannels. Here, we focus on acoustic manipulation in disposable, plastic microchannels in order to enable a low-cost processing tool for point-of-care diagnostics. Unfortunately, the performance of resonant acoustofluidic devices in plastic is hampered by a lack of a predictive model. In this paper, we build and test a plastic blood-bacteria separation device informed by a design of experiments approach, parametric rapid prototyping, and screening by image-processing. We demonstrate that the new device geometry can separate bacteria from blood while operating at 275% greater flow rate as well as reduce the power requirement by 82%, while maintaining equivalent separation performance and resolution when compared to the previously published plastic acoustofluidic separation device.

  1. Rapid point-of-care concentration of bacteria in a disposable microfluidic device using meniscus dragging effect.

    Science.gov (United States)

    Zhang, Jane Yuqian; Do, Jaephil; Premasiri, W Ranjith; Ziegler, Lawrence D; Klapperich, Catherine M

    2010-12-07

    We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from liquid samples using enhanced evaporation targeted at downstream detection using surface enhanced Raman spectroscopy (SERS). The device is composed of a poly(dimethylsiloxane) (PDMS) liquid sample flow layer, a reusable metal airflow layer, and a porous PTFE (Teflon™) membrane sandwiched in between the liquid and air layers. The concentration capacity of the device was successfully demonstrated with fluorescently tagged Escherichia coli (E. coli). The recovery concentration was above 85% for all initial concentrations lower than 1 × 10(4) CFU mL(-1). In the lowest initial concentration cases, 100 µL initial volumes of bacteria solution at 100 CFU mL(-1) were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min. Subsequent tests with SERS on clinically relevant Methicillin-Sensitive Staphylococcus aureus (MSSA) after concentration in this device proved more than 100-fold enhancement in SERS signal intensity compared to the signal obtained from the unconcentrated sample. The concentration device is straightforward to design and use, and as such could be used in conjunction with a number of detection technologies.

  2. Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

    Science.gov (United States)

    Barnini, Simona; Ghelardi, Emilia; Brucculeri, Veronica; Morici, Paola; Lupetti, Antonella

    2015-06-18

    Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

  3. Rapid detection of EBOLA VP40 in microchip immunofiltration assay

    Science.gov (United States)

    Miethe, Peter; Gary, Dominik; Hlawatsch, Nadine; Gad, Anne-Marie

    2015-05-01

    In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.

  4. Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens

    Directory of Open Access Journals (Sweden)

    Bukola Rhoda Aremu

    2015-09-01

    Full Text Available Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI. These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation.

  5. Rapid colorimetric sensing platform for the detection of Listeria monocytogenes foodborne pathogen.

    Science.gov (United States)

    Alhogail, Sahar; Suaifan, Ghadeer A R Y; Zourob, Mohammed

    2016-12-15

    Listeria monocytogenes is a serious cause of human foodborne infections worldwide, which needs spending billions of dollars for inspection of bacterial contamination in food every year. Therefore, there is an urgent need for rapid, in-field and cost effective detection techniques. In this study, rapid, low-cost and simple colorimetric assay was developed using magnetic nanoparticles for the detection of listeria bacteria. The protease from the listeria bacteria was detected using D-amino acid substrate. D-amino acid substrate was linked to the carboxylic acid on the magnetic nanoparticles using EDC/NHS chemistry. The cysteine residue at the C-terminal of the substrate was used for the self-assembled monolayer formation on the gold sensor surface, which in turn the black magnetic nanobeads will mask the golden color. The color will change from black to golden color upon the cleavage of the specific peptide sequence by the Listeria protease. The sensor was tested with serial dilutions of Listeria bacteria. It was found that the appearance of the gold surface area is proportional to the bacterial concentrations in CFU/ml. The lowest detection limit of the developed sensor for Listeria was found to be 2.17×10(2) colony forming unit/ml (CFU/ml). The specificity of the biosensor was tested against four different foodborne associated bacteria (Escherichia coli, Salmonella, Shigella flexnerii and Staphylococcus aureus). Finally, the sensor was tested with artificially spiked whole milk and ground meat spiked with listeria. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens

    DEFF Research Database (Denmark)

    This book provides a new focus on the rapid detection of food–borne pathogens, employing food production chains as a starting point instead of a specific method or various detection technologies. This reference is organized by production chains or contamination scenarios, and offers a unique...... in implementation. It also provides guidelines for faster, more user–friendly, and cost–effective enumeration of pathogens....

  7. A rapid ultrasound particle agglutination method for HIV antibody detection: Comparison with conventional rapid HIV tests.

    Science.gov (United States)

    Bystryak, Simon; Ossina, Natalya

    2017-11-01

    We present the results of the feasibility and preliminary studies on analytical performance of a rapid test for detection of human immunodeficiency virus (HIV) antibodies in human serum or plasma that is an important advance in detecting HIV infection. Current methods for rapid testing of antibodies against HIV are qualitative and exhibit poor sensitivity (limit of detection). In this paper, we describe an ultrasound particle agglutination (UPA) method that leads to a significant increase of the sensitivity of conventional latex agglutination tests for HIV antibody detection in human serum or plasma. The UPA method is based on the use of: 1) a dual mode ultrasound, wherein a first single-frequency mode is used to accelerate the latex agglutination process, and then a second swept-frequency mode of sonication is used to disintegrate non-specifically bound aggregates; and 2) a numerical assessment of results of the agglutination process. The numerical assessment is carried out by optical detection and analysis of moving patterns in the resonator cell during the swept-frequency mode. The single-step UPA method is rapid and more sensitive than the three commercial rapid HIV test kits analyzed in the study: analytical sensitivity of the new UPA method was found to be 510-, 115-, and 80-fold higher than that for Capillus™, Multispot™ and Uni-Gold™ Recombigen HIV antibody rapid test kits, respectively. The newly developed UPA method opens up additional possibilities for detection of a number of clinically significant markers in point-of-care settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Detection of carotenoids in psychrotrophic bacteria by spectroscopic approach

    Directory of Open Access Journals (Sweden)

    Kirti Kushwaha

    2014-12-01

    Full Text Available The combination of Raman and Infrared spectroscopic signatures were used to find the different vibrational modes of individual carotenoid as their spectral fingerprint. Both have been previously demonstrated to be highly useful methodology for the identification and/or typing of microorganisms. In this study, we set out to evaluate whether these technologies could be applied to detect the presence of carotenoids in psychrotrophic bacterial isolates. FTIR and Raman spectra of four psychrotrophic bacteria viz. Kocuria rosea, K. turfanensis, Sanguibacter suarezii and Planococcus maritimus were examined during the investigation. FTIR spectra bands at 1653-1661cm-1 in different samples were assigned as part of chlorophyll, 1424-1426 cm-1 as -C-H- (CH2 bending vibration from methylene of carotenoids or lycopene, 1366-1367 cm-1 band as the -ionone ring of β-carotene due to the C-H, (–CH3 symmetrical bending. Interestingly, Raman spectra revealed intense Raman bands in the range of 1511-1530, 1153-1159 and 1003-1010 cm-1 representing bacterial carotenoids. We hypothesize the biosynthesis of carotenoid as adaptive strategy to cope up inhospitable cold environments of Leh and Ladakh. The strong, scattering bands by different isolates attributable to ν(C=C phase stretching, ν(C-C and δ(C-CH3 methyl components systems, which could be probably membrane-associated C50 carotenoids. Their high intensities are due to resonance enhancement. It can be concluded that Raman spectroscopy is a sensitive and convenient detection tool for typing of the bacterial biomarkers with less time consumption.

  9. Trichomonas spp. in pigeons: detection by OSOM Trichomonas Rapid Test.

    Science.gov (United States)

    Valek, Petr; Kunca, Tomas; Langrova, Iva; Hartlova, Helena; Brozova, Adela; Jankovska, Ivana; Kudrnacova, Marie; Sloup, Vladislav

    2013-12-01

    The efficacy of the OSOM Trichomonas Rapid Test (developed for rapid diagnosis of human Trichomonas vaginalis) in detection of Trichomonas spp. in pigeons (Columba livia) was investigated. Two oral cavity swabs were taken from 50 farm pigeons. Cultivation in Diamond Trichomonas medium was used as a reference method. According to a morphological determination, Trichomonas gallinae was the only protozoan found; however, no further molecular analysis was conducted. The OSOM Trichomonas test was positive in 39 oral swabs. In comparison with the cultivation method three samples were identified as false negative and one as false positive. Test specificity and sensitivity were established as 93% and 90%, respectively. Using Cohen's Kappa, the concordance between the two testing methods was found to be strong (kappa = 0.7506, 95% CI = 0.5162-0.9850). The OSOM Trichomonas test is not able to distinguish between Trichomonas species; however, results suggest that the test is suitable for the rapid detection of Trichomonas spp. infection in pigeons.

  10. Palm kernel agar: An alternative culture medium for rapid detection ...

    African Journals Online (AJOL)

    Palm kernel agar: An alternative culture medium for rapid detection of aflatoxins in agricultural commodities. ... a pink background and blue or blue green fluorescence of palm kernel agar Under long wave UV light (366nm) as against the white background of DCA, which often interferes with fluorescence with corresponding ...

  11. Rapid detection of methicillin-resistant staphylococci by multiplex PCR

    African Journals Online (AJOL)

    A rapid and sensitive method for excluding the presence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples was developed. The combination of MRSA detection by mecA coaA PCR with prior enrichment in selective broth was tested for 300 swabs. PCR identified 26 MRSApositive samples, ...

  12. Evaluation of a direct colorimetric assay for rapid detection of ...

    African Journals Online (AJOL)

    Yemane Berhane

    bromide (MTT) for a rapid detection of rifampicin resistance. Methods: Sputum was inoculated directly into 7H9 .... a loopful of the corresponding broth on nutrient agar and incubating it at 370C for 24 hours before performing the .... and Training in Tropical Diseases (TDR). This study was part of the MSc thesis of DW at Addis ...

  13. A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples

    Directory of Open Access Journals (Sweden)

    Jiyun Li

    2017-10-01

    Full Text Available The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 102 cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.

  14. Detecting bacteria and Determining Their Susceptibility to Antibiotics by Stochastic Confinement in Nanoliter Droplets using Plug-Based Microfluidics

    Energy Technology Data Exchange (ETDEWEB)

    Boedicker, J.; Li, L; Kline, T; Ismagilov, R

    2008-01-01

    This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminating the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as stochastic confinement. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.

  15. Radiometric method for the rapid detection of Leptospira organisms

    Energy Technology Data Exchange (ETDEWEB)

    Manca, N.; Verardi, R.; Colombrita, D.; Ravizzola, G.; Savoldi, E.; Turano, A.

    1986-02-01

    A rapid and sensitive radiometric method for detection of Leptospira interrogans serovar pomona and Leptospira interrogans serovar copenhageni is described. Stuart's medium and Middlebrook TB (12A) medium supplemented with bovine serum albumin, catalase, and casein hydrolysate and labeled with /sup 14/C-fatty acids were used. The radioactivity was measured in a BACTEC 460. With this system, Leptospira organisms were detected in human blood in 2 to 5 days, a notably shorter time period than that required for the majority of detection techniques.

  16. Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Jiang, Dongneng; Pu, Xiaoyun; Wu, Jiehong; Li, Meng; Liu, Ping

    2013-01-01

    Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

  17. 9 CFR 113.25 - Culture media for detection of bacteria and fungi.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Culture media for detection of bacteria and fungi. 113.25 Section 113.25 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Standard Procedures § 113.25 Culture media for detection of bacteria and fungi. (a...

  18. Dead/alive bacteria detection using an all-fibre optical system

    Science.gov (United States)

    Bogomolny, E.; Swift, S.; Cheng, M.; van Binsbergen, S.; Vanholsbeeck, F.

    2014-03-01

    Accurate monitoring of microbial viability plays an essential role in pharmacodynamic studies such as in estimating the efficiency of antimicrobial agents. Traditionally, bacterial viability is determined by their ability to form colonies on solid growth medium or to proliferate in liquid nutrient broths but, with these culture-based methods, the live bacterial population can only be estimated retrospectively. To address this challenge, we have employed differential fluorescence staining and an all-fiber optical system developed by our group. The detection is based on the collection of the fluorescence from commercial dyes that produce a substantially increased signal upon binding with bacterial nucleic acids. The dyes allow discrimination between alive and dead cells through differential membrane permeability and fluorescence wavelength. The respective fluorescence signal is correlated to the number of bacterial cells present in the sample. Our setup uses DPSS lasers and a sensitive CCD-based spectrometer over the 400-800 nm wavelength range. A laser shutter allows the sample exposure time and acquisition time to be synchronized to minimize the effect of photobleaching. As a model, bacteria (Escherichia coli or Staphylococcus aureus) killed with isopropyl alcohol were mixed with live cells at different ratios. The population ratios of alive and dead cells were accurately quantified by our optical setup providing a rapid method for the estimation of bactericidal treatments. In summary, our optical system may offer a robust, accurate and fast alternative for detection of dead/alive bacteria in turbid solution opening the new avenues for pharmacodynamic studies.

  19. An FPGA-based rapid wheezing detection system.

    Science.gov (United States)

    Lin, Bor-Shing; Yen, Tian-Shiue

    2014-01-29

    Wheezing is often treated as a crucial indicator in the diagnosis of obstructive pulmonary diseases. A rapid wheezing detection system may help physicians to monitor patients over the long-term. In this study, a portable wheezing detection system based on a field-programmable gate array (FPGA) is proposed. This system accelerates wheezing detection, and can be used as either a single-process system, or as an integrated part of another biomedical signal detection system. The system segments sound signals into 2-second units. A short-time Fourier transform was used to determine the relationship between the time and frequency components of wheezing sound data. A spectrogram was processed using 2D bilateral filtering, edge detection, multithreshold image segmentation, morphological image processing, and image labeling, to extract wheezing features according to computerized respiratory sound analysis (CORSA) standards. These features were then used to train the support vector machine (SVM) and build the classification models. The trained model was used to analyze sound data to detect wheezing. The system runs on a Xilinx Virtex-6 FPGA ML605 platform. The experimental results revealed that the system offered excellent wheezing recognition performance (0.912). The detection process can be used with a clock frequency of 51.97 MHz, and is able to perform rapid wheezing classification.

  20. Rapid identification of bio-molecules applied for detection of biosecurity agents using rolling circle amplification.

    Directory of Open Access Journals (Sweden)

    Jenny Göransson

    Full Text Available Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification. The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans and spores (Bacillus atrophaeus released in field.

  1. Rapid determination of the toxicity of quantum dots with luminous bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Wang Lingling [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, No. 2 Tiansheng Road, Beibei District, Chongqing 400715 (China); Zheng Huzhi, E-mail: zhenghz@swu.edu.cn [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, No. 2 Tiansheng Road, Beibei District, Chongqing 400715 (China); Long Yijuan; Gao Mei; Hao Jianyu; Du Juan; Mao Xiaojiao; Zhou Dongbo [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, No. 2 Tiansheng Road, Beibei District, Chongqing 400715 (China)

    2010-05-15

    In this paper, a novel method so-called bioluminescence inhibition assay with luminous bacteria (Photobacterium phosphoreum) was introduced to evaluate the toxicity of quantum dots. The bioassay was based on measuring the decrease of the light emitted by luminous bacteria. With obvious advantages of simplicity, rapidity and sensitivity, it can dramatically improve the efficiency of probing the toxicity of QDs. Based on this method, we systemically explored the effect of the composition and surface modification on QDs' toxicity. The experiment of composition effect was performed using three kinds of QDs, namely CdSe, CdTe and ZnS-AgInS{sub 2} QDs with the same stabilizer - dihydrolipoic acid. As for the effect of different stabilizers, mercaptoacetic acid, L-cysteine and dihydrolipoic acid stabilized CdSe were researched, respectively. Our results demonstrated that both the composition and surface modification were the important factors affecting the toxicity of QDs. In addition, a concentration dependence of toxicity was also found.

  2. Use of predictive models and rapid methods to nowcast bacteria levels at coastal beaches

    Science.gov (United States)

    Francy, Donna S.

    2009-01-01

    The need for rapid assessments of recreational water quality to better protect public health is well accepted throughout the research and regulatory communities. Rapid analytical methods, such as quantitative polymerase chain reaction (qPCR) and immunomagnetic separation/adenosine triphosphate (ATP) analysis, are being tested but are not yet ready for widespread use.Another solution is the use of predictive models, wherein variable(s) that are easily and quickly measured are surrogates for concentrations of fecal-indicator bacteria. Rainfall-based alerts, the simplest type of model, have been used by several communities for a number of years. Deterministic models use mathematical representations of the processes that affect bacteria concentrations; this type of model is being used for beach-closure decisions at one location in the USA. Multivariable statistical models are being developed and tested in many areas of the USA; however, they are only used in three areas of the Great Lakes to aid in notifications of beach advisories or closings. These “operational” statistical models can result in more accurate assessments of recreational water quality than use of the previous day's Escherichia coli (E. coli)concentration as determined by traditional culture methods. The Ohio Nowcast, at Huntington Beach, Bay Village, Ohio, is described in this paper as an example of an operational statistical model. Because predictive modeling is a dynamic process, water-resource managers continue to collect additional data to improve the predictive ability of the nowcast and expand the nowcast to other Ohio beaches and a recreational river. Although predictive models have been shown to work well at some beaches and are becoming more widely accepted, implementation in many areas is limited by funding, lack of coordinated technical leadership, and lack of supporting epidemiological data.

  3. Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment.

    Science.gov (United States)

    Josefsen, M H; Löfström, C; Hansen, T B; Christensen, L S; Olsen, J E; Hoorfar, J

    2010-08-01

    A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C(T)) values (R(2) = 0.993), with a quantification range of 1 x 10(2) to 1 x 10(7) CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R(2) = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.

  4. Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

    Directory of Open Access Journals (Sweden)

    Sascha D Braun

    Full Text Available Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL and narrow spectrum (NSBL beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13, blaGIM (2/2, blaKPC (27/27, blaNDM (5/5, blaIMP-2/4/7/8/13/14/15/16/31 (10/10, blaOXA-23 (12/13, blaOXA-40-group (7/7, blaOXA-48-group (32/33, blaOXA-51 (1/1 and blaOXA-58 (1/1. Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16, blaOXA-2 (4/4, blaOXA-9 (33/33, OXA-10 (3/3, blaOXA-51 (25/25, blaOXA-58 (2/2, CTX-M1/M15 (17/17 and blaVIM (1/1]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4% isolates, including Acinetobacter baumannii (28/28, Enterobacter spec. (5/5, Escherichia coli (4/4, Klebsiella pneumoniae (62/63, Klebsiella oxytoca (0/2, Pseudomonas aeruginosa (12/12, Citrobacter freundii (1/1 and Citrobacter

  5. Simultaneous detection of pathogenic bacteria using agglutination test based on colored silica nanoparticles.

    Science.gov (United States)

    Yu, Hui; Zhao, Guangying; Dou, Wenchao

    2015-01-01

    Aimed to explore an agglutination test which can simultaneously detect two pathogenic bacteria, an agglutination test based on colored silica nanoparticles (colored-SiNps) was established in this work. Monodisperse colored-SiNps were used as agglutination test carriers; red-SiNps and blue-SiNps were prepared by reverse microemulsion with C.I. Reactive red 136 and C.I. Reactive Blue 14. Then the red-SiNps were sensitized with antibodies against E. sakazaki and denoted as IgG-red-SiNps; The blue-SiNps were coated with antibodies against S. pullorum and S. Gallinarum and denoted as IgGblue- SiNps. The mixture solution of IgG-red-SiNps and IgG-blue-SiNps could simultaneously agglutinate with E. sakazakii and S. pullorum and S. gallinarum on glass slide. The E. sakazakii and S. pullorum and S. gallinarum could be simultaneously detected by agglutination test with obvious agglutination phenomena. The E. sakazakii and S. pullorum and S. gallinarum could both be detected in a range from 4×10(3) to 4×10(9) CFU/mL. The pullorum and S. gallinarum and E. sakazakii in the infected food sample were detected by mixture solution of IgG-red-SiNps and IgG-blue-SiNps too. This agglutination test was easy and rapid, it might be useful for in situ rapid detection method for simultaneously screening different pathogenic microorganisms of foods and feeds in the field.

  6. Individual differences in detecting rapidly presented fearful faces.

    Directory of Open Access Journals (Sweden)

    Dandan Zhang

    Full Text Available Rapid detection of evolutionarily relevant threats (e.g., fearful faces is important for human survival. The ability to rapidly detect fearful faces exhibits high variability across individuals. The present study aimed to investigate the relationship between behavioral detection ability and brain activity, using both event-related potential (ERP and event-related oscillation (ERO measurements. Faces with fearful or neutral facial expressions were presented for 17 ms or 200 ms in a backward masking paradigm. Forty-two participants were required to discriminate facial expressions of the masked faces. The behavioral sensitivity index d' showed that the detection ability to rapidly presented and masked fearful faces varied across participants. The ANOVA analyses showed that the facial expression, hemisphere, and presentation duration affected the grand-mean ERP (N1, P1, and N170 and ERO (below 20 Hz and lasted from 100 ms to 250 ms post-stimulus, mainly in theta band brain activity. More importantly, the overall detection ability of 42 subjects was significantly correlated with the emotion effect (i.e., fearful vs. neutral on ERP (r = 0.403 and ERO (r = 0.552 measurements. A higher d' value was corresponding to a larger size of the emotional effect (i.e., fearful--neutral of N170 amplitude and a larger size of the emotional effect of the specific ERO spectral power at the right hemisphere. The present results suggested a close link between behavioral detection ability and the N170 amplitude as well as the ERO spectral power below 20 Hz in individuals. The emotional effect size between fearful and neutral faces in brain activity may reflect the level of conscious awareness of fearful faces.

  7. Rapid Visual Tests: Fast and Reliable Detection of Ochratoxin A

    Directory of Open Access Journals (Sweden)

    Miguel Lopez-Ferber

    2010-08-01

    Full Text Available This paper reviews the early detection strategies that have been employed for the rapid monitoring of ochratoxin A (OTA contamination of food. OTA, a mycotoxin mainly produced by some Aspergillus and Penicillium species, is found in cereals, coffee, wine, pork and grapes. To minimize the entry of this mycotoxin into the food chain, rapid diagnostic tools are required. To this end, the potential use of lateral flow devices has also been developed. In this study, we analyze the robustness of test strips using published methods for colorimetric detection. Different test formats are discussed, and challenges in the development of lateral flow devices for on-site determination of OTA, with requirements such as robustness, speed, and cost-effectiveness, are discussed.

  8. Rapid and Sensitive Detection of BLAD in Cattle Population

    Directory of Open Access Journals (Sweden)

    Daniela Elena Ilie

    2014-05-01

    Full Text Available Bovine leukocyte adhesion deficiency (BLAD is an autosomal recessive disorder with negative impact on dairy cattle breeding. The molecular basis of BLAD is a single point mutation (A→G, resulting in a single amino acid substitution (aspartic acid → glycine at amino acid 128 in the adhesion molecule CD18. The object of this study was to establish a fast and sensitive molecular genotyping assay to detect BLAD carriers using high-resolution melting (HRM curve analysis. We tested animals with known genotypes for BLAD that were previously confirmed by PCR-RFLP method, and then examined the sensitivity of mutation detection using PCR followed by HRM curve analysis. BLAD carriers were readily detectable using HRM assay. Thus, the PCR-HRM genotyping method is a rapid, easily interpretable, reliable and cost-effective assay for BLAD mutant allele detection. This assay can be useful in cattle genotyping and genetic selection.

  9. Rapid detection of methanol in artisanal alcoholic beverages

    Science.gov (United States)

    de Goes, R. E.; Muller, M.; Fabris, J. L.

    2015-09-01

    In the industry of artisanal beverages, uncontrolled production processes may result in contaminated products with methanol, leading to risks for consumers. Owing to the similar odor of methanol and ethanol, as well as their common transparency, the distinction between them is a difficult task. Contamination may also occur deliberately due to the lower price of methanol when compared to ethanol. This paper describes a spectroscopic method for methanol detection in beverages based on Raman scattering and Principal Component Analysis. Associated with a refractometric assessment of the alcohol content, the method may be applied in field for a rapid detection of methanol presence.

  10. [Rapid detection of Shigella dysenteriae by PCR assay].

    Science.gov (United States)

    Chen, Hongyuan; Zhong, Qingping; Wang, Li; Sun, Yuanming

    2010-09-01

    Based on the invasive plasmid antigen H gene (ipaH) of S. dysenteriae, one pair of specific primers was designed for PCR assays in this study. The concentrations of dNTP, Mg2+ and primer, dosage of Taq DNA polymerase, annealing temperature and circulating parameter in the PCR amplification system were optimized. In this way, a rapid and stable method of PCR assay for the detection of S. dysenteriae was established. The specificity and sensitivity of PCR were also analyzed. The detection limits of pure culture and genomic DNA in the PCR assay were 1.06 x 10(2) cfu/ml and 106.34 pg/PCR system, respectively. The detection limit for S. dysenteriae in artificially contaminated food samples was 3.21 x 10(4) cfu/ml. These results indicated that the PCR method for S. dysenteriae detection was simple, rapid, high in specificity and sensitivity and suitable for the detection of pathogens in foods caused by Shigella dysenteriae.

  11. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    Science.gov (United States)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  12. Rapid Detection and Characterization of Emerging Foreign Animal Disease Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-11-18

    To best safeguard human and animal health requires early detection and characterization of disease events. This must include effective surveillance for emerging infectious diseases. Both deliberate and natural outbreaks have enormous economic and public health impacts, and can present serious threats to national security. In this project, we developed novel next generation detection technologies to protect the agricultural economy and biosecurity. The first technology is a multiplexed assay to simultaneously detection 10 swine viral and bacterial pathogens. The second one is the Lawrence Livermore Microbial Detection Array (LLMDA) which can detect more than 10,000 microbial species including 4219 viruses, 5367 bacteria, 265 fungi, 117 protozoa and 293 archaea. We analyzed a series of swine clinical samples from past disease events to demonstrate the utility of the assays for faster and cheaper detection of emerging and foreign animal disease pathogens, and their utility as s routine diagnosis and surveillance tool. A second goal of the study is to better understand mechanisms of African swine fever virus (ASFV) infection in pigs to aid the development of countermeasures and diagnostics. There is no vaccine available for ASF. ASF outbreak is on the rise on several European countries. Though ASF is not currently in the U.S., a potential outbreak in the U.S. would be detrimental to the swine industry and the US agricultural economy. We pursued a genome-wide approach to characterize the pig immune responses after ASFV infection. We used RNA sequencing and bioinformatics methods to identify genes and pathways that are affected during ASF infection. We have identified a list of most differentially expressed genes that are in the immune response pathways.

  13. Rapid detection, characterization, and enumeration of foodborne pathogens.

    Science.gov (United States)

    Hoorfar, J

    2011-11-01

    As food safety management further develops, microbiological testing will continue to play an important role in assessing whether Food Safety Objectives are achieved. However, traditional microbiological culture-based methods are limited, particularly in their ability to provide timely data. The present review discusses the reasons for the increasing interest in rapid methods, current developments in the field, the research needs, and the future trends. The advent of biotechnology has introduced new technologies that led to the emergence of rapid diagnostic methods and altered food testing practices. Rapid methods are comprised of many different detection technologies, including specialized enzyme substrates, antibodies and DNA, ranging from simple differential plating media to the use of sophisticated instruments. The use of non-invasive sampling techniques for live animals especially came into focus with the 1990s outbreak of bovine spongiform encephalopathy that was linked to the human outbreak of Creutzfeldt Jakob's Disease. Serology is still an important tool in preventing foodborne pathogens to enter the human food supply through meat and milk from animals. One of the primary uses of rapid methods is for fast screening of large number of samples, where most of them are expected to be test-negative, leading to faster product release for sale. This has been the main strength of rapid methods such as real-time Polymerase Chain Reaction (PCR). Enrichment PCR, where a primary culture broth is tested in PCR, is the most common approach in rapid testing. Recent reports show that it is possible both to enrich a sample and enumerate by pathogen-specific real-time PCR, if the enrichment time is short. This can be especially useful in situations where food producers ask for the level of pathogen in a contaminated product. Another key issue is automation, where the key drivers are miniaturization and multiple testing, which mean that not only one instrument is flexible

  14. COMPOSITE SAMPLING FOR DETECTION OF COLIFORM BACTERIA IN WATER SUPPLY

    Science.gov (United States)

    Low densities of coliform bacteria introduced into distribution systems may survive in protected habitats. These organisms may interfere with and cause confusion in the use of the coliforms as indicators of sewage contamination of drinking water. Methods of increasing the probabi...

  15. Presenting a rapid method for detection of Bacillus cereus, Listeria monocytogenes and Campylobacter jejuni in food samples

    Directory of Open Access Journals (Sweden)

    Ali Razei

    2017-08-01

    Full Text Available Objective(s: Listeria monocytogens, Bacillus cereus and Campylobacter jejuni are three toxin producing bacteria over the world, especially in Iran, and it is essential to find a certain, rapid procedure to identify these microorganisms. In this research, these bacteria were simultaneously detected by multiplex PCR technique in foods. Materials and Methods: The primary approval of bacterial strains was performed by biochemical tests. PCR primers were designed based on the nucleotide sequences of the NHEB/NHEC gene of B. cereus, the hly gene of L. monocytogenes and the C gene of C. jejuni. The specificity of Multiplex PCR method was determined using seven food poisoning bacteria including Salmonella typhi, Shigella dysentery, Yersinia pestis, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum and Vibrio cholerae. To confirm the reaction, DNA extraction was performed from 30 food samples (milk, and gene amplification was performed by PCR. The length of amplified fragments was 300 bp, 210 bp and 160 bpfor NHEB/NHEC, hly and C genes, respectively. Results: The detection limits of the PCR method were 5, 4 and 3 pg for L. monocytogenes, B. cereus and C. jejuni, respectively. Specifisity test showed that this reaction is spesific to these 3 bacteria. Conclusion: In this study, we  introduced a new multiplex PCR method for simultsnus detection of L. monocytogens, B. cereus and C. jejuni. These results can be used  for detection of other toxin producing bacteria in food.

  16. Rapid detection, characterization, and enumeration of foodborne pathogens

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey

    2011-01-01

    into focus with the 1990s outbreak of bovine spongiform encephalopathy that was linked to the human outbreak of Creutzfeldt Jakob's Disease. Serology is still an important tool in preventing foodborne pathogens to enter the human food supply through meat and milk from animals. One of the primary uses...... broth is tested in PCR, is the most common approach in rapid testing. Recent reports show that it is possible both to enrich a sample and enumerate by pathogen-specific real-time PCR, if the enrichment time is short. This can be especially useful in situations where food producers ask for the level...... following a short log-phase enrichment, (iv) detection of foodborne pathogens in air samples, and finally (v) biotracing of pathogens based on mathematical modeling, even in the absence of isolate. Rapid methods are discussed in a broad global health perspective, international food supply...

  17. Rapid detection of Brucella spp. using loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Chen, Shouyi; Li, Xunde; Li, Juntao; Atwill, Edward R

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Livestock that are most vulnerable to brucellosis include cattle, goats, and pigs. Brucella spp. cause serious health problems to humans and animals and economic losses to the livestock industry. Traditional methods for detection of Brucella spp. take 48-72 h (Kumar et al., J Commun Dis 29:131-137, 1997; Barrouin-Melo et al., Res Vet Sci 83:340-346, 2007) that do not meet the food industry's need of rapid detection. Therefore, there is an urgent need of fast, specific, sensitive, and inexpensive method for diagnosing of Brucella spp. Loop-mediated isothermal amplification (LAMP) is a method to amplify nucleic acid at constant temperatures. Amplification can be detected by visual detection, fluorescent stain, turbidity, and electrophoresis. We targeted at the Brucella-specific gene omp25 and designed LAMP primers for detection of Brucella spp. Amplification of DNA with Bst DNA polymerase can be completed at 65 °C in 60 min. Amplified products can be detected by SYBR Green I stain and 2.0% agarose gel electrophoresis. The LAMP method is feasible for detection of Brucella spp. from blood and milk samples.

  18. Modified agar dilution method for rapid antibiotic susceptibility testing of anaerobic bacteria.

    Science.gov (United States)

    Hanson, C W; Martin, W J

    1978-01-01

    A simplified method has been developed for agar dilution antimicrobial susceptibility testing of anaerobic bacteria, designed to economize on time and money when only a few isolates need to be tested. The procedure is based on the principle of using filter paper disks as carriers of the antibiotic and 35- by 10-mm petri dishes which, when inoculated with the Steers replicator, can test up to four organisms per plate. The procedure was run in parallel with conventional agar dilution techniques and showed 95% agreement to within one dilution for all minimal inhibitory concentrations recorded on fresh anaerobic isolates from clinical specimens. The technique was further simplified by using commercially available antibiotic-containing disks, thereby alleviating the tedious and time-consuming procedure of preparing the disks. The data indicated that 48- to 72-h diffusion periods were sufficient to achieve a uniform concentration of the antibiotic in the petri plate and that the antibiotics were stable at room temperature for that period of time. In terms of applicability and relevance to the needs of the clinical microbiology laboratory, the modified agar dilution method for rapid antimicrobial susceptibility testing of individual anaerobic isolates was found to be superior to the broth dilution method since it was easier to read and required considerably less set up time. PMID:400819

  19. Rapid in situ detection of chromosome 21 by PRINS technique

    Energy Technology Data Exchange (ETDEWEB)

    Pellestor, F.; Girardet, A.; Andreo, B. [CNRS UPR 9008, Montpellier (France)] [and others

    1995-05-08

    The {open_quotes}PRimed IN Situ labeling{close_quotes} (PRINS) method is an interesting alternative to in situ hybridization for chromosomal detection. In this procedure, chromosome labeling is performed by in situ annealing of specific oligonucleotide primers, followed by primer elongation by a Taq polymerase in the presence of labeled nucleotides. Using this process, we have developed a simple and semi-automatic method for rapid in situ detection of human chromosome 21. The reaction was performed on a programmable temperature cycler, with a chromosome 21 specific oligonucleotide primer. Different samples of normal and trisomic lymphocytes and amniotic fluid cells were used for testing the method. Specific labeling of chromosome 21 was obtained in both metaphases and interphase nuclei in a 1 hour reaction. The use of oligonucleotide primer for in situ labeling overcomes the need for complex preparations of specific DNA probes. The present results demonstrate that PRINS may be a simple and reliable technique for rapidly detecting aneuploidies. 18 refs., 1 fig.

  20. Rapid shape detection signals in area V4.

    Science.gov (United States)

    Weiner, Katherine F; Ghose, Geoffrey M

    2014-01-01

    Vision in foveate animals is an active process that requires rapid and constant decision-making. For example, when a new object appears in the visual field, we can quickly decide to inspect it by directing our eyes to the object's location. We studied the contribution of primate area V4 to these types of rapid foveation decisions. Animals performed a reaction time task that required them to report when any shape appeared within a peripherally-located noisy stimulus by making a saccade to the stimulus location. We found that about half of the randomly sampled V4 neurons not only rapidly and precisely represented the appearance of this shape, but they were also predictive of the animal's saccades. A neuron's ability to predict the animal's saccades was not related to the specificity with which the cell represented a single type of shape but rather to its ability to signal whether any shape was present. This relationship between sensory sensitivity and behavioral predictiveness was not due to global effects such as alertness, as it was equally likely to be observed for cells with increases and decreases in firing rate. Careful analysis of the timescales of reliability in these neurons implies that they reflect both feedforward and feedback shape detecting processes. In approximately 7% of our recorded sample, individual neurons were able to predict both the delay and precision of the animal's shape detection performance. This suggests that a subset of V4 neurons may have been directly and causally contributing to task performance and that area V4 likely plays a critical role in guiding rapid, form-based foveation decisions.

  1. Rapid Antemortem Detection of CWD Prions in Deer Saliva

    Science.gov (United States)

    Haley, Nicholas J.; Denkers, Nathaniel D.; Nalls, Amy V.; Mathiason, Candace K.; Caughey, Byron; Hoover, Edward A.

    2013-01-01

    Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids, now identified in 22 United States, 2 Canadian provinces and Korea. One hallmark of CWD is the shedding of infectious prions in saliva, as demonstrated by bioassay in deer. It is also clear that the concentration of prions in saliva, blood, urine and feces is much lower than in the nervous system or lymphoid tissues. Rapid in vitro detection of CWD (and other) prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA) and the presence of inhibitors in these complex biological materials that hamper detection. Here we use real-time quaking induced conversion (RT-QuIC) to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3%) diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%). In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1%) of the above saliva samples. Bioassay in Tg[CerPrP] mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination. PMID:24040235

  2. Rapid antemortem detection of CWD prions in deer saliva.

    Directory of Open Access Journals (Sweden)

    Davin M Henderson

    Full Text Available Chronic wasting disease (CWD is an efficiently transmitted prion disease of cervids, now identified in 22 United States, 2 Canadian provinces and Korea. One hallmark of CWD is the shedding of infectious prions in saliva, as demonstrated by bioassay in deer. It is also clear that the concentration of prions in saliva, blood, urine and feces is much lower than in the nervous system or lymphoid tissues. Rapid in vitro detection of CWD (and other prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA and the presence of inhibitors in these complex biological materials that hamper detection. Here we use real-time quaking induced conversion (RT-QuIC to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3% diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%. In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1% of the above saliva samples. Bioassay in Tg[CerPrP] mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination.

  3. Surface enhanced Raman scattering (SERS) with biopolymer encapsulated silver nanosubstrates for rapid detection of foodborne pathogens.

    Science.gov (United States)

    Sundaram, Jaya; Park, Bosoon; Kwon, Yongkuk; Lawrence, Kurt C

    2013-10-01

    A biopolymer encapsulated with silver nanoparticles was prepared using silver nitrate, polyvinyl alcohol (PVA) solution, and trisodium citrate. It was deposited on a mica sheet to use as SERS substrate. Fresh cultures of Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus and Listeria innocua were washed from chicken rinse and suspended in 10 ml of sterile deionized water. Approximately 5 μl of the bacterial suspensions was placed on the substrate individually and exposed to 785 nm HeNe laser excitation. SERS spectral data were recorded over the Raman shift between 400 and 1800 cm(-1) from 15 different spots on the substrate for each sample; and three replicates were done on each bacteria type. Principal component analysis (PCA) model was developed to classify foodborne bacteria types. PC1 identified 96% of the variation among the given bacteria specimen, and PC2 identified 3%, resulted in a total of 99% classification accuracy. Soft Independent Modeling of Class Analogies (SIMCA) of validation set gave an overall correct classification of 97%. Comparison of the SERS spectra of different types of gram-negative and gram-positive bacteria indicated that all of them have similar cell walls and cell membrane structures. Conversely, major differences were noted around the nucleic acid and amino acid structure information between 1200 cm(-1) and 1700 cm(-1) and at the finger print region between 400 cm(-1) and 700 cm(-1). Silver biopolymer nanoparticle substrate could be a promising SERS tool for pathogen detection. Also this study indicates that SERS technology could be used for reliable and rapid detection and classification of food borne pathogens. Published by Elsevier B.V.

  4. Method and apparatus for detecting phycocyanin-pigmented algae and bacteria from reflected light

    Science.gov (United States)

    Vincent, Robert (Inventor)

    2013-01-01

    The present invention relates to a method of detecting phycocyanin algae or bacteria in water from reflected light, and also includes devices for the measurement, calculation and transmission of data relating to that method.

  5. Photonic Crystal Biosensor Chip for Label-Free Detection of Bacteria

    DEFF Research Database (Denmark)

    Kristensen, Martin; Krüger, Asger Christian; Groothoff, Nathaniel

    Narrow polarization-mixing resonances in planar photonic crystals are studied as candidate components for label-free refractive index sensors for detecting bacteria causing sepsis through the identification of DNA strands....

  6. Detection of Biomaterials and Bacteria Using Functionalized Nano- and Micro-spaces

    National Research Council Canada - National Science Library

    Shiho TOKONAMI; Takuya IIDA; Hiroshi SHIIGI; Tsutomu NAGAOKA

    2015-01-01

      The detection systems of biomaterials, for example, DNA and protein, which are important in genetic and allergy tests, or those of bacteria causing various disease, are essential tools in preventive...

  7. Rapid detection of Listeria monocytogenes in milk using confocal micro-Raman spectroscopy and chemometric analysis.

    Science.gov (United States)

    Wang, Junping; Xie, Xinfang; Feng, Jinsong; Chen, Jessica C; Du, Xin-jun; Luo, Jiangzhao; Lu, Xiaonan; Wang, Shuo

    2015-07-02

    Listeria monocytogenes is a facultatively anaerobic, Gram-positive, rod-shape foodborne bacterium causing invasive infection, listeriosis, in susceptible populations. Rapid and high-throughput detection of this pathogen in dairy products is critical as milk and other dairy products have been implicated as food vehicles in several outbreaks. Here we evaluated confocal micro-Raman spectroscopy (785 nm laser) coupled with chemometric analysis to distinguish six closely related Listeria species, including L. monocytogenes, in both liquid media and milk. Raman spectra of different Listeria species and other bacteria (i.e., Staphylococcus aureus, Salmonella enterica and Escherichia coli) were collected to create two independent databases for detection in media and milk, respectively. Unsupervised chemometric models including principal component analysis and hierarchical cluster analysis were applied to differentiate L. monocytogenes from Listeria and other bacteria. To further evaluate the performance and reliability of unsupervised chemometric analyses, supervised chemometrics were performed, including two discriminant analyses (DA) and soft independent modeling of class analogies (SIMCA). By analyzing Raman spectra via two DA-based chemometric models, average identification accuracies of 97.78% and 98.33% for L. monocytogenes in media, and 95.28% and 96.11% in milk were obtained, respectively. SIMCA analysis also resulted in satisfied average classification accuracies (over 93% in both media and milk). This Raman spectroscopic-based detection of L. monocytogenes in media and milk can be finished within a few hours and requires no extensive sample preparation. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Rapid detection of anti-Vaccinia virus neutralizing antibodies

    Directory of Open Access Journals (Sweden)

    Lichtfuss Gregor F

    2011-03-01

    Full Text Available Abstract Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR.

  9. Rapid and Reliable Diagnostic Algorithm for Detection of Clostridium difficile▿

    Science.gov (United States)

    Fenner, Lukas; Widmer, Andreas F.; Goy, Gisela; Rudin, Sonja; Frei, Reno

    2008-01-01

    We evaluated a two-step algorithm for detection of Clostridium difficile in 1,468 stool specimens. First, specimens were screened by an immunoassay for C. difficile glutamate dehydrogenase antigen (C.DIFF CHEK-60). Second, screen-positive specimens underwent toxin testing by a rapid toxin A/B assay (TOX A/B QUIK CHEK); toxin-negative specimens were subjected to stool culture. This algorithm allowed final results for 92% of specimens with a turnaround time of 4 h. PMID:18032627

  10. Development of Rapid Detection and Genetic Characterization of Salmonella in Poultry Breeder Feeds

    Directory of Open Access Journals (Sweden)

    Steven C. Ricke

    2009-07-01

    Full Text Available Salmonella is a leading cause of foodborne illness in the United States, with poultry and poultry products being a primary source of infection to humans. Poultry may carry some Salmonella serovars without any signs or symptoms of disease and without causing any adverse effects to the health of the bird. Salmonella may be introduced to a flock by multiple environmental sources, but poultry feed is suspected to be a leading source. Detecting Salmonella in feed can be challenging because low levels of the bacteria may not be recovered using traditional culturing techniques. Numerous detection methodologies have been examined over the years for quantifying Salmonella in feeds and many have proven to be effective for Salmonella isolation and detection in a variety of feeds. However, given the potential need for increased detection sensitivity, molecular detection technologies may the best candidate for developing rapid sensitive methods for identifying small numbers of Salmonella in the background of large volumes of feed. Several studies have been done using polymerase chain reaction (PCR assays and commercial kits to detect Salmonella spp. in a wide variety of feed sources. In addition, DNA array technology has recently been utilized to track the dissemination of a specific Salmonella serotype in feed mills. This review will discuss the processing of feeds and potential points in the process that may introduce Salmonella contamination to the feed. Detection methods currently used and the need for advances in these methods also will be discussed. Finally, implementation of rapid detection for optimizing control methods to prevent and remove any Salmonella contamination of feeds will be considered.

  11. Development of Rapid Detection and Genetic Characterization of Salmonella in Poultry Breeder Feeds

    Science.gov (United States)

    Jarquin, Robin; Hanning, Irene; Ahn, Soohyoun; Ricke, Steven C.

    2009-01-01

    Salmonella is a leading cause of foodborne illness in the United States, with poultry and poultry products being a primary source of infection to humans. Poultry may carry some Salmonella serovars without any signs or symptoms of disease and without causing any adverse effects to the health of the bird. Salmonella may be introduced to a flock by multiple environmental sources, but poultry feed is suspected to be a leading source. Detecting Salmonella in feed can be challenging because low levels of the bacteria may not be recovered using traditional culturing techniques. Numerous detection methodologies have been examined over the years for quantifying Salmonella in feeds and many have proven to be effective for Salmonella isolation and detection in a variety of feeds. However, given the potential need for increased detection sensitivity, molecular detection technologies may the best candidate for developing rapid sensitive methods for identifying small numbers of Salmonella in the background of large volumes of feed. Several studies have been done using polymerase chain reaction (PCR) assays and commercial kits to detect Salmonella spp. in a wide variety of feed sources. In addition, DNA array technology has recently been utilized to track the dissemination of a specific Salmonella serotype in feed mills. This review will discuss the processing of feeds and potential points in the process that may introduce Salmonella contamination to the feed. Detection methods currently used and the need for advances in these methods also will be discussed. Finally, implementation of rapid detection for optimizing control methods to prevent and remove any Salmonella contamination of feeds will be considered. PMID:22346699

  12. Detection of gfp expression from gfp-labelled bacteria spot ...

    African Journals Online (AJOL)

    SERVER

    Valdivia et al., 1998; Compant et al., 2005). However, there is one major limiting factor in the detection of GFP in living organisms whose cells or tissues emit background autofluorescence such that it becomes difficult to detect the GFP's fluorescence.

  13. Rapid detection of autosomal aneuploidy using microsatellite markers

    Energy Technology Data Exchange (ETDEWEB)

    Ray, P.N.; Teshima, I.E. [Hospital for Sick Children, Ontario (Canada); Winsor, E.J.T. [Toronto Hospital, Ontario (Canada)] [and others

    1994-09-01

    Trisomy occurs in at least 4% of all clinically recognized pregnancies, making it the most common type of chromosome abnormality in humans. The most commonly occurring trisomies are those of chromosomes 13, 18, 21 and aneuploidy of X and Y, accounting for about 0.3% of all newborns and a much higher percentage of conceptuses. In Canada, prenatal chromosome analysis by amniocentesis is offered to those women {ge} 35 years of age at the time of delivery or equivalent risk by maternal serum screen. We are developing a rapid molecular diagnostic test to detect the most common autosomal aneuploidies in prenatal and neonatal samples. The tests makes use of highly polymorphic short tandem repeat markers labeled with fluorescent tags which allow analysis on a GENESCANNER automated fragment analyzer (ABI). Multiple polymorphic markers have been selected on each of chromosomes 13, 18 and 21. At a given locus, trisomic fetuses/neonates will have either three alleles or two alleles with one allele having twice the intensity of the other. Unaffected individuals have two equal intensity alleles. We are conducting a blind study that will compare the detection efficiencies of FISH analysis on uncultured cells and the molecular method on confirmation amniotic fluid samples collected at the time of termination of affected fetuses. Results on cultured amniocytes from one such patient confirmed that trisomy 21 can be detected. FISH was not done on this sample. In addition, detection efficiency of the molecular method in whole blood samples from affected neonates is also being studied. To date, two such samples have been tested, one with trisomy 13 and one with trisomy 18, and both samples were diagnosed correctly. Preliminary results suggest that this method may provide a valuable tool for the rapid diagnosis of aneuploidy.

  14. Quartz crystal microbalance biosensor for rapid detection of aerosolized microorganisms

    Science.gov (United States)

    Farka, Zdenĕk.; Kovár, David; Skládal, Petr

    2015-05-01

    Biological warfare agents (BWAs) represent the current menace of the asymmetric war. The early detection of BWAs, especially in the form of bioaerosol, is a challenging task for governments all around the world. Label-free quartz crystal microbalance (QCM) immunosensor and electrochemical immunosensor were developed and tested for rapid detection of BWA surrogate (E. coli) in the form of bioaerosol. Two immobilization strategies for the attachment of antibody were tested; the gold sensor surface was activated by cysteamine and then antibody was covalently linked either using glutaraldehyde, or the reduced antibodies were attached via Sulfo-SMCC. A portable bioaerosol chamber was constructed and used for safe manipulation with aerosolized microorganisms. The dissemination was done using a piezoelectric humidifier, distribution of bioaerosol inside the chamber was ensured using three 12-cm fans. The whole system was controlled remotely using LAN network. The disseminated microbial cells were collected and preconcentrated using the wetted-wall cyclone SASS 2300, the analysis was done using the on-line linked immunosensors. The QCM immunosensor had limit of detection 1×104 CFU·L-1 of air with analysis time 16 min, the whole experiment including dissemination and sensor surface regeneration took 40 min. In case of blank (disseminated sterile buffer), no signal change was observed. The electrochemical immunosensor was able to detect 150 CFU·L-1 of air in 20 min; also in this case, no interferences were observed. Reference measurements were done using particle counter Met One 3400 and by cultivation method on agar plates. The sensors have proved to be applicable for rapid screening of microorganisms in air.

  15. Rapid Detection Strategies for the Global Threat of Zika Virus: Current State, New Hypotheses and Limitations

    Directory of Open Access Journals (Sweden)

    Shruti Shukla

    2016-10-01

    Full Text Available The current scenario regarding the widespread Zika virus (ZIKV has resulted in numerous diagnostic studies, specifically in South America and in locations where there is frequent entry of travelers returning from ZIKV-affected areas, including pregnant women with or without clinical symptoms of ZIKV infection. The World Health Organization, WHO, announced that millions of cases of ZIKV are likely to occur in the United States of America in the near future. This situation has created an alarming public health emergency of international concern requiring the detection of this life-threatening viral candidate due to increased cases of newborn microcephaly associated with ZIKV infection. Hence, this review reports possible methods and strategies for the fast and reliable detection of ZIKV with particular emphasis on current updates, knowledge and new hypotheses that might be helpful for medical professionals in poor and developing countries that urgently need to address this problem. In particular, we emphasize liposome-based biosensors. Although these biosensors are currently among the less popular tools for human disease detection, they have become useful tools for the screening and detection of pathogenic bacteria, fungi and viruses because of their versatile advantageous features compared to other sensing devices. This review summarizes the currently available methods employed for the rapid detection of ZIKV and suggests an innovative approach involving the application of a liposome-based hypothesis for the development of new strategies for ZIKV detection and their use as effective biomedicinal tools.

  16. [Rapid test for detection of susceptibility to cefotaxime in Enterobacteriaceae].

    Science.gov (United States)

    Jiménez-Guerra, Gemma; Hoyos-Mallecot, Yannik; Rodríguez-Granger, Javier; Navarro-Marí, José María; Gutiérrez-Fernández, José

    In this work an "in house" rapid test based on the change in pH that is due to hydrolysis for detecting Enterobacteriaceae susceptible to cefotaxime is evaluated. The strains of Enterobacteriaceae from 1947 urine cultures were assessed using MicroScan panels and the "in house" test. This rapid test includes red phenol solution and cefotaxime. Using MicroScan panels, 499 Enterobacteriaceae isolates were evaluated, which included 27 isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBL), 16 isolates of Klebsiella pneumoniae ESBL and 1 isolate of Klebsiella oxytoca ESBL. The "in house" test offers the following values: sensitivity 98% and specificity 97%, with negative predictive value 100% and positive predictive value 78%. The "in house" test based on the change of pH is useful in our area for detecting presumptively cefotaxime-resistant Enterobacteriaceae strains. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  17. Rapid and robust traffic accident detection based on orientation map

    Science.gov (United States)

    Zhou, Jinglei; Ye, Mao; Ding, Jian; Mao, Songan; Zhang, Huixiong John

    2012-11-01

    Video-based rapid traffic accident detection is very important for intelligent transport systems. Traditional methods are either not fast enough or not stable with working environments. A rapid and environment-adaptive method is proposed. The inspiration of the method is originated from the key observation that the traffic accident brings abundant information on motion directions. This method includes three steps. First, the orientation map for each video frame is constructed based on the optical flows. Then, for each orientation map, the connected regions are formed. An entropy-like energy function is used to measure the orientation information of the connected region. The higher the energy value, the more moving directions exist. The highest measure of these connected regions in each orientation map is considered as its energy measure. Finally, based on the energy sequence of orientation maps, a Gaussian model is established to learn the normal energy fluctuation range for some environment. In the detection process, if the energy of one orientation map burst out of the normal range, it means there exists a traffic accident. The advantages of our method include the fast processing speed, a compact parameter set, and the robustness to the different environments and illuminations. Experimental results confirm the above advantages of the proposed approach.

  18. Detection of Streptococcus pyogenes using rapid visual molecular assay.

    Science.gov (United States)

    Zhao, Xiangna; He, Xiaoming; Li, Huan; Zhao, Jiangtao; Huang, Simo; Liu, Wei; Wei, Xiao; Ding, Yiwei; Wang, Zhaoyan; Zou, Dayang; Wang, Xuesong; Dong, Derong; Yang, Zhan; Yan, Xiabei; Huang, Liuyu; Du, Shuangkui; Yuan, Jing

    2015-09-01

    Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64°C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Improved method for detection of methanotrophic bacteria in forest soils by PCR.

    Science.gov (United States)

    Steinkamp, R; Zimmer, W; Papen, H

    2001-05-01

    A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the alpha-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils.

  20. Carbapenemase producing bacteria in the food supply escaping detection.

    Directory of Open Access Journals (Sweden)

    Beverly J Morrison

    Full Text Available Carbapenem antimicrobials are critically important to human health and they are often the only remaining effective antibiotics for treating serious infections. Resistance to these drugs mediated by acquired carbapenemase enzymes is increasingly encountered in gram-negative bacteria and is considered a public health emergency. Animal origin food products are recognized as a potential source of resistant organisms, although carbapenem resistance has only recently been reported. In western countries there are active resistance surveillance programs targeting food animals and retail meat products. These programs primarily target beef, pork and poultry and focus exclusively on E. coli, Salmonella, Campylobacter spp. and Enterococcus spp. This global surveillance strategy does not capture the diversity of foods available nor does it address the presence of resistance gene-bearing mobile genetic elements in non-pathogenic bacterial taxa. To address this gap, a total of 121 seafood products originating in Asia purchased from retail groceries in Canada were tested. Samples were processed using a taxa-independent method for the selective isolation of carbapenem resistant organisms. Isolates were characterized by phenotypic antimicrobial susceptibility testing, PCR and DNA sequencing. Carbapenemase producing bacteria, all blaOXA-48, were isolated from 4 (3.3% of the samples tested. Positive samples originated from China (n=2 and Korea (n=2 and included squid, sea squirt, clams and seafood medley. Carbapenemase producing organisms found include Pseudomonas, Stenotrophomonas and Myroides species. These findings suggest that non-pathogenic bacteria, excluded from resistance surveillance programs, in niche market meats may serve as a reservoir of carbapenemase genes in the food supply.

  1. Rapid Monitoring of Bacteria and Fungi aboard the International Space Station (ISS)

    Science.gov (United States)

    Gunter, D.; Flores, G.; Effinger, M.; Maule, J.; Wainwright, N.; Steele, A.; Damon, M.; Wells, M.; Williams, S.; Morris, H.; hide

    2009-01-01

    Microorganisms within spacecraft have traditionally been monitored with culture-based techniques. These techniques involve growth of environmental samples (cabin water, air or surfaces) on agar-type media for several days, followed by visualization of resulting colonies or return of samples to Earth for ground-based analysis. Data obtained over the past 4 decades have enhanced our understanding of the microbial ecology within space stations. However, the approach has been limited by the following factors: i) Many microorganisms (estimated > 95%) in the environment cannot grow on conventional growth media; ii) Significant time lags (3-5 days for incubation and up to several months to return samples to ground); iii) Condensation in contact slides hinders colony counting by crew; and iv) Growth of potentially harmful microorganisms, which must then be disposed of safely. This report describes the operation of a new culture-independent technique onboard the ISS for rapid analysis (within minutes) of endotoxin and beta-1, 3-glucan, found in the cell walls of gramnegative bacteria and fungi, respectively. The technique involves analysis of environmental samples with the Limulus Amebocyte Lysate (LAL) assay in a handheld device, known as the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS). LOCADPTS was launched to the ISS in December 2006, and here we present data obtained from Mach 2007 until the present day. These data include a comparative study between LOCADPTS analysis and existing culture-based methods; and an exploratory survey of surface endotoxin and beta-1, 3-glucan throughout the ISS. While a general correlation between LOCAD-PTS and traditional culture-based methods should not be expected, we will suggest new requirements for microbial monitoring based upon culture-independent parameters measured by LOCAD-PTS.

  2. Rapid classification of heavy metal-exposed freshwater bacteria by infrared spectroscopy coupled with chemometrics using supervised method

    Science.gov (United States)

    Gurbanov, Rafig; Gozen, Ayse Gul; Severcan, Feride

    2018-01-01

    Rapid, cost-effective, sensitive and accurate methodologies to classify bacteria are still in the process of development. The major drawbacks of standard microbiological, molecular and immunological techniques call for the possible usage of infrared (IR) spectroscopy based supervised chemometric techniques. Previous applications of IR based chemometric methods have demonstrated outstanding findings in the classification of bacteria. Therefore, we have exploited an IR spectroscopy based chemometrics using supervised method namely Soft Independent Modeling of Class Analogy (SIMCA) technique for the first time to classify heavy metal-exposed bacteria to be used in the selection of suitable bacteria to evaluate their potential for environmental cleanup applications. Herein, we present the powerful differentiation and classification of laboratory strains (Escherichia coli and Staphylococcus aureus) and environmental isolates (Gordonia sp. and Microbacterium oxydans) of bacteria exposed to growth inhibitory concentrations of silver (Ag), cadmium (Cd) and lead (Pb). Our results demonstrated that SIMCA was able to differentiate all heavy metal-exposed and control groups from each other with 95% confidence level. Correct identification of randomly chosen test samples in their corresponding groups and high model distances between the classes were also achieved. We report, for the first time, the success of IR spectroscopy coupled with supervised chemometric technique SIMCA in classification of different bacteria under a given treatment.

  3. A biosensor platform for rapid detection of E. coli in drinking water.

    Science.gov (United States)

    Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza

    2016-02-01

    There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or

  4. A C. elegans-based foam for rapid on-site detection of residual live virus.

    Energy Technology Data Exchange (ETDEWEB)

    Negrete, Oscar A.; Branda, Catherine; Hardesty, Jasper O. E. (Sandia National Laboratories, Albuquerque, NM); Tucker, Mark David (Sandia National Laboratories, Albuquerque, NM); Kaiser, Julia N. (Global Product Management, Hilden, Germany); Kozina, Carol L.; Chirica, Gabriela S.

    2012-02-01

    In the response to and recovery from a critical homeland security event involving deliberate or accidental release of biological agents, initial decontamination efforts are necessarily followed by tests for the presence of residual live virus or bacteria. Such 'clearance sampling' should be rapid and accurate, to inform decision makers as they take appropriate action to ensure the safety of the public and of operational personnel. However, the current protocol for clearance sampling is extremely time-intensive and costly, and requires significant amounts of laboratory space and capacity. Detection of residual live virus is particularly problematic and time-consuming, as it requires evaluation of replication potential within a eukaryotic host such as chicken embryos. The intention of this project was to develop a new method for clearance sampling, by leveraging Sandia's expertise in the biological and material sciences in order to create a C. elegans-based foam that could be applied directly to the entire contaminated area for quick and accurate detection of any and all residual live virus by means of a fluorescent signal. Such a novel technology for rapid, on-site detection of live virus would greatly interest the DHS, DoD, and EPA, and hold broad commercial potential, especially with regard to the transportation industry.

  5. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

    Science.gov (United States)

    García, L; Tercero, J C; Legido, B; Ramos, J A; Alemany, J; Sanz, M

    1998-01-01

    The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens.

  6. A rapid DNA extraction method suitable for human papillomavirus detection.

    Science.gov (United States)

    Brestovac, Brian; Wong, Michelle E; Costantino, Paul S; Groth, David

    2014-04-01

    Infection with oncogenic human papillomavirus (HPV) genotypes is necessary for the development of cervical cancer. Testing for HPV DNA from liquid based cervical samples can be used as an adjunct to traditional cytological screening. In addition there are ongoing viral load, genotyping, and prevalence studies. Therefore, a sensitive DNA extraction method is needed to maximize the efficiency of HPV DNA detection. The XytXtract Tissue kit is a DNA extraction kit that is rapid and so could be useful for HPV testing, particularly in screening protocols. This study was undertaken to determine the suitability of this method for HPV detection. DNA extraction from HeLa and Caski cell lines containing HPV 18 and 16 respectively together with DNA from five liquid based cervical samples were used in a HPV PCR assay. DNA was also extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as a comparison. DNA extracts were serially diluted and assayed. HPV DNA was successfully detected in cell lines and cervical samples using the XytXtract Tissue kit. In addition, the XytXtract method was found to be more sensitive than the QIAmp method as determined by a dilution series of the extracted DNA. While the XytXtract method is a closed, the QIAamp method uses a spin column with possible loss of DNA through DNA binding competition of the matrix, which could impact on the final extraction efficiency. The XytXtract is a cheap, rapid and efficient method for extracting HPV DNA from both cell lines and liquid based cervical samples. © 2014 Wiley Periodicals, Inc.

  7. Detection of bacteria in porous media using x-ray computed micro tomography

    Science.gov (United States)

    Polak, A.; Landon, M.; Grader, A. S.; Elsworth, D.

    2003-04-01

    Results are reported on experiments to determine if bacteria containing naturally occurring magnetite or magnetite that had been conjugated to the bacteria could be detected with CMT (Computed Micro Tomography). In-situ monitoring was done on Magnetospirillum, microaerophillic bacteria containing magnetite particles (Fe_3O_4) that were successfully grown in the lab. The bacteria were scanned in MSGM revised medium with and without ferric quinate and in a sample of sand grains. In another experiment, monoclonal anti-E. coli antibody that was immobilized onto BSA coated ferromagnetite particles and mixed with an aliquot of x-ray resistant E. coli bacteria was used. The sample was scanned in solution and in a Berea rock sample. We found that the Magnetospirillum can be detected in the porous media if the concentration of the bacteria is high enough, as the amount of the magnetite particles inside the bacteria is small. In the second experiment, the tagged E. coli was detected in a solution and within the Berea sample using the Computed Micro Tomography.

  8. A rapid Raman detection of deoxynivalenol in agricultural products.

    Science.gov (United States)

    Yuan, Jing; Sun, Chuanwen; Guo, Xiaoyu; Yang, Tianxi; Wang, Hui; Fu, Shuyue; Li, Chuanchuan; Yang, Haifeng

    2017-04-15

    Mycotoxin results in financial damage and considerable safety risks. In this paper, the possibility of portable Raman system-based surface-enhanced Raman scattering (SERS) for a rapid detection of deoxynivalenol (DON) a mycotoxin in cereals was investigated. Under an optimized condition, SERS analysis for pure DON solution has a wide dynamic concentration range from 10-7M to 10-2M with the limit of detection (LOD) down to 100nM. Density functional theory (DFT) analysis at the level of B3LYP/6-311++G(d, p) was also preformed for vibrational assignment. For practical application, the LOD of the proposed Raman method for both DON-contaminated corns and kidney beans were validated as 10-6M and the LOD for DON-contaminated oats was 10-4M. As a perspective, the SERS-based technology could be developed into an alternatively promising assay for on-field detection of DON residues at various cereals due to it high sensitivity and selectivity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Rapid detection of radiation-induced hydrocarbons in cooked ham.

    Science.gov (United States)

    Barba, C; Santa-María, G; Herraiz, M; Calvo, M M

    2012-03-01

    Solid phase microextraction (SPME) coupled with either gas chromatography-ionization flame detector (CG-FID) or multidimensional gas chromatography-mass spectrometry (MDGC-MS) was evaluated for its ability to detect volatile hydrocarbons produced during the irradiation of cooked ham. The chromatogram of an irradiated sample obtained using GC-FID showed a complex pattern of peaks, with several co-eluting peaks superimposed, indicating that the method was unlikely to resolve adequately the volatile hydrocarbons formed during irradiation. Using SPME-MDGC-MS 1-tetradecene (C(1-14:1)), n-pentadecane (C(15:0)), 1-hexadecene (C(1-16:1)), n-heptadecane (C(17:0)) and 8-heptadecene (C(8-17:1)) were detected in cooked ham irradiated at 0.5, 2, 4 and 8kGy. This method allows the detection of most n-alkanes and n-alkenes produced during the irradiation of the majority of fatty acids in cooked ham, namely oleic acid, stearic acid and palmitic acid. SPME is rapid and inexpensive and does not require organic solvents. The proposed SPME-MDGC-MS method allows the determination of radiolytic markers in cooked ham in less than 115min. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Detection of rapid-eye movements in sleep studies.

    Science.gov (United States)

    Agarwal, Rajeev; Takeuchi, Tomoka; Laroche, Suzie; Gotman, Jean

    2005-08-01

    One of the key features of rapid-eye movement (REM) sleep is the presence of bursts of REMs. Sleep studies routinely use REMs to classify sleep stages. Moreover, REM count or density has been used in studies involving learning and various psychiatric disorders. Most of these studies have been based on the visual identification of REMs, which is generally a very time-consuming task. This and the varying definitions of REMs across scorers have warranted the development of automatic REM detection methodologies. In this paper, we present a new detection scheme that combines many of the intrinsic properties of REMs and requires minimal parameter adjustments. In the proposed method, a single parameter can be used to control the REM detection sensitivity and specificity tradeoff. Manually scored training data are used to develop the method. We assess the performance of the method against manual scoring of individual REM events and present validation results using a separate data set. The ability of the method to discriminate fast horizontal ocular movement in REM sleep from other types of events is highlighted. A key advantage of the presented method is the minimal a priori information requirement. The results of training data (recordings from five subjects) show an overall sensitivity of 78.8% and specificity of 81.6%. The performance on the testing data (recording from five subjects different from the training data) showed overall sensitivity of 67.2% and specificity of 77.5%.

  11. Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform.

    Science.gov (United States)

    Kalsi, Sumit; Valiadi, Martha; Tsaloglou, Maria-Nefeli; Parry-Jones, Lesley; Jacobs, Adrian; Watson, Rob; Turner, Carrie; Amos, Robert; Hadwen, Ben; Buse, Jonathan; Brown, Chris; Sutton, Mark; Morgan, Hywel

    2015-07-21

    The widespread dissemination of CTX-M extended spectrum β-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16,800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes.

  12. Rapid immunochromatographic malarial antigen detection unreliable for detecting Plasmodium malariae and Plasmodium ovale

    NARCIS (Netherlands)

    Grobusch, M. P.; Hänscheid, T.; Zoller, T.; Jelinek, T.; Burchard, G. D.

    2002-01-01

    In order to determine the reliability of two commercial tests for the rapid detection of plasmodial antigen in cases of infection with Plasmodium ovale and Plasmodium malariae, the products were evaluated in four centers and a search of the relevant literature was performed. The results of the

  13. Rapid detection of cryptococcal antigen by a flow assay

    Directory of Open Access Journals (Sweden)

    Graziano Bargiggia

    2017-10-01

    Full Text Available Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii. Tests for quick detection of the cryptococcal antigen are needed. This study compares the performance of a lateral flow assay (LFA to the latex agglutination method. Thirty-five cryopreserved positive samples (sera and cerebrospinal fluids plus three negative sera for control have been examined. LFA does not need high-temperature incubation or enzyme pre-treatment. All the results, except for one serum, agree with previous obtained with latex agglutination method. LFA has an important clinical utility for its rapidity and sensitivity, and it also can be used as a point-of-care test.

  14. Rapid methods for detection of bacterial resistance to antibiotics.

    Science.gov (United States)

    March-Rosselló, Gabriel Alberto

    2017-03-01

    The most widely used antibiotic susceptibility testing methods in Clinical Microbiology are based on the phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. These conventional methods take typically 24hours to obtain results. Here we review the main techniques for rapid determination of antibiotic susceptibility. Data obtained with different methods such as molecular techniques, microarrays, commercial methods used in work routine, immunochromatographic methods, colorimetric methods, image methods, nephelometry, MALDI-TOF mass spectrometry, flow cytometry, chemiluminescence and bioluminescence, microfluids and methods based on cell disruption are analysed in detail. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  15. Concentration and detection of bacteria in virtual environmental samples based on non-immunomagnetic separation and quantum dots by using a laboratory-made system

    Science.gov (United States)

    Cheng, Zhi; Wu, Taihu; Chen, Feng; Du, Yaohua; Gu, Biao; Li, Chao; Yang, Zijian

    2012-03-01

    This study investigated a method that simultaneously detects three bacteria, Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus via an approach that combines un-immunized magnetic nanoparticles for the enrichment and antibody-conjugated quantum dots (QDs) as fluorescence markers, by using a laboratory-made system. In the enrichment procedure, the un-immunized superparamagnetic polymer nanoparticles and the three bacteria formed "beadcell" complex. Magnetic nanoparticles with different size were used and some interferents were added into the bacteria suspension respectively to check the influence on concentration efficiency. In the immuno-fluorescence labeling procedure, QDs with different emission wavelenghs were immobilized with antibody. Antibody conjugated QDs capture the bacteria selectively and specifically so that "sandwich" complex were formed. The suspension of the labeled bacteria was trickled onto a microporous membrane. A 450nm semiconductor laser was used as a part of the laboratory-made system to excite the QDs. Three PMT detectors were utilized to detect the fluorescence intensity. These un-immunized magnetic nanoparticles can be applied in nonspecific separation and enrichment of bacteria from environmental samples, and this method, of which the detection procedures are completed within 2 h, can be applied to the cost-effective and rapid detecting of bacterial contamination.

  16. Microbial agent detection using near-IR electrophoretic and spectral signatures (MADNESS) for rapid identification in detect-to-warn applications.

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, Anthony Lee; Bambha, Ray P.; VanderNoot, Victoria A.; Fruetel, Julia A.; Renzi, Ronald F.; Krafcik, Karen Lee

    2009-10-01

    Rapid identification of aerosolized biological agents following an alarm by particle triggering systems is needed to enable response actions that save lives and protect assets. Rapid identifiers must achieve species level specificity, as this is required to distinguish disease-causing organisms (e.g., Bacillus anthracis) from benign neighbors (e.g., Bacillus subtilis). We have developed a rapid (1-5 minute), novel identification methodology that sorts intact organisms from each other and particulates using capillary electrophoresis (CE), and detects using near-infrared (NIR) absorbance and scattering. We have successfully demonstrated CE resolution of Bacillus spores and vegetative bacteria at the species level. To achieve sufficient sensitivity for detection needs ({approx}10{sup 4} cfu/mL for bacteria), we have developed fiber-coupled cavity-enhanced absorbance techniques. Using this method, we have demonstrated {approx}two orders of magnitude greater sensitivity than published results for absorbing dyes, and single particle (spore) detection through primarily scattering effects. Results of the integrated CE-NIR system for spore detection are presented.

  17. Rapid detection of listeria spp. using an internalin A aptasensor based on carbon-metal nanohybrid structures

    Science.gov (United States)

    Vanegas, D. C.; Rong, Yue; Schwalb, N.; Hills, K. D.; Gomes, C.; McLamore, E. S.

    2015-05-01

    Foodborne outbreaks caused by Listeria monocytogenes continue to raise major public health concerns worldwide. In the United States alone, the centers for disease control and prevention have confirmed the occurrence of 183 cases of listeriosis with 39 fatalities within the last 3 years. Standard methods for the detection of pathogenic strains require up to 7 days to yield results, thus faster techniques with the same level of reliability for bacteria detection are desirable. This study reports on the development of a rapid, accurate, and sensitive electrochemical biosensor for rapid testing of Listeria spp. based on the selective binding of InlA aptamers to internalins in the cell membrane of the target bacteria. Hybrid nanomaterial platforms based on reduced graphene oxide and nanoplatinum were deposited onto Pt/Ir electrodes for enhancing electrochemical transduction during the recognition events. InlA aptamers were immobilized onto the nanomaterial platforms via metal-thiol adsorption. Aptamer loading onto different platform nanostructures was investigated through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The detection mechanism was evaluated by recording the electrochemical response to several bacterial dilutions in PBS buffer using the non-pathogenic species Listeria innocua. These preliminary results show that the aptasensor can be tuned for detection of Listeria concentrations as low as 100 CFU/ml in less than 3 hours (including incubation time and data analysis). The developed aptasensor opens a promising direction for rapid testing of Listeria monocytogenes in food products.

  18. Survival and detection of bacteria in an aquatic environment.

    OpenAIRE

    Amy, P. S.; Hiatt, H D

    1989-01-01

    A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable c...

  19. Rapid detection of flooded areas after Tohoku March 2011 tsunami

    Science.gov (United States)

    Faruolo, M.; Coviello, I.; Falconieri, A.; Lacava, T.; Pergola, N.; Tramutoli, V.

    2012-04-01

    In the recent years there has been a continuous increase of natural disasters affecting the world. Their catastrophic consequences generally have extreme impacts both from an economic and social point of view. The effects are the more severe the higher are the population density and concentration of industrial facilities and infrastructures in disaster-affected areas. Systems able to provide timely information about the affected areas may help in supporting decision makers to manage the crisis. In this context, satellite data may give a useful and effectively support. The devastating earthquake occurred on March 11, 2011 off the Japan coasts produced a huge tsunami which strongly affected the municipality of Miyagi, where more than two millions of inhabitants were used to live. In this paper, the Robust Satellite Techniques (RST) approach was used to detect areas affected by the flood due to such a tsunami. RST have been already applied with satisfactory results for the detection and monitoring of flooded area by using data acquired both from polar (NOAA-AVHRR and EOS-MODIS) and geostationary (MSG-SEVIRI) system. The potential of such data acquired in the visible and infrared regions of the electromagnetic spectrum has already been verified, allowing the development of an all-day detection and monitoring system of flooded areas. In particular, the potential of RST when implemented on optical data acquired by the Japan geostationary MT-SAT series satellites for rapid detection of flooded areas within Sendai district will be investigated in this study. MT-SAT, guaranteeing a temporal resolution of 30 minutes and a spatial resolution of up to 1km in the visible channel, together with the high sensitivity to detecting changes, offered by RST approach, should assure the capability for a prompt and effective detection, allowing for a near real time identification of the dynamics and the evolution of the disaster. Results and main achievements of this study will be

  20. Technique for rapid detection of phthalates in water and beverages

    KAUST Repository

    Zia, Asif I.

    2013-05-01

    ), USA. Results showed that the new sensor was able to detect different concentrations of phthalates in energy drinks. The experimental outcomes provided sufficient indication to favour the development of a low cost detection system for rapid quantification of phthalates in beverages for industrial use. © 2012 Elsevier Ltd. All rights reserved.

  1. Bacteria detected in the honeybee parasitic mite Varroa destructor collected from beehive winter debris.

    Science.gov (United States)

    Hubert, J; Erban, T; Kamler, M; Kopecky, J; Nesvorna, M; Hejdankova, S; Titera, D; Tyl, J; Zurek, L

    2015-09-01

    The winter beehive debris containing bodies of honeybee parasitic mite Varroa destructor is used for veterinary diagnostics. The Varroa sucking honeybee haemolymph serves as a reservoir of pathogens including bacteria. Worker bees can pick up pathogens from the debris during cleaning activities and spread the infection to healthy bees within the colony. The aim of this study was to detect entomopathogenic bacteria in the Varroa collected from the winter beehive debris. Culture-independent approach was used to analyse the mite-associated bacterial community. Total DNA was extracted from the samples of 10 Varroa female individuals sampled from 27 different sites in Czechia. The 16S rRNA gene was amplified using universal bacterial primers, cloned and sequenced, resulting in a set of 596 sequences representing 29 operational taxonomic units (OTU97). To confirm the presence of bacteria in Varroa, histological sections of the mites were observed. Undetermined bacteria were observed in the mite gut and fat tissue. Morganella sp. was the most frequently detected taxon, followed by Enterococcus sp., Pseudomonas sp., Rahnella sp., Erwinia sp., and Arsenophonus sp. The honeybee putative pathogen Spiroplasma sp. was detected at one site and Bartonella-like bacteria were found at four sites. PCR-based analysis using genus-specific primers enabled detection of the following taxa: Enterococcus, Bartonella-like bacteria, Arsenophonus and Spiroplasma. We found potentially pathogenic (Spiroplasma) and parasitic bacteria (Arsenophonus) in mites from winter beehive debris. The mites can be reservoirs of the pathogenic bacteria in the apicultures. © 2015 The Society for Applied Microbiology.

  2. Impedimetric detection of pathogenic bacteria with bacteriophages using gold nanorod deposited graphite electrodes

    OpenAIRE

    Moghtader, Farzaneh; Çongur, Gülşah; Zareie, Hadi M.; Erdem, Arzum; Pişkin, Erhan

    2016-01-01

    Electrochemical impedance spectroscopy (EIS) is applied for the detection of bacteria using bacteriophages as a bioprobe together with gold nanorods (GNRs). Escherichia coli-E. coli K12 was used as a model target bacteria and also for the propagation of its specific T4-phages. Gold nanorods (GNRs) were synthesized via a two-step protocol and characterized using different techniques. EIS measurements were conducted in an electrochemical cell consisting of a three electrode system. Single-use p...

  3. Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria.

    Science.gov (United States)

    Weber, D G; Sahm, K; Polen, T; Wendisch, V F; Antranikian, G

    2008-10-01

    The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

  4. Detection of sulphate-reducing bacteria in human saliva.

    Science.gov (United States)

    Heggendorn, Fabiano Luiz; Gonçalves, Lucio Souza; Dias, Eliane Pedra; Silva Junior, Arley; Galvão, Mariana Machado; Lutterbach, Márcia T S

    2013-11-01

    The aim of the current study was to investigate the presence of sulphate-reducing bacteria (SRB) in human saliva and correlate with oral and systemic conditions. Saliva samples were collected from 118 patients and inoculated in 2 ml of modified Postgate's E medium culture. After 28 days of incubation at 30°C the presence of SRB was identified by the production of sulphide. Of 118 saliva samples collected, 35 were positive for the presence of SRB. Three positive samples were randomly chosen to identify the species of SRB by PCR and sequenced. The three selected samples were identified as Desulfovibrio fairfieldensis, Desulfovibrio desulfuricans and Raoultella ornithinolytica. Gastritis (14.4%) was the most prevalent systemic disease, followed by diabetes (3.4%), while periodontitis (11%) and traumatic fibroma (4.2%) were the oral manifestations most frequently found. A bivariate analysis was performed to examine for the presence of SRB and the most prevalent systemic and oral manifestations. Only periodontitis showed a statistically significant association (p = 0.0003). The results showed SRB can be found in oral microbiota of healthy patients. Regarding the several conditions studied, there was a higher prevalence of SRB in patients with gastritis and patients with periodontal disease, with a possible correlation between the presence of SRB in the oral microbiota and periodontal disease.

  5. Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

    Directory of Open Access Journals (Sweden)

    Kirill V Sergueev

    Full Text Available BACKGROUND: Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3 CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

  6. Rapid Electrochemical Detection and Identification of Microbiological and Chemical Contaminants for Manned Spaceflight Project

    Science.gov (United States)

    Pierson, Duane; Botkin, Douglas; Gazda, Daniel

    2014-01-01

    Microbial control in the spacecraft environment is a daunting task, especially in the presence of human crew members. Currently, assessing the potential crew health risk associated with a microbial contamination event requires return of representative environmental samples that are analyzed in a ground-based laboratory. It is therefore not currently possible to quickly identify microbes during spaceflight. This project addresses the unmet need for spaceflight-compatible microbial identification technology. The electrochemical detection and identification platform is expected to provide a sensitive, specific, and rapid sample-to-answer capability for in-flight microbial monitoring that can distinguish between related microorganisms (pathogens and non-pathogens) as well as chemical contaminants. This will dramatically enhance our ability to monitor the spacecraft environment and the health risk to the crew. Further, the project is expected to eliminate the need for sample return while significantly reducing crew time required for detection of multiple targets. Initial work will focus on the optimization of bacterial detection and identification. The platform is designed to release nucleic acids (DNA and RNA) from microorganisms without the use of harmful chemicals. Bacterial DNA or RNA is captured by bacteria-specific probe molecules that are bound to a microelectrode, and that capture event can generate a small change in the electrical current (Lam, et al. 2012. Anal. Chem. 84(1): 21-5.). This current is measured, and a determination is made whether a given microbe is present in the sample analyzed. Chemical detection can be accomplished by directly applying a sample to the microelectrode and measuring the resulting current change. This rapid microbial and chemical detection device is designed to be a low-cost, low-power platform anticipated to be operated independently of an external power source, characteristics optimal for manned spaceflight and areas where power

  7. AOTF hyperspectral microscope imaging for foodborne bacteria detection

    Science.gov (United States)

    Food safety is an important public health issue worldwide. Researchers have developed many different methods for detecting foodborne pathogens; however, most technologies currently being used have limitations, in terms of speed, sensitivity and selectivity, for practical use in the food industry. Ac...

  8. Detection and quantification of probiotic bacteria using optimized ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-08

    Mar 8, 2010 ... Fish and shrimp sauce were used as a model for complex microbial community. Directly form samples, 4 ... highest yield, quality and performance. Moreover, the specificity of the primer set specific for ... superior in the detection and enumeration of many microorganisms. Nevertheless, their use has been ...

  9. Detection of gfp expression from gfp-labelled bacteria spot ...

    African Journals Online (AJOL)

    Green fluorescent protein (GFP) as a marker gene has facilitated biological research in plant-microbe interactions. However, there is one major limiting factor in the detection of GFP in living organisms whose cells emit background autofluorescence. In this study, Herbaspirillum sp. B501gfp1 bacterial cells were spot ...

  10. Rapid and sensitive detection of bisphenol a from serum matrix.

    Science.gov (United States)

    Lin, Xiaogang; Cheng, Cheng; Terry, Paul; Chen, Jiangang; Cui, Haochen; Wu, Jayne

    2017-05-15

    Bisphenol A (BPA) is an endocrine disrupting compound that may have adverse developmental, reproductive, neurological, and immune system effects. Low-level exposure to BPA is ubiquitous in human populations due to its widespread use in consumer products. Therefore, highly sensitive methods are needed to quantify BPA in various matrices including water, serum, and food products. In this study, we developed a simple, rapid, highly sensitive and specific sensor based on an aptamer probe and AC electrokinetics capacitive sensing method that successfully detected BPA at femto molar (fM) levels, which is an improvement over prior work by a factor of 10. We were able to detect BPA spiked in human serum as well as in maternal and cord blood within 30s. The sensor is responsive to BPA down to femto molar levels, but not to structurally similar compounds including bisphenol F (BPF) or bisphenol S (BPS) even at much higher concentration. Further development of this platform may prove useful in monitoring exposure to BPA and other small molecules in various matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Rapid detection of intracellular nanoparticles by electron microscopy

    Directory of Open Access Journals (Sweden)

    Kyoung Hwan Lee

    2010-03-01

    Full Text Available Recently, a number of nanoparticle carriers have provided new platforms for research in biotechnology and biomedicine. A particularly interest in these fields is the monitoring of nanoparticle delivery to target cells. Since the structures involved are on a nanometer scale, high resolution imaging, such as electron microscopy, is required. Aside from assessing the structural characteristics of the target sites localized with the nanoparticles, an electron microscope can also be used to observe the biological effects of the nanoparticles on the cells. It can also be used to test and detect a wide range of fluorescent nanoparticles and nanoassemblies. Although this approach has many advantages, most researchers are unwilling to try electron microscopy due to the complicated specimen preparation procedures and time-consuming process. Here, we developed a method to simplify the sample preparation and shorten the total processing time. In particular, double staining was removed, and cryo-preparation was included. Using this simple and rapid sample preparation, we were able to observe nanoparticles with high-contrast images of the cellular organelles. This efficient detection method can be applied to studies on nanoparticle drug delivery systems and nanoparticle-cell interactions.

  12. Designing Multicomponent Nanosystems for Rapid Detection of Circulating Tumor Cells.

    Science.gov (United States)

    Banerjee, Shashwat S; Khobragade, Vrushali; Khandare, Jayant

    2017-01-01

    Detection of circulating tumor cells (CTCs) in the blood circulation holds immense promise as it predicts the overall probability of patient survival. Therefore, CTC-based technologies are gaining prominence as a "liquid biopsy" for cancer diagnostics and prognostics. Here, we describe the design and synthesis of two distinct multicomponent magnetic nanosystems for rapid capture and detection of CTCs. The multifunctional Magneto-Dendrimeric Nano System (MDNS) composed of an anchoring dendrimer that is conjugated to multiple agents such as near infrared (NIR) fluorescent cyanine 5 NHS (Cy5), glutathione (GSH), transferrin (Tf), and iron oxide (Fe3O4) magnetic nanoparticle (MNP) for simultaneous tumor cell-specific affinity, multimodal high resolution confocal imaging, and cell isolation. The second nanosystem is a self-propelled microrocket that is composed of carbon nanotube (CNT), chemically conjugated with targeting ligand such as transferrin on the outer surface and Fe3O4 nanoparticles in the inner surface. The multicomponent nanosystems described here are highly efficient in targeting and isolating cancer cells thus benefiting early diagnosis and therapy of cancer.

  13. Tetrodotoxin-Producing Bacteria: Detection, Distribution and Migration of the Toxin in Aquatic Systems

    Directory of Open Access Journals (Sweden)

    Timur Yu. Magarlamov

    2017-05-01

    Full Text Available This review is devoted to the marine bacterial producers of tetrodotoxin (TTX, a potent non-protein neuroparalytic toxin. In addition to the issues of the ecology and distribution of TTX-producing bacteria, this review examines issues relating to toxin migration from bacteria to TTX-bearing animals. It is shown that the mechanism of TTX extraction from toxin-producing bacteria to the environment occur through cell death, passive/active toxin excretion, or spore germination of spore-forming bacteria. Data on TTX microdistribution in toxic organs of TTX-bearing animals indicate toxin migration from the digestive system to target organs through the transport system of the organism. The role of symbiotic microflora in animal toxicity is also discussed: despite low toxin production by bacterial strains in laboratory conditions, even minimal amounts of TTX produced by intestinal microflora of an animal can contribute to its toxicity. Special attention is paid to methods of TTX detection applicable to bacteria. Due to the complexity of toxin detection in TTX-producing bacteria, it is necessary to use several methods based on different methodological approaches. Issues crucial for further progress in detecting natural sources of TTX investigation are also considered.

  14. Development a rapid and accurate multiplex real time PCR method for the detection Chlamydia trachomatis and Mycoplasma hominis.

    Science.gov (United States)

    Safarkar, Roya; Mehrabadi, Jalil Fallah; Noormohammadi, Zahra; Mirnejad, Reza

    2017-11-01

    Sexually transmitted diseases easily spread among sexually active people and often have no symptoms. Rapid and accurate method for detecting these infections are necessary in early stages. The traditional detection methods of them are difficult and time-consuming. In this study, multiplex real time PCR was optimized for rapid identification of Chlamydia trachomatis and Mycoplasma hominis in a single tube and was performed with our designed primers. The sensitivity test was carried out to designed primers with diluted genomic DNA. To defined the specificity, non STD bacteria were used as DNA template. This study indicated that the developed multiplex real time PCR can be an effective alternative procedure to the conventional methods for rapid and accurate identification of C Chlamydia trachomatis and Mycoplasma hominis. Multiplex real-time PCR Results of them were checked with melting curves. The sensitivity of our designed primer by multiplex real time PCR for Chlamydia trachomatis and Mycoplasma hominis were 4.78×1010 and 8.35×1010 , respectively, Which the primers did not amplify any product from a non-STD species. Multiplex real time PCR by our new primers and analysis of melting curves were successfully usable for rapid and accurate detection of Chlamydia trachomatis and Mycoplasma hominis. This assay instead of traditional culture method, has considerable potential to be rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous and direct detection. © 2017 Wiley Periodicals, Inc.

  15. Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp.

    Science.gov (United States)

    Brewster, Jeffrey D; Paul, Moushumi

    2016-05-01

    Centrifugation is widely used to isolate and concentrate bacteria from dairy products before assay. We found that more than 98% of common pathogenic bacteria added to pasteurized, homogenized, or pasteurized homogenized milk were recovered in the pellet after centrifugation, whereas less than 7% were recovered from raw milk. The remaining bacteria partitioned into the cream layer of raw milk within 5 min, and half-saturation of the cream layer required a bacterial load of approximately 5×10(8) cfu/mL. Known treatments (e.g., heat, enzymes or solvents) can disrupt cream layer binding and improve recovery from raw milk, but can also damage bacteria and compromise detection. We developed a simple, rapid agitation treatment that disrupted bacteria binding to the cream layer and provided more than 95% recovery without affecting bacteria viability. Combining this simple agitation treatment with a previously developed real-time quantitative PCR assay allowed the detection of Salmonella spp. in raw milk at 4 cfu/mL within 3 h. To our knowledge, this is the first report of an effective method for achieving high centrifugal recovery of bacteria from raw milk without impairing bacterial viability. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. Rapid Detection of the Varicella Zoster Virus in Saliva

    Science.gov (United States)

    Pierson, Duane L.; Mehta, Satish K.; Cohrs, Randall J.; Gilden, Don H.; Harding, Robert E.

    2011-01-01

    Varicella zoster virus (VZV) causes chicken pox on first exposure (usually in children), and reactivates from latency causing shingles (usually in adults). Shingles can be extremely painful, causing nerve damage, organ damage, and blindness in some cases. The virus can be life-threatening in immune-compromised individuals. The virus is very difficult to culture for diagnosis, requiring a week or longer. This invention is a rapid test for VZV from a saliva sample and can be performed in a doctor s office. The kit is small, compact, and lightweight. Detec tion is sensitive, specific, and noninvasive (no needles); only a saliva sample is required. The test provides results in minutes. The entire test is performed in a closed system, with no exposure to infectious materials. The components are made mostly of inexpensive plastic injection molded parts, many of which can be purchased off the shelf and merely assembled. All biological waste is contained for fast, efficient disposal. This innovation was made possible because of discovery of a NASA scientists flight experiment showing the presence of VZV in saliva during high stress periods and disease. This finding enables clinicians to quickly screen patients for VZV and treat the ones that show positive results with antiviral medicines. This promotes a rapid recovery, easing of pain and symptoms, and reduces chances of complications from zoster. Screening of high-risk patients could be incorporated as part of a regular physical exam. These patients include the elderly, pregnant women, and immune-compromised individuals. In these patients, VZV can be a life-threatening disease. In both high- and low-risk patients, early detection and treatment with antiviral drugs can dramatically decrease or even eliminate the clinical manifestation of disease.

  17. A nanoporous membrane-based impedimetric immunosensor for label-free detection of pathogenic bacteria in whole milk.

    Science.gov (United States)

    Joung, Cho-Kyung; Kim, Han-Nah; Lim, Min-Cheol; Jeon, Tae-Joon; Kim, Hae-Yeong; Kim, Young-Rok

    2013-06-15

    We introduce a nanoporous membrane based impedimetric immunosensor for the label-free detection of bacterial pathogens in whole milk. A simple and rapid method to modify a commercially available alumina nanoporous membrane with hyaluronic acid (HA) effectively reduced the non-specific binding of biomolecules and other cells, and permitted successful immobilization of antibodies. Escherichia coli O157:H7, one of the most harmful food-borne pathogenic bacteria, was tested as a model pathogen in this study. The ionic impedance of electrolytes through nanopores, due to antibody-pathogen interactions, was monitored by impedance spectra and analyzed by normalized impedance change (NIC). The regression equation for the NIC at 1 kHz versus concentration of E. coli O157:H7 (10-10(5)cfu/ml) was obtained, and the detection limit found to be as low as 10 cfu/ml. In addition, the proposed immunosensor was successfully used for the detection of E. coli O157:H7 in whole milk samples with the detection limit as low as 83.7 cfu/ml with 95% probability. The specificity of the immunosensor was also demonstrated using non-target bacteria, including Staphylococcus aureus, Bacillus cereus, and non pathogenic E. coli DH5α. This study shows that a HA-functionalized nanoporous membrane-based impedimetric sensor is capable of detecting pathogenic bacteria in whole milk without any pretreatment. This is a significant step for evaluating the safety of food and environmental samples and other medical diagnostics. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Multifunctional magnetic-plasmonic nanoparticles for fast concentration and sensitive detection of bacteria using SERS.

    Science.gov (United States)

    Zhang, Lei; Xu, Jiajie; Mi, Luo; Gong, Heng; Jiang, Shaoyi; Yu, Qiuming

    2012-01-15

    Multifunctional magnetic-plasmonic Fe(3)O(4)-Au core-shell nanoparticles (Au-MNPs) were prepared for simultaneous fast concentration of bacterial cells by applying an external point magnetic field, and sensitive detection and identification of bacteria using surface-enhanced Raman spectroscopy (SERS). We demonstrated that a spread of a 10 μL drop of a mixture of 10(5) cfu/mL bacteria and 3 μg/mL Au-MNPs on a silicon surface can be effectively condensed into a highly compact dot within 5 min by applying an external point magnetic field, resulting in 60 times more concentrated bacteria in the dot area than on the spread area without concentration. Surrounded by dense uniformly packed Au-MNPs, bacteria can be sensitively and reproducibly detected directly using SERS. The principle component analysis (PCA) showed that three different Gram-negative bacterial strains can be clearly differentiated. We also demonstrated that the condensed multifunctional Au-MNPs dot can be used as a highly sensitive SERS-active substrate and a limit of detection better than 0.1 ppb was obtained in detection of small molecules such as 4-mercaptopyrine. This novel platform significantly simplifies the concentration and detection process, which holds great promise for applications in food safety, environmental monitoring, medical diagnoses, and chemical and biological threat detections. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Rapid quantification of rice root-associated bacteria by flow cytometry.

    Science.gov (United States)

    Valdameri, G; Kokot, T B; Pedrosa, F de O; de Souza, E M

    2015-03-01

    To understand the mechanism of plant-bacterium interaction, it is critical to enumerate epiphytic bacteria colonizing the roots of the host. We developed a new approach, based on flow cytometry, for enumerating these bacteria and used it with rice plants, 7 and 20 days after colonization with Herbaspirillum rubrisubalbicans and Azospirillum brasilense. The results were compared with those obtained with the traditional plate count method. Both methods gave similar numbers of H. rubrisubalbicans associated with rice roots (c. 10(9) CFU g(-1) ). However, flow cytometry gave a number of viable cells of rice-associated A. brasilense that was approx. 10-fold greater than that obtained with the plate count method. These results suggest that the plate count method can underestimate epiphytic populations. Flow cytometry has the additional advantage that it is more precise and much faster than the plate count method. Determination of precise number of root-associated bacteria is critical for plant-bacteria interaction studies. We developed a flow cytometry approach for counting bacteria and compared it with the plate count method. Our flow cytometry assay solves two major limitations of the plate count method, namely that requires long incubation times of up to 48 h and only determines culturable cells. This flow cytometry assay provides an efficient, precise and fast tool for enumerating epiphytic cells. © 2014 The Society for Applied Microbiology.

  20. Dopamine coated Fe3O4 nanoparticles as enzyme mimics for the sensitive detection of bacteria.

    Science.gov (United States)

    Mumtaz, Shazia; Wang, Li-Sheng; Hussain, Syed Zajif; Abdullah, Muhammad; Huma, Zille; Iqbal, Zafar; Creran, Brian; Rotello, Vincent M; Hussain, Irshad

    2017-11-02

    We report a simple and economical colorimetric bacterial sensing strategy with catalytic amplification using dopamine-capped iron oxide (Dop-Fe3O4) nanoparticles. These nanoparticles catalyse the oxidation of a chromogenic substrate in the presence of H2O2 into a green colored product. The catalytic activity of the nanoparticles is inhibited in the presence of bacteria, providing naked eye detection of bacteria at 10(4) cfu mL(-1) and by spectrophotometric detection down to 10(2) cfu mL(-1).

  1. Rapid Detection of Small Movements with GNSS Doppler Observables

    Science.gov (United States)

    Hohensinn, Roland; Geiger, Alain

    2017-04-01

    High-alpine terrain reacts very sensitively to varying environmental conditions. As an example, increasing temperatures cause thawing of permafrost areas. This, in turn causes an increasing threat by natural hazards like debris flow (e.g. rock glaciers) or rockfalls. The Institute of Geodesy and Photogrammetry is contributing to alpine mass-movement monitoring systems in different project areas in the Swiss Alps. A main focus lies on providing geodetic mass-movement information derived from GNSS static solutions on a daily and a sub-daily basis, obtained with low-cost and autonomous GNSS stations. Another focus is set on rapidly providing reliable geodetic information in real-time i.e. for an integration in early warning systems. One way to achieve this is the estimation of accurate station velocities from observations of range rates, which can be obtained as Doppler observables from time derivatives of carrier phase measurements. The key for this method lies in a precise modeling of prominent effects contributing to the observed range rates, which are satellite velocity, atmospheric delay rates and relativistic effects. A suitable observation model is then devised, which accounts for these predictions. The observation model, combined with a simple kinematic movement model forms the basis for the parameter estimation. Based on the estimated station velocities, movements are then detected using a statistical test. To improve the reliablity of the estimated parameters, another spotlight is set on an on-line quality control procedure. We will present the basic algorithms as well as results from first tests which were carried out with a low-cost GPS L1 phase receiver. With a u-blox module and a sampling rate of 5 Hz, accuracies on the mm/s level can be obtained and velocities down to 1 cm/s can be detected. Reliable and accurate station velocities and movement information can be provided within seconds.

  2. Development of highly sensitive handheld device for real-time detection of bacteria in food

    Science.gov (United States)

    Zhang, Kewei; Zhang, Anxue; Fu, Liling; Chin, Bryan A.; Cheng, Z.-Y.

    2010-04-01

    To ensure the safety of food, a detection device, which can detect/monitor the present of bacteria in a real-time manner and can be easily used for in-field tests, is highly desirable. Recently, magnetostrictive particles (MSPs) as a new type of high-performance biosensor have been developed. The detection of various bacteria and spores in food with high sensitivity has already been experimentally demonstrated. To fully use the technique for food safety, two miniaturized interrogation systems based on frequency-domain and time-domain technique are developed to fabricate a handheld detection device. The detection of Salmonella typhimurium (S. typhimurium) in liquid using a time-domain based interrogation system was demonstrated.

  3. Rapid Detection of Human Norovirus in Frozen Raspberries.

    Science.gov (United States)

    Summa, Maija; Maunula, Leena

    2017-10-10

    Raspberries have lately caused several human norovirus (HuNoV) outbreaks in Europe. In this study, we developed and evaluated for HuNoV reverse transcription (RT)-PCR detection in frozen raspberries extraction methods that have equal sensitivity but are less time-consuming than widely used methods based on polyethylene glycol (PEG) precipitation and chloroform-butanol purification. One method was applied to stored frozen raspberries linked to previous HuNoV outbreaks and berries on sale. In the virus elution-based Method 1, sparkling water eluted viruses most efficiently from the berries. Method 2, based on direct nucleic acid extraction with minor PEG supplement, yielded the highest number of positive findings (4 out of 9) at low virus concentration level of 100 genome copies HuNoV genogroup II per 25 g raspberries. Both methods showed approximately equal sensitivity to a method including PEG precipitation and chloroform-butanol purification. Two naturally contaminated berry samples linked to HuNoV outbreaks in 2006 and 2009 were still positive for HuNoV genogroup I, but all berry products purchased from a local store remained negative for HuNoV. In conclusion, this study presents two efficient and rapid methods which can be used in urgent HuNoV outbreak investigations, since the results of the virus analysis are available in a few hours.

  4. Rapid detection of biothreat agents based on cellular machinery.

    Energy Technology Data Exchange (ETDEWEB)

    Lane, Todd W.; Gantt, Richard W.

    2004-12-01

    This research addresses rapid and sensitive identification of biological agents in a complex background. We attempted to devise a method by which the specificity of the cellular transcriptional machinery could be used to detect and identify bacterial bio-terror agents in a background of other organisms. Bacterial cells contain RNA polymerases and transcription factors that transcribe genes into mRNA for translation into proteins. RNA polymerases in conjunction with transcription factors recognize regulatory elements (promoters) upstream of the gene. These promoters are, in many cases, recognized by the polymerase and transcription factor combinations of one species only. We have engineered a plasmid, for Escherichia coli, containing the virA promoter from the target species Shigella flexneri. This promoter was fused to a reporter gene Green Fluorescent Protein (GFP). In theory the indicator strain (carrying the plasmid) is mixed with the target strain and the two are lysed. The cellular machinery from both cells mixes and the GFP is produced. This report details the results of testing this system.

  5. Detection of bacteria in suspension using a superconducting Quantum interference device

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, H.L.; Myers, W.R.; Vreeland, V.J.; Alper, J.D.; Bertozzi, C.R.; Clarke, J.

    2003-06-09

    We demonstrate a technique for detecting magnetically-labeled Listeria monocytogenes and for measuring the binding rate between antibody-linked magnetic particles and bacteria. This assay, which is both sensitive and straightforward to perform, can quantify specific bacteria in a sample without the need to immobilize the bacteria or wash away unbound magnetic particles. In the measurement, we add 50 nm diameter superparamagnetic particles, coated with antibodies, to a liquid sample containing L. monocytogenes. We apply a pulsed magnetic field to align the magnetic dipole moments and use a high transition temperature Superconducting Quantum Interference Device (SQUID), an extremely sensitive detector of magnetic flux, to measure the magnetic relaxation signal when the field is turned off. Unbound particles randomize direction by Brownian rotation too quickly to be detected. In contrast, particles bound to L. monocytogenes are effectively immobilized and relax in about 1 s by rotation of the internal dipole moment. This Neel relaxation process is detected by the SQUID. The measurements indicate a detection limit of (5.6 {+-} 1.1) x 10{sup 6} L. monocytogenes for a 20 {micro}L sample volume. If the sample volume were reduced to 1 nL, we estimate that the detection limit could be improved to 230 {+-} 40 L. monocytogenes cells. Time-resolved measurements yield the binding rate between the particles and bacteria.

  6. Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria.

    Science.gov (United States)

    Sandetskaya, N; Engelmann, B; Brandenburg, K; Kuhlmeier, D

    2015-08-01

    The molecular detection of microorganisms in liquid samples generally requires their enrichment or isolation. The aim of our study was to evaluate the capture and pre-concentration of bacteria by immobilized particular cationic antimicrobial peptides, called synthetic anti-lipopolysaccharide peptides (SALP). For the proof-of-concept and screening of different SALP, the peptides were covalently immobilized on glass slides, and the binding of bacteria was confirmed by microscopic examination of the slides or their scanning, in case of fluorescent bacterial cells. The most efficient SALP was further tethered to magnetic beads. SALP beads were used for the magnetic capture of Escherichia coli in liquid samples. The efficiency of this strategy was evaluated using polymerase chain reaction (PCR). Covalently immobilized SALP were capable of capturing bacteria in liquid samples. However, PCR was hampered by the unspecific binding of DNA to the positively charged peptide. We developed a method for DNA recovery by the enzymatic digestion of the peptide, which allowed for a successful PCR, though the method had its own adverse impact on the detection and, thus, did not allow for the reliable quantitative analysis of the pathogen enrichment. Immobilized SALP can be used as capture molecules for bacteria in liquid samples and can be recommended for the design of the assays or decontamination of the fluids. For the accurate subsequent detection of bacteria, DNA-independent methods should be used.

  7. Seasonality of Coliform Bacteria Detection Rates in New Jersey Domestic Wells.

    Science.gov (United States)

    Atherholt, Thomas B; Procopio, Nicholas A; Goodrow, Sandra M

    2017-05-01

    It is important that indicators of fecal pollution are reliable. Coliform bacteria are a commonly used indicator of fecal pollution. As other investigators have reported elsewhere, we observed a seasonal pattern of coliform bacteria detections in domestic wells in New Jersey. Examination of a statewide database of 10 years of water quality data from 93,447 samples, from 78,207 wells, generated during real estate transactions, revealed that coliform bacteria were detected in a higher proportion of wells during warm weather months. Further examination of the seasonal pattern of other data, including well water pH, precipitation, ground and surface water temperatures, surface water coliform bacteria concentrations, and vegetation, resulted in the hypothesis that these bacteria may be derived from nonfecal (or environmentally adapted) as well as fecal sources. We provide evidence that the coliform seasonality may be the result of seasonal changes in groundwater extraction volumes (and to a lesser extent precipitation), and temperature-driven changes in the concentration of surface or near-surface coliform sources. Nonfecal coliform sources may not indicate the presence of fecal wastes and hence the potential presence of pathogens, or do so in an inconsistent fashion. Additional research is needed to identify the sources of the coliforms detected in groundwater. © 2016, National Ground Water Association.

  8. Scientists Detect Radio Emission from Rapidly Rotating Cosmic Dust Grains

    Science.gov (United States)

    2001-11-01

    Astronomers have made the first tentative observations of a long-speculated, but never before detected, source of natural radio waves in interstellar space. Data from the National Science Foundation's 140 Foot Radio Telescope at the National Radio Astronomy Observatory in Green Bank, W.Va., show the faint, tell-tale signals of what appear to be dust grains spinning billions of times each second. This discovery eventually could yield a powerful new tool for understanding the interstellar medium - the immense clouds of gas and dust that populate interstellar space. The NRAO 140 Foot Radio Telescope The NRAO 140-Foot Radio Telescope "What we believe we have found," said Douglas P. Finkbeiner of Princeton University's Department of Astrophysics, "is the first hard evidence for electric dipole emission from rapidly rotating dust grains. If our studies are confirmed, it will be the first new source of continuum emission to be conclusively identified in the interstellar medium in nearly the past 20 years." Finkbeiner believes that these emissions have the potential in the future of revealing new and exciting information about the interstellar medium; they also may help to refine future studies of the Cosmic Microwave Background Radiation. The results from this study, which took place in spring 1999, were accepted for publication in Astrophysical Journal. Other contributors to this paper include David J. Schlegel, department of astrophysics, Princeton University; Curtis Frank, department of astronomy, University of Maryland; and Carl Heiles, department of astronomy, University of California at Berkeley. "The idea of dust grains emitting radiation by rotating is not new," comments Finkbeiner, "but to date it has been somewhat speculative." Scientists first proposed in 1957 that dust grains could emit radio signals, if they were caused to rotate rapidly enough. It was believed, however, that these radio emissions would be negligibly small - too weak to be of any impact to

  9. Porphyrin-modified antimicrobial peptide indicators for detection of bacteria

    Directory of Open Access Journals (Sweden)

    Brandy J. Johnson

    2016-05-01

    Full Text Available This study demonstrates the potential of porphyrin modified antimicrobial peptides for indication of bacterial targets on the basis of changes in the spectrophotometric characteristics of the construct. Detection is a result of changes in the structure of the antimicrobial peptide upon target binding. Those constructs comprised of peptides that offer little or no change in conformation upon interaction with bacterial cells demonstrated negligible changes in absorbance and fluorescence when challenged using Escherichia coli or Bacillus cereus. CD analysis confirms the presence/absence of conformational changes in the porphyrin-peptide constructs. Differing spectrophotometric responses were observed for constructs utilizing different peptides. The incorporation of metals into the porphyrin component of the constructs was shown to alter their spectrophotometric characteristics as well as the resulting absorbance and fluorescence changes noted upon interaction with a target. The described constructs offer the potential to enable a new type of biosensing approach in which the porphyrin-peptide indicators offer both target recognition and optical transduction, requiring no additional reagents.

  10. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes

    NARCIS (Netherlands)

    Jansen, GJ; Mooibroek, M; Idema, J; Harmsen, HJM; Welling, GW; Degener, JE

    The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial

  11. Novel Simplified and Rapid Method for Screening and Isolation of Polyunsaturated Fatty Acids Producing Marine Bacteria

    Directory of Open Access Journals (Sweden)

    Ashwini Tilay

    2012-01-01

    Full Text Available Bacterial production of polyunsaturated fatty acids (PUFAs is a potential biotechnological approach for production of valuable nutraceuticals. Reliable method for screening of number of strains within short period of time is great need. Here, we report a novel simplified method for screening and isolation of PUFA-producing bacteria by direct visualization using the H2O2-plate assay. The oxidative stability of PUFAs in growing bacteria towards added H2O2 is a distinguishing characteristic between the PUFAs producers (no zone of inhibition and non-PUFAs producers (zone of inhibition by direct visualization. The confirmation of assay results was performed by injecting fatty acid methyl esters (FAMEs produced by selected marine bacteria to Gas Chromatography-Mass Spectrometry (GCMS. To date, this assay is the most effective, inexpensive, and specific method for bacteria producing PUFAs and shows drastically reduction in the number of samples thus saves the time, effort, and cost of screening and isolating strains of bacterial PUFAs producers.

  12. Construction of xylose dehydrogenase displayed on the surface of bacteria using ice nucleation protein for sensitive D-xylose detection.

    Science.gov (United States)

    Liang, Bo; Li, Liang; Mascin, Marco; Liu, Aihua

    2012-01-03

    A novel method was developed to detect d-xylose (INS 967) sensitively and selectively, which is based on a xylose dehydrogenase (XDH) cell-surface displaying system using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. With coenzyme NAD(+), the XDH-displayed bacteria facilitates the catalysis of the oxidization of xylose and the resultant NADH can be detected spectrometrically at 340 nm. The fusion protein was characterized by proteinase accessibility, Western blot, and enzyme activity assays. The established XDH surface displaying system did not inhibit the growth of the recombinant Escherichia coli strain. The XDH was mainly displayed on the surface of host cells, which is of high XDH activity and high D-xylose specificity. The optimal temperature and pH of cell displayed XDH were found at 30 °C and pH 8.0, respectively. The XDH-displayed bacteria can be used directly without further enzyme extraction and purification, and it improved the stability of the enzyme. Moreover, the cell-surface-displayed-protein-based approach showed a wide linear range (5-900 μM) and a low detection limit of 2 μM of d-xylose. More importantly, the recombinant cells could be used for precise detection of D-xylose from the real samples such as foods and degradation products of lignocellulose. The method shown here provides a simple, rapid, and low-cost strategy for the sensitive and selective measurement of D-xylose. In addition, the XDH-displayed bacteria showed an interesting response in developing electrochemical biosensors. Thus, the genetically engineered cells may find broad application in such biosensors and biocatalysts. Similarly, this type of genetic approach may be used for the expression of other intracellular enzymes of interest for certain purposes. © 2011 American Chemical Society

  13. An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk.

    Science.gov (United States)

    Soejima, Takashi; Minami, Jun-ichi; Yaeshima, Tomoko; Iwatsuki, Keiji

    2012-07-01

    Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451 bp) following EMA treatment to completely suppress the amplification of DNA of up to 7 logs (10(7) cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25 U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1 colony forming unit (CFU)/2.22 ml within 7.5 h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1 CFU/2.22 ml).

  14. Detection of multiple potentially pathogenic bacteria in Matang mangrove estuaries, Malaysia.

    Science.gov (United States)

    Ghaderpour, Aziz; Mohd Nasori, Khairul Nazrin; Chew, Li Lee; Chong, Ving Ching; Thong, Kwai Lin; Chai, Lay Ching

    2014-06-15

    The deltaic estuarine system of the Matang Mangrove Forest Reserve of Malaysia is a site where several human settlements and brackish water aquaculture have been established. Here, we evaluated the level of fecal indicator bacteria (FIB) and the presence of potentially pathogenic bacteria in the surface water and sediments. Higher levels of FIB were detected at downstream sampling sites from the fishing village, indicating it as a possible source of anthropogenic pollution to the estuary. Enterococci levels in the estuarine sediments were higher than in the surface water, while total coliforms and E. coli in the estuarine sediments were not detected in all samples. Also, various types of potentially pathogenic bacteria, including Klebsiella pneumoniae, Serratia marcescens and Enterobacter cloacae were isolated. The results indicate that the Matang estuarine system is contaminated with various types of potential human bacterial pathogens which might pose a health risk to the public. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Development of immunochromatographic strip test using fluorescent, micellar silica nanosensors for rapid detection of B. abortus antibodies in milk samples.

    Science.gov (United States)

    Vyas, Swati S; Jadhav, Sushma V; Majee, Sharmila B; Shastri, Jayanthi S; Patravale, Vandana B

    2015-08-15

    Presence of bacteria such as Brucella spp. in dairy products is an immense risk to public health. Point of care immunoassays are rapid in that they can quickly screen various samples in a relatively short amount of time, are sensitive, specific and offer a great advantage in accurate and fast diagnosis of infectious diseases. We have fabricated a point of care rapid diagnostic assay that employs fluorescent, micellar silica nanosensors capable of specifically detecting Brucella IgG antibodies in milk samples of afflicted animals. Currently, point of care detection assays are not commercially available for field testing of farm animals using milk samples. The nanosensing allows precise detection of antibodies with low sample volumes (50 μl). We demonstrate recognition of B. abortus antibodies through capture by fluorescent silica nanosensors using spiked and raw milk samples validated by ELISA and PCR. The test results are accurate and repeatable with high sensitivity and specificity, and a short assay time of 10 min for antigenic recognition and do not require any sample processing procedures such as isolation and separation. Additionally, well defined antigenic components and surface biomarkers of various disease causing microbes can be broadly incorporated within the purview of this technology for accurate and rapid detection of suspected bovine pathological conditions, and can largely enable rapid field testing that can be implemented in farms and food industry. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. ATP bioluminescence rapid detection of total viable count in soy sauce.

    Science.gov (United States)

    Yan, Shou-Lei; Miao, Su-Na; Deng, Shao-Ya; Zou, Min-Juan; Zhong, Fo-Sheng; Huang, Wen-Biao; Pan, Si-Yi; Wang, Qing-Zhang

    2012-01-01

    The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10(2) -3 × 10(4) CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB-941) and 96-well plates and could analyse 50-100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce. Copyright © 2011 John Wiley & Sons, Ltd.

  17. A novel gold nanoparticle-DNA aptamer-based plasmonic chip for rapid and sensitive detection of bacterial pathogens

    DEFF Research Database (Denmark)

    Sun, Yi; Phuoc Long, Truong; Wolff, Anders

    2016-01-01

    Gold nanoparticles (AuNPs)-based biosensors are emerging technologies for rapid detection of pathogens. However, it is very challenging to develop chip-based AuNP-biosensors for whole cells. This paper describes a novel AuNPs-DNA aptamer-based plasmonic assay which allows DNA aptamers to be detac......Gold nanoparticles (AuNPs)-based biosensors are emerging technologies for rapid detection of pathogens. However, it is very challenging to develop chip-based AuNP-biosensors for whole cells. This paper describes a novel AuNPs-DNA aptamer-based plasmonic assay which allows DNA aptamers...... to be detached from AuNPs when interacting with bacteria. The new strategy greatly increases the sensitivity and specificity of chip-based whole-cell biosensing....

  18. 9 CFR 113.26 - Detection of viable bacteria and fungi except in live vaccine.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Detection of viable bacteria and fungi except in live vaccine. 113.26 Section 113.26 Animals and Animal Products ANIMAL AND PLANT HEALTH... in live vaccine. Each serial and subserial of biological product except live vaccines shall be tested...

  19. Fluorescence techniques to detect and to assess viability of plant pathogenic bacteria

    NARCIS (Netherlands)

    Chitarra, L.G.

    2001-01-01

    Plant pathogenic bacteria cause major economic losses in commercial crop production worldwide every year. The current methods used to detect and to assess the viability of bacterial pathogens and to test seed lots or plants for contamination are usually based on plate assays or on

  20. Polydiacetylene-Based Liposomes: An "Optical Tongue" for Bacteria Detection and Identification

    Science.gov (United States)

    West, Matthew R.; Hanks, Timothy W.; Watson, Rhett T.

    2009-01-01

    Food- and water-borne bacteria are a major health concern worldwide. Current detection methods are time-consuming and require sophisticated equipment that is not always readily available. However, new techniques based on nanotechnology are under development that will result in a new generation of sensors. In this experiment, liposomes are…

  1. Detection and characterization of foodborne pathogenic bacteria with hyperspectral microscope imaging

    Science.gov (United States)

    Rapid detection and identification of pathogenic microorganisms naturally occurring during food processing are important in developing intervention and verification strategies. In the poultry industry, contamination of poultry meat with foodborne pathogens (especially, Salmonella and Campylobacter) ...

  2. Rapid Methods for detection of Veterinary Drug residues in Meat

    Directory of Open Access Journals (Sweden)

    Chandan

    2010-10-01

    Full Text Available The use of substances having hormonal or thyreostatic action as well as b-agonists is banned in many countries. However, sometimes forbidden drugs may be added to feeds for illegal administration to farm animals for promoting increased muscle development or increased water retention and thus obtain an economical benefit. The result is a fraudulent overweight of meat but, what is worse, residues of these substances may remain in meat and may pose a real threat to the consumer either through exposure to the residues, transfer of antibiotic resistance or allergy risk. This has exerted a great concern among the meat consumers. The control of the absence of these forbidden substances in animal foods and feeds is regulated in the European Union by Directive 96/23/EC on measures to monitor certain substances and residues in live animals and animal products. Analytical methodology, including criteria for identification and confirmation, for the monitoring of compliance was also given in Decisions 93/256/EEC and 93/257/EEC. More recently, Decision 2002/657/EC provided rules for the analytical methods to be used in testing of official samples. New substances with anabolic properties are being detected year by year increasing the list of forbidden compounds to be tested. Furthermore, the extended practice consisting in the use of “cocktails” (mixtures of low amounts of several substances that exert a synergistic effect to have a similar growth promotion, reduces the margin for an effective analytical detection. Thus, the evolution of the “black market” is making really difficult to have an effective analytical control of the residues of these substances in foods of animal origin. Control laboratories must face an increasing demand of analysis like the growing number of residues to be analysed in different types of samples, the strict guidelines for analytical methodologies according to the latest Directives, the increased costs of such new

  3. Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.

    Directory of Open Access Journals (Sweden)

    Robert J Clifford

    Full Text Available Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.

  4. Rapid detection of fluorescent and chemiluminescent total coliforms and Escherichia coli on membrane filters.

    Science.gov (United States)

    Van Poucke, S O; Nelis, H J

    2000-11-01

    The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-beta-D-glycosides as enzyme substrates and a UV-lamp for visualization. The most rapid procedures based on this approach for the demonstration of these indicator bacteria in water take 6-7.5 h to complete. As part of efforts to further reduce the detection time, an improved two-step procedure for the fluorescence or chemiluminescence labelling of microcolonies of E. coli/TC on a membrane filter has been developed. Essential features of this approach include a separation of the bacterial propagation and target enzyme induction from the actual enzymatic labelling, the use of improved fluorogenic, i.e., 4-trifluoromethylumbelliferyl-beta-D-glycosides and fluorescein-di-beta-D-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for beta-glucuronidase/beta-galactosidase, of enzyme inducers, of special membrane filters and of polymyxin B to promote the cellular uptake of the substrate. This labelling procedure has been applied in conjunction with different detection devices including a UV-lamp, CCD-cameras, X-ray film and the ChemScan((R)) RDI. Using the former three, microcolonies of pure cultures could be detected within 5.5-6.5 h, but waterborne E. coli/TC may fail to form microcolonies in this short time period, thus yielding poor sensitivity and a high false-negative rate. In contrast, a quantitative enumeration was feasible in less than 4 h with the ChemScan((R)) RDI, owing to its ability to detect both microcolonies and non-dividing single cells.

  5. A new method to extract dental pulp DNA: application to universal detection of bacteria.

    Directory of Open Access Journals (Sweden)

    Lam Tran-Hung

    Full Text Available BACKGROUND: Dental pulp is used for PCR-based detection of DNA derived from host and bacteremic microorganims. Current protocols require odontology expertise for proper recovery of the dental pulp. Dental pulp specimen exposed to laboratory environment yields contaminants detected using universal 16S rDNA-based detection of bacteria. METHODOLOGY/PRINCIPAL FINDINGS: We developed a new protocol by encasing decontaminated tooth into sterile resin, extracting DNA into the dental pulp chamber itself and decontaminating PCR reagents by filtration and double restriction enzyme digestion. Application to 16S rDNA-based detection of bacteria in 144 teeth collected in 86 healthy people yielded a unique sequence in only 14 teeth (9.7% from 12 individuals (14%. Each individual yielded a unique 16S rDNA sequence in 1-2 teeth per individual. Negative controls remained negative. Bacterial identifications were all confirmed by amplification and sequencing of specific rpoB sequence. CONCLUSIONS/SIGNIFICANCE: The new protocol prevented laboratory contamination of the dental pulp. It allowed the detection of bacteria responsible for dental pulp colonization from blood and periodontal tissue. Only 10% such samples contained 16S rDNA. It provides a new tool for the retrospective diagnostic of bacteremia by allowing the universal detection of bacterial DNA in animal and human, contemporary or ancient tooth. It could be further applied to identification of host DNA in forensic medicine and anthropology.

  6. A high speed detection platform based on surface-enhanced Raman scattering for monitoring antibiotic-induced chemical changes in bacteria cell wall.

    Directory of Open Access Journals (Sweden)

    Ting-Ting Liu

    Full Text Available Rapid and accurate diagnosis for pathogens and their antibiotic susceptibility is critical for controlling bacterial infections. Conventional methods for determining bacterium's sensitivity to antibiotic depend mostly on measuring the change of microbial proliferation in response to the drug. Such "biological assay" inevitably takes time, ranging from days for fast-growing bacteria to weeks for slow-growers. Here, a novel tool has been developed to detect the "chemical features" of bacterial cell wall that enables rapid identification of drug resistant bacteria within hours. The surface-enhanced Raman scattering (SERS technique based on our newly developed SERS-active substrate was applied to assess the fine structures of the bacterial cell wall. The SERS profiles recorded by such a platform are sensitive and stable, that could readily reflect different bacterial cell walls found in Gram-positive, Gram-negative, or mycobacteria groups. Moreover, characteristic changes in SERS profile were noticed in the drug-sensitive bacteria at the early period (i.e., approximately 1 hr of antibiotic exposure, which could be used to differentiate them from the drug-resistant ones. The SERS-based diagnosis could be applied to a single bacterium. The high-speed SERS detection represents a novel approach for microbial diagnostics. The single-bacterium detection capability of SERS makes possible analyses directly on clinical specimen instead of pure cultured bacteria.

  7. Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom Tricholoma matsutake.

    Science.gov (United States)

    Kataoka, Ryota; Siddiqui, Zaki Anwar; Kikuchi, Junichi; Ando, Masaki; Sriwati, Rina; Nozaki, Ai; Futai, Kazuyoshi

    2012-04-01

    The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a 'shiro'. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.

  8. Rapid whole genome sequencing for the detection and characterization of microorganisms directly from clinical samples

    DEFF Research Database (Denmark)

    Hasman, Henrik; Saputra, Dhany; Sicheritz-Pontén, Thomas

    2014-01-01

    Whole genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples this could further reduce diagnostic time and thereby improve control and treatment. A major bottle-neck is the availability of fast and reliable bioinformatics...... microbiology, WGS of isolated bacteria and by directly sequencing on pellets from the urine. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples, but only in pure culture from 17. WGS improved the identification of the cultivated bacteria and almost complete...

  9. Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

    Science.gov (United States)

    Bushon, R.N.; Brady, A.M.; Likirdopulos, C.A.; Cireddu, J.V.

    2009-01-01

    Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

  10. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks.

    Science.gov (United States)

    Wei, B; Cha, S-Y; Kang, M; Park, I-J; Moon, O-K; Park, C-K; Jang, H-K

    2013-05-01

    Infections with Pasteurella multocida, Salmonella enterica, Riemerella anatipestifer, and Escherichia coli result in high morbidity and mortality, which cause significant economic loss in the poultry industry. It can be difficult to distinguish these pathogens based on clinical signs because these pathogens can cause similar clinical signs and coinfections can occur. Thus, rapid and sensitive detection of these 4 major bacterial pathogens are important in ducks. The aim of this study was to develop a multiplex PCR (mPCR) assay for simultaneously detecting and identifying these 4 pathogenic bacteria in a single tube reaction. The target genes used were KMT1 of P. multocida, the invasion protein gene of S. enterica, 16S rDNA of R. anatipestifer, and the alkaline phosphatase gene of E. coli. The detection limit of the assay for all bacterial DNA was 10 pg. The mPCR did not produce any nonspecific amplification products when tested against other related pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Mycoplasma gallinarum, Mycoplasma synoviae, and Mycoplasma gallisepticum, which can also infect ducks. We applied mPCR to field samples, and the results were the same as the single PCR results. These results suggest that mPCR for the 4 bacteria is a useful and rapid technique to apply to field samples.

  11. Development of a Calcium Phosphate Nanocomposite for Fast Fluorogenic Detection of Bacteria

    Directory of Open Access Journals (Sweden)

    Claudio R. Martínez

    2014-09-01

    Full Text Available Current procedures for the detection and identification of bacterial infections are laborious, time-consuming, and require a high workload and well-equipped laboratories. Therefore the work presented herein developed a simple, fast, and low cost method for bacterial detection based on hydroxyapatite nanoparticles with a nutritive mixture and the fluorogenic substrate. Calcium phosphate ceramic nanoparticles were characterized and integrated with a nutritive mixture for the early detection of bacteria by visual as well as fluorescence spectroscopy techniques. The composite was obtained by combining calcium phosphate nanoparticles (Ca:P ratio, 1.33:1 with a nutritive mixture of protein hydrolysates and carbon sources, which promote fast bacterial multiplication, and the fluorogenic substrate 4-methylumbellipheryl-β-d-glucuronide (MUG. The composite had an average particle size of 173.2 nm and did not show antibacterial activity against Gram-negative or Gram-positive bacteria. After an Escherichia coli suspension was in contact with the composite for 60–90 min, fluorescence detected under UV light or by fluorescence spectrophotometer indicated the presence of bacteria. Intense fluorescence was observed after incubation for a maximum of 90 min. Thus, this calcium phosphate nanocomposite system may be useful as a model for the development of other nanoparticle composites for detection of early bacterial adhesion.

  12. A rapid phospholipase A2 bioassay using 14C-oleate-labelled E. coli bacterias.

    Science.gov (United States)

    Meyer, T; von Wichert, P; Weins, D

    1989-02-01

    Two methods of phospholipase A2 determination using 14C-labelled E. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved 14C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A2 regarding pH and Ca2+ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A2 levels, but that the filter method is superior in terms of sensitivity and workload.

  13. [Significance of bacteria detection with filter paper method on diagnosis of diabetic foot wound infection].

    Science.gov (United States)

    Zou, X H; Zhu, Y P; Ren, G Q; Li, G C; Zhang, J; Zou, L J; Feng, Z B; Li, B H

    2017-02-20

    Objective: To evaluate the significance of bacteria detection with filter paper method on diagnosis of diabetic foot wound infection. Methods: Eighteen patients with diabetic foot ulcer conforming to the study criteria were hospitalized in Liyuan Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from July 2014 to July 2015. Diabetic foot ulcer wounds were classified according to the University of Texas diabetic foot classification (hereinafter referred to as Texas grade) system, and general condition of patients with wounds in different Texas grade was compared. Exudate and tissue of wounds were obtained, and filter paper method and biopsy method were adopted to detect the bacteria of wounds of patients respectively. Filter paper method was regarded as the evaluation method, and biopsy method was regarded as the control method. The relevance, difference, and consistency of the detection results of two methods were tested. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of filter paper method in bacteria detection were calculated. Receiver operating characteristic (ROC) curve was drawn based on the specificity and sensitivity of filter paper method in bacteria detection of 18 patients to predict the detection effect of the method. Data were processed with one-way analysis of variance and Fisher's exact test. In patients tested positive for bacteria by biopsy method, the correlation between bacteria number detected by biopsy method and that by filter paper method was analyzed with Pearson correlation analysis. Results: (1) There were no statistically significant differences among patients with wounds in Texas grade 1, 2, and 3 in age, duration of diabetes, duration of wound, wound area, ankle brachial index, glycosylated hemoglobin, fasting blood sugar, blood platelet count, erythrocyte sedimentation rate, C-reactive protein, aspartate aminotransferase, serum creatinine, and

  14. Turnbull - Early Detection and Rapid Response Team 2007

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Biocontrol agents and chemicals to facilitate the rapid response phase of the project will be purchased and applied and a Washington Service Corps AmeriCorps member...

  15. Turnbull - Early Detection and Rapid Response Team 2010

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Funding from this grant will allow for the purchase of biocontrol agents and chemicals to facilitate the rapid response phase of the project and to provide funds to...

  16. Turnbull - Early Detection and Rapid Response Team 2008

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Funding from this grant will allow for the purchase of biocontrol agents and chemicals to facilitate the rapid response phase of the project and to provide match for...

  17. Comparison of two methods for detection of fecal indicator bacteria used in water quality monitoring of the Three Gorges Reservoir.

    Science.gov (United States)

    Wang, Zhaodan; Xiao, Guosheng; Zhou, Nong; Qi, Wenhua; Han, Lin; Ruan, Yu; Guo, Dongqin; Zhou, Hong

    2015-12-01

    Scientifically sound methods to rapidly measure fecal indicator bacteria are important to ensure safe water for drinking and recreational purposes. A total of 200 water samples obtained from the Three Gorges Reservoir during three successive one-year study periods (October 2009 to September 2012) were analyzed using multiple-tube fermentation (MTF) and most probable numbers combined with polymerase chain reaction (MPN-PCR). The MPN-PCR method was found to be significantly more sensitive than the MTF method for detecting Escherichia coli and Enterococcus spp., and of equal sensitivity for detecting total coliforms when all surface water samples were grouped together. The two analytical methods had a strong, significant relationship, but MPN-PCR took only 12-18hr, compared with the 3-8days needed using the MTF method. Bacterial concentrations varied per sampling site but were significantly lower in the mainstream of the Yangtze River than those in the backwater areas of tributaries. The water quality of 85.8% of water samples from the mainstream was suitable for use as a centralized potable water source, while the water quality of 52.5% of water samples from the backwater areas was unsuitable for recreational activities. Relationships between fecal indicator bacteria showed significant correlation (r=0.636-0.909, pcoliforms, E. coli, and Enterococcus spp. in surface water. Copyright © 2015. Published by Elsevier B.V.

  18. Lanthanide-labeled immunochromatographic strips for the rapid detection of Pantoea stewartii subsp. stewartii.

    Science.gov (United States)

    Zhang, Fan; Zou, Mingqiang; Chen, Yan; Li, Jinfeng; Wang, Yanfei; Qi, Xiaohua; Xue, Qiang

    2014-01-15

    The lateral flow immunoassay is used in commercial pregnancy detection, and is an accepted point-of-care testing technique. The most widely used format for lateral flow immunochromatographic strips uses gold nanoparticles for colorimetric detection. However, this method often suffers from poor quantitative discrimination and low analytical sensitivity. To address these limitations, lanthanide chelate-loaded silica nanoparticles have been used as fluorescent labels. The fluorescent nanoparticles can easily bind to antibodies, with dextran as a linker. The strip reader described here was based on a sandwich immunoreaction performed on a strip, using lanthanide-labeled antibodies that served as signal vehicles for the fluorescent readout. The strip reader was used as a quantitative test system. In this work, Pantoea stewartii subsp. stewartii (Pss) was used as a model analyte to demonstrate the use of the strip reader. Under optimal conditions, the detection limit was determined as 10(3)cfu/mL. The quantification limit was calculated to be 10(4)cfu/mL. The detection limit for Pss was 100 times lower than those displayed by colloidal gold-labeled strips or ELISAs. No cross-reactions were observed with the other nine strains, indicating the good specificity of the Pss strip. This strip showed good stability in repeated tests. The tests using the fluorescence immunochromatographic strip were easy to perform, rapid, and sensitive. Methods using fluorescence strips and a strip reader have the potential to be a powerful tool for the quantification of bacteria. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Application of Reverse Transcriptase -PCR (RT-PCR) for rapid detection of viable Escherichia coli in drinking water samples.

    Science.gov (United States)

    Molaee, Neda; Abtahi, Hamid; Ghannadzadeh, Mohammad Javad; Karimi, Masoude; Ghaznavi-Rad, Ehsanollah

    2015-01-01

    Polymerase chain reaction (PCR) is preferred to other methods for detecting Escherichia coli (E. coli) in water in terms of speed, accuracy and efficiency. False positive result is considered as the major disadvantages of PCR. For this reason, reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to solve this problem. The aim of present study was to determine the efficiency of RT-PCR for rapid detection of viable Escherichia coli in drinking water samples and enhance its sensitivity through application of different filter membranes. Specific primers were designed for 16S rRNA and elongation Factor II genes. Different concentrations of bacteria were passed through FHLP and HAWP filters. Then, RT-PCR was performed using 16srRNA and EF -Tu primers. Contamination of 10 wells was determined by RT-PCR in Arak city. To evaluate RT-PCR efficiency, the results were compared with most probable number (MPN) method. RT-PCR is able to detect bacteria in different concentrations. Application of EF II primers reduced false positive results compared to 16S rRNA primers. The FHLP hydrophobic filters have higher ability to absorb bacteria compared with HAWB hydrophilic filters. So the use of hydrophobic filters will increase the sensitivity of RT-PCR. RT-PCR shows a higher sensitivity compared to conventional water contamination detection method. Unlike PCR, RT-PCR does not lead to false positive results. The use of EF-Tu primers can reduce the incidence of false positive results. Furthermore, hydrophobic filters have a higher ability to absorb bacteria compared to hydrophilic filters.

  20. Cassette bacteria detection system. [for monitoring the sterility of regenerated water in spacecraft

    Science.gov (United States)

    1974-01-01

    The design, fabrication, and testing of an automatic bacteria detection system, with a zero-g capability, based on the filter-capable approach, and intended for monitoring the sterility of regenerated water in spacecraft is discussed. The principle of detection is based on measuring the increase in chemiluminescence produced by the action of bacterial porphyrins on a luminol-hydrogen peroxide mixture. Viable organisms are detected by comparing the signal of an incubated water sample with an unincubated control. High signals for the incubated water sample indicate the presence of viable organisms.

  1. Monitoring of Water Spectral Pattern Reveals Differences in Probiotics Growth When Used for Rapid Bacteria Selection.

    Directory of Open Access Journals (Sweden)

    Aleksandar Slavchev

    Full Text Available Development of efficient screening method coupled with cell functionality evaluation is highly needed in contemporary microbiology. The presented novel concept and fast non-destructive method brings in to play the water spectral pattern of the solution as a molecular fingerprint of the cell culture system. To elucidate the concept, NIR spectroscopy with Aquaphotomics were applied to monitor the growth of sixteen Lactobacillus bulgaricus one Lactobacillus pentosus and one Lactobacillus gasseri bacteria strains. Their growth rate, maximal optical density, low pH and bile tolerances were measured and further used as a reference data for analysis of the simultaneously acquired spectral data. The acquired spectral data in the region of 1100-1850nm was subjected to various multivariate data analyses - PCA, OPLS-DA, PLSR. The results showed high accuracy of bacteria strains classification according to their probiotic strength. Most informative spectral fingerprints covered the first overtone of water, emphasizing the relation of water molecular system to cell functionality.

  2. A Rapid Detection Method of Brucella with Quantum Dots and Magnetic Beads Conjugated with Different Polyclonal Antibodies

    Science.gov (United States)

    Song, Dandan; Qu, Xiaofeng; Liu, Yushen; Li, Li; Yin, Dehui; Li, Juan; Xu, Kun; Xie, Renguo; Zhai, Yue; Zhang, Huiwen; Bao, Hao; Zhao, Chao; Wang, Juan; Song, Xiuling; Song, Wenzhi

    2017-03-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Traditional methods for detection of Brucella spp. take 48-72 h that does not meet the need of rapid detection. Herein, a new rapid detection method of Brucella was developed based on polyclonal antibody-conjugating quantum dots and antibody-modified magnetic beads. First, polyclonal antibodies IgG and IgY were prepared and then the antibody conjugated with quantum dots (QDs) and immunomagnetic beads (IMB), respectively, which were activated by N-(3-dimethylaminopropyl)- N'-ethylcar-bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form probes. We used the IMB probe to separate the Brucella and labeled by the QD probe, and then detected the fluorescence intensity with a fluorescence spectrometer. The detection method takes 105 min with a limit of detection of 103 CFU/mL and ranges from 10 to 105 CFU/mL ( R 2 = 0.9983), and it can be well used in real samples.

  3. Detection, isolation, and preliminary characterization of bacteria contaminating plant tissue cultures

    Directory of Open Access Journals (Sweden)

    Monika Kałużna

    2014-01-01

    Full Text Available In order to limit the contamination problem in plant tissue cultures experiments on selection of media suitable for detection and isolation of bacteria contaminating plant tissue explants, and preliminary characterization of isolates were made. In the first experiment aiming at detection of bacteria in plant explants four strains representing genera most often occurring at our survey of plant tissue cultures, and earlier isolated and identified (Bacillus, Methylobacterium, Pseudomonas and Xanthomonas were streaked on five bacteriological media (NA, King B, K, R2A and 523 and on the medium used for plant culture initiation – ½ MS with milk albumin (IM. All strains grew on all media but on K and IM at the slowest rate and on 523 medium at the fastest. The IM medium proved to be useful for immediate bacteria detection at the initial stage of culture. In the second experiment, aiming at characterization of isolates on the basis of colony growth and morphology 14 strains (Agrobacterium, Bacillus, Curtobacterium, Flavobacterium, Lactobacillus, Methylobacterium – 2 strains Mycobacterium, Paenibacillus, Plantibacterium, Pseudomonas, Stenotrophomonas, Xanthomonas, and species Serratia marcescens were streaked on five microbiological media: KB, NBY, YDC, YNA and YPGA. All strains grew on all those media but at different rates. The only exception was the strain of Lactobacillus spp., which did not grow on King B medium. This medium allowed the detection of such characteristic traits as fluorescence (Pseudomonas and secretion of inclusions (Stenotrophomonas. The third experiment was focussed on assessment of the sensitivity of detection of specific bacteria in pure cultures and in plant tis- sue cultures using standard PCR and BIO-PCR techniques with genus specific primers and 2 methods of DNA isolation. Results showed that the use of Genomic Mini kit enabled an increase of the sensitivity by 100 times as compared to extraction of DNA by boiling

  4. Rapid detection of genetic modification for GMO monitoring in agriculture

    Directory of Open Access Journals (Sweden)

    Petrović Sofija

    2015-01-01

    Full Text Available Transgenic technology has expanded the ways of new genetic variability creation. Genetically modified organisms (GMOs are organisms which total genome is altered in a way that could not happen in nature. GM crops recorded a steady increase in its share in agricultural production. However, for the most part, GMO in agriculture has been limited to two cultivars - soy and corn, and the two genetic modifications, the total herbicide resistance and pest of the Lepidoptera genus. In order to monitor cultivation and trade of GMOs, tests of different precision are used, qualitatively and/or quantitatively determining the presence of genetic modification. Tests for the rapid determination of the presence of GM are suitable, since they can be implemented quickly and accurately, in terms of declared sensitivity, outside or in the laboratory. The example of the use of rapid tests demonstrates their value in use for rapid and efficient monitoring.

  5. Rapid evolutionary adaptation to elevated salt concentrations in pathogenic freshwater bacteria Serratia marcescens

    OpenAIRE

    Ketola, Tarmo; Hiltunen, Teppo

    2014-01-01

    Rapid evolutionary adaptions to new and previously detrimental environmental conditions can increase the risk of invasion by novel pathogens. We tested this hypothesis with a 133-day-long evolutionary experiment studying the evolution of the pathogenic Serratia marcescens bacterium at salinity niche boundary and in fluctuating conditions. We found that S. marcescens evolved at harsh (80 g/L) and extreme (100 g/L) salt conditions had clearly improved salt tolerance than those evolved in the ot...

  6. Rapid Detection & Identification of Bacillus Species using MALDI-TOF/TOF and Biomarker Database

    Science.gov (United States)

    2006-06-01

    Dieckmann, R., Graeber, I., Kaesler, I., Szewzyk, U., and von D6hren, H. (2005). Rapid screening and dereplication of bacterial isolates from marine sponges...Bacteriology, vol. 2. Gram- positive bacteria other than Actinomycetes . 1’ ed. Baltimore: Williams & Wilkins, section 13, p 1104-1139. 18 DRDC Suffield

  7. Rapid Biosynthesis of Silver Nanoparticles Using Culture Supernatant of Bacteria with Microwave Irradiation

    Directory of Open Access Journals (Sweden)

    N. Saifuddin

    2009-01-01

    Full Text Available The development of rapid and reliable processes for the synthesis of nanosized materials is of great importance in the field of nanotechnology. Synthesis of silver nanoparticles using microorganism have been reported, but the process is rather slow. In this paper, we describe a novel combinatorial synthesis approach which is rapid, simple and “green” for the synthesis of metallic nanostructures of noble metals such as silver (Ag, by using a combination of culture supernatanant of Bacillus subtilis and microwave (MW irradiation in water in absence of a surfactant or soft template. It was found that exposure of culture supernatanant of Bacillus subtilis and microwave irradiation to silver ion lead to the formation of silver nanoparticles. The silver nanoparticles were in the range of 5-60 nm in dimension. The nanoparticles were examined using UV-Visible Spectroscopy, and Transmission Electron Microscopy (TEM analyses. The formation of nanoparticles by this method is extremely rapid, requires no toxic chemicals and the nanoparticles are stable for several months. The main conclusion is that the bio-reduction method to produce nanoparticles is a good alternative to the electrochemical methods.

  8. A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA).

    Science.gov (United States)

    Kuo, Jong-Tar; Cheng, Chiu-Yu; Huang, Hsiao-Han; Tsao, Chia-Fen; Chung, Ying-Chien

    2010-03-01

    Methods to detect the presence of coliform bacteria in drinking water usually involve a series of complex cultivating steps that are time-consuming and subject to external influences. For this reason, the new 16S rRNA probe has been developed in this study as an alternative detector PCR-ELISA technique that does not involve the culture of bacteria and that is able to detect, identify, and quantify the representative coliform species present in water samples. Our results indicate that this technique is both rapid (detection time of 4 h) and accurate (1.4% error rate). The limit of detection (LOD) was 5 CFU/100 ml for total coliforms, which meets the standards set by most countries for drinking water. Our comparative study demonstrated that this PCR-ELISA method is superior to current conventional methods in terms of detection time, LOD, and accuracy.

  9. Lab-on-a-chip for rapid electrochemical detection of nerve agent Sarin

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin; Loke, Weng Keong; Nguyen, Nam-Trung

    2014-01-01

    This paper reports a lab-on-a-chip for the detection of Sarin nerve agent based on rapid electrochemical detection. The chemical warfare agent Sarin (C4H10FO2P, O-isopropyl methylphosphonofluoridate) is a highly toxic organophosphate that induces rapid respiratory depression, seizures and death w...

  10. DETECTION OF AEROSOLIZED BACTERIA IN EXPIRED AIR SAMPLES FROM ASIAN ELEPHANTS (ELEPHAS MAXIMUS).

    Science.gov (United States)

    Burke, Sophie M; Vogelnest, Larry; Thompson, Paul; Tovey, Euan R; Williamson, Peter

    2017-06-01

    Elephant-mediated transmission of tuberculosis is assumed to be similar to human models, which state close and prolonged contact with an infected individual is required for transmission. Although considered a risk factor for infection, several case studies have reported that close contact with an elephant is not always necessary for transmission, and the role of aerosolized bacteria remains unclear. To investigate aerosol-mediated transmission of pathogenic bacteria from elephants, a method for the detection of aerosols using an adapted sampling system was developed. A commensal bacterium was isolated from the upper respiratory tract of elephants ( Elephas maximus ) and was used as a proxy organism to detect aerosolized droplets in the sampling system. It was found that elephants are capable of producing aerosolized bacterial particles of a size small enough to remain airborne for prolonged periods and penetrate the lower regions of the human respiratory tract.

  11. Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama by PCR

    Directory of Open Access Journals (Sweden)

    María E Cerón Cucchi

    Full Text Available The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.

  12. Detection of malaria parasites by microscopy and rapid diagnostic ...

    African Journals Online (AJOL)

    The effectiveness of Rapid Diagnostic Test Kit (RDT) was compared with microscopy for the evaluation of malaria infection in children and pregnant women attending two selected health facilities in Lagos State, south-western, Nigeria. A total of 482 patients comprising 252 pregnant women (mean age: 26.86±4.46 years) ...

  13. An ultrasensitive young interferometer handheld sensor for rapid virus detection

    NARCIS (Netherlands)

    Ymeti, Aurel; Subramaniam, Vinod; Beumer, Tom A M; Kanger, Johannes S

    Future viral outbreaks are a major threat to societal and economic development throughout the world. A rapid, sensitive and easy-to-use test for viral infections is essential to prevent and control such viral pandemics. Furthermore, a compact, portable device is potentially very useful in remote or

  14. Rapid detection of methicillin-resistant staphylococci by multiplex PCR

    African Journals Online (AJOL)

    Administrator

    2010-11-08

    Nov 8, 2010 ... Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16: 11141-11156. Fang H, Hedin G (2003). Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay.

  15. Development of rapid, specific and sensitive detection of Cucumber ...

    African Journals Online (AJOL)

    detection of the CMV in infected plants using a monoclonal and polyclonal antibodies. Dotimmunobinding assays (DIBA) are useful alternatives to microtitre plate enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies were readily used for detected CMV by TAS-ELISA and DIBA of infected plants.

  16. Rapid detection of Ganoderma lucidum and assessment of inhibition ...

    African Journals Online (AJOL)

    Molecular and immunological methods have been applied for detecting the Ganoderma disease of coconut. Polyclonal antibodies (PAbs) raised against basidiocarp protein of Ganoderma were used for detection. For polymerase chain reaction (PCR) test, the primer generated from the internal transcribed spacer region one ...

  17. Fluorescence techniques to detect and to assess viability of plant pathogenic bacteria

    OpenAIRE

    Chitarra, L.G.

    2001-01-01

    Plant pathogenic bacteria cause major economic losses in commercial crop production worldwide every year. The current methods used to detect and to assess the viability of bacterial pathogens and to test seed lots or plants for contamination are usually based on plate assays or on serological techniques. Plating methods provide information about cell viability, but are generally laborious and time-consuming. Serological techniques, such as immunofluorescence microscopy (IF) and enzym...

  18. DGGE detection and screening of lignocellulolytic bacteria from the termite gut of Coptotermes formosanus

    Directory of Open Access Journals (Sweden)

    Mathew, G.M.

    2011-01-01

    Full Text Available Aims: Termites thrive in terrestrial ecosystems and play an important role in the bio-recycling of lignocellulose. The objective of this study is to isolate and detect bacteria from the termite gut of Coptotermes formosanus and to screen their various enzyme activities by qualitative methods. In addition, this study was aimed to isolate lignin and furfural tolerant strains for various industrial bioprocesses.Methodology and Results: In this study, 50 worker termites of Coptotermes formosanus were collected from dead trees, from a forest in Taichung, Taiwan in June 2008 and the composition of the microbial flora from the termite guts was analyzed by DGGE analysis. The results proved that anaerobic and facultatively anaerobic bacteria consisting of Acinetobacter, Bacteroides thetaiotaomicron, Escherichia coli, and Caulobacter readily existed in the guts of termites. Although the majority of these gut symbionts have not yet been cultivated or identified, some related bacteria were isolated. Two isolates 1-8 and 2-2 of Genus Bacillus, exhibited endocellulase, protease, lipase, amylase, peroxidase and lignin peroxidase activity. Under aerobic conditions, the growth density of isolate 1-8 cultured in 1000 ppm lignin containing MSM medium was two-folds higher than cultured in MSM medium without lignin. Furthermore, the isolate 1-8 was tolerant to 20 mM furfural supplemented in the MSM medium. HPLC analysis confirmed Bacillus isolate 1-8 could degrade up to 15 mM furfural.Conclusion, significance and impact of study: Hind gut bacteria from C. formosanus were detected by culture independent DGGE method. Also, Bacillus isolates 1-8 and 2-2 obtained by culture dependent methods could withstand higher concentration of furfural and as well as lignin. These isolates may be co-cultured with ethanologenic bacteria and be used as an industrial biocatalyst for biofuel production.

  19. Rapid Detection of Pediatric Bacteriuria Using Narrow Angle Forward Laser Scattering Technology (NAFLST) with Bacterioscan

    Science.gov (United States)

    Cline, Adriana; Jhaveri, Ravi; Levinson, Kara; Miller, Melissa

    2017-01-01

    Abstract Background Pediatric urinary tract infections (UTI) are common, but culture-based diagnosis can take up to 48 hours. This time delay means patients are exposed to potentially unnecessary antibiotics. The sensitivity of screening urinalysis can vary, so rapid detection of UTI by another means would be beneficial. Narrow Angle Forward Laser Scattering Technology (NAFLST) with Bacterioscan can rapidly detect bacteriuria by shining a laser continuously through a liquid sample containing replicating bacteria, and graphing the degree of light refraction over time. Higher degrees of light refraction represent higher initial bacterial load and continued bacterial growth. After 3 hours, the optical scatter classifies a sample as either Likely Positive or Likely Negative. We compared Bacterioscan results to culture data in pediatric patients to assess the ability to diagnose UTI and avoid unnecessary urine culture. Methods This protocol was approved by the UNC Biomedical Institutional Review Board. Over one month, 169 pediatric (<18 yo) urine cultures were collected as part of routine patient care. An individual urine sample and 2.5mL of Sterile Tryptic Soy Broth were pipetted into a Bacterioscan micro-curette. Bacterioscan labeled the specimen as Likely Positive or Likely Negative after a 3 hour period. Results were then compared with urine culture results obtained by routine microbiologic methods. Results Of the 169 urine cultures, 96 were positive, but only 27 were positive for uropathogens. Bacterioscan was 100% sensitive and 58.4% specific in predicting clinically relevant/pathogenic bacterial growth in culture (PPV 31.3%, NPV 100%), and 70.8% sensitive and 75.3% specific in predicting any bacterial growth (PPV 79.0%, NPV 66.2%). If a “Likely Positive” Bacterioscan result had been used in our study population to screen urine samples for culture, then 58% (83/142) of negative urine cultures would have been eliminated with no UTIs missed. Conclusion By

  20. Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species

    Energy Technology Data Exchange (ETDEWEB)

    Siddiqi, S.H.; Hwangbo, C.C.; Silcox, V.; Good, R.C.; Snider, D.E. Jr.; Middlebrook, G.

    1984-10-01

    Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on /sup 14/CO/sub 2/ evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of /sup 14/CO/sub 2/ evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or ''no viable organisms'' within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

  1. Multiplexed plasmon sensor for rapid label-free analyte detection.

    Science.gov (United States)

    Rosman, Christina; Prasad, Janak; Neiser, Andreas; Henkel, Andreas; Edgar, Jonathan; Sönnichsen, Carsten

    2013-07-10

    Efficient and cost-effective multiplexed detection schemes for proteins in small liquid samples would bring drastic advances to fields like disease detection or water quality monitoring. We present a novel multiplexed sensor with randomly deposited aptamer functionalized gold nanorods. The spectral position of plasmon resonances of individual nanorods, monitored by dark-field spectroscopy, respond specifically to different proteins. We demonstrate nanomolar sensitivity, sensor recycling, and the potential to upscale to hundreds or thousands of targets.

  2. Bioluminescence-based identification of nisin producers - a rapid and simple screening method for nisinogenic bacteria in food samples.

    Science.gov (United States)

    Virolainen, Nina; Guglielmetti, Simone; Arioli, Stefania; Karp, Matti

    2012-08-17

    We present a simple and rapid method for screening nisin producers that directly identifies nisinogenic bacteria by induction of bioluminescence within the Lactococcus lactis NZ9800lux biosensor strain (Immonen and Karp, 2007, Biosensors and Bioelectronics 22, 1982-7). An overlay of putative nisinogenic colonies with the biosensor strain gives identification results within 1h. Functionality and specificity of the method were verified by screening nisin producers among 144 raw milk colonies and a panel of 91 lactococcal strains. Studies performed on strains and colonies that did not induce bioluminescence but inhibited growth of the biosensor demonstrated that only nisinogenic bacteria can cause induction. Bacteria known to produce bacteriocins other than nisin failed to induce bioluminescence, further verifying the specificity of the assay. We discovered a non-inducing but inhibitory lactococcal strain harboring a modified nisin Z gene, and demonstrated that the source of the inhibitory action is not a non-inducing variant of nisin, but a bacteriocin of lower molecular weight. The concentration of nisin producers in a raw milk sample was 1.3 × 10(2)CFU/ml. We identified from raw milk a total of seven nisin Z producing L. lactis subsp. lactis colonies, which were shown by genetic fingerprinting to belong to three different groups. Among the panel of 91 lactococci, four strains were nisin A producers, and one strain harbored the modified nisin Z gene. The method presented here is robust, cost-effective and simple to perform, and avoids the pitfalls of traditional screening methods by directly specifying the identity of the inhibitory substance. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Improved detection of Escherichia coli and coliform bacteria by multiplex PCR.

    Science.gov (United States)

    Molina, Felipe; López-Acedo, Elena; Tabla, Rafael; Roa, Isidro; Gómez, Antonia; Rebollo, José E

    2015-06-04

    The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified. In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively. The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

  4. Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry

    Science.gov (United States)

    Massad-Ivanir, Naama; Shtenberg, Giorgi; Raz, Nitzan; Gazenbeek, Christel; Budding, Dries; Bos, Martine P.; Segal, Ester

    2016-11-01

    Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >107 cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps.

  5. Control of coliform bacteria detected from diarrhea associated patients by extracts of Moringa oleifera.

    Science.gov (United States)

    Rahman, M M; Rahman, M M; Akhter, S; Jamal, M A H M; Pandeya, D R; Haque, M A; Alam, M F; Rahman, A

    2010-03-01

    The aims of this study were to determine the total population of coliform bacteria in the samples collected from diarrhea associated patients from the local area of Bangladesh and to examine the antibacterial efficacy of leaf extracts of Moringa oleifera (Moringaceae) against the isolated coliform bacteria. The coliform bacteria detected in these samples by some microbial-biochemical tests such as Escherichia coli, Shigella dysenteriae, Salmonella sp., Enterobacter sp., Klebsiella pneumoniae and Serratia marcescens. The total isolation rate of coliform bacterial species was ranged from 38.01-3.51%. At the concentration of 300 ig/disc, the organic extracts of hexane, chloroform, ethyl acetate and methanol extracts of Moringa oleifera leaf exhibited a remarkable antibacterial effect against all the tested bacterial pathogens. The zones of inhibition against all the tested bacterial pathogens were found in the range of 8.0 to 23.2 mm, along with their respective minimum inhibitory concentration (MIC) values ranging from 62.5-1000 ig/mL. The results obtained in this study suggest that the extracts from Moringa oleifera leaf can be a source of natural antimicrobials with potential applications in pharmaceutical industry to control coliform bacteria.

  6. High sensitive virus and bacteria detection using plasma-surface-functionalized and antibody-integrated carbon nanomaterials

    Science.gov (United States)

    Nagatsu, Masaaki

    2015-09-01

    In this study we will present our recent results on the virus and bacteria detection system using the surface-functionalized carbon-encapsulated magnetic nanoparticles (NPs) fabricated by dc arc discharge, and carbon nanotube(CNT) dot-array prepared with a combined thermal and plasma CVD system. Surface functionalization of their surfaces has been carried out by plasma chemical modification using a low-pressure RF plasma for carbon-encapsulated magnetic NPs, and an ultrafine atmospheric pressure plasma jet(APPJ) for CNT dot-array substrate. After immobilization of the relevant biomolecules onto the surface of nano-structured materials, we have carried out the experiments on virus or bacteria detection using these surface-functionalized nano-structured materials. From the preliminary experiments with carbon-encapsulated magnetic NPs, we confirmed that influenza A (H1N1) virus concentration of 17.3-fold was achieved by using anti-influenza A virus hemagglutinin (HA) antibody. We have also confirmed a rapid and sensitive detection of Salmonella using the proposed method. The feasibility of CNT dot-array as a microarray biosensor has been studied by maskless functionalization of amino (-NH2) and carboxyl (-COOH) groups onto CNTs by using a ultrafine APPJ with a micro-capillary. The experimental results of chemical derivatization with the fluorescent dye showed that the CNT dot-array was not only functionalized with amino group and carboxyl group, but was also functionalized without any interference between functional groups. The success of maskless functionalization in the line pattern provides a feasibility of a multi-functionalization CNT dot-array device for future application of a microarray biosensor. This work has been supported in part by Grant-in-Aid for Scientific Research (Nos. 21110010 and 25246029) from the JSPS and the International Research Collaboration and Scientific Publication Grant (DIPA-23.04.1.673453/2015) from DGHE Indonesia.

  7. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array‐based multiplex assay

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.

    2011-01-01

    for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV‐1, CyHV‐2 and CyHV‐3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were......Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been...... one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad‐range PCR amplification combined with DNA array hybridization...

  8. Evaluation of a quantitative real-time PCR for rapid detection of Riemerella Anatipestifer infection in birds.

    Science.gov (United States)

    Zhang, Qingshan; Wan, Chunhe; Li, Chenxi; Bai, Xiaofei; Liu, Ming; Liu, Siguo; Zhang, Yun

    2017-08-05

    To establish an accurate, rapid, and a quantifiable method for the detection of Riemerella anatipestifer infection, a widespread infectious disease in birds, we developed a TaqMan-based real-time PCR assay by using DtxR gene-specific primers and a TaqMan probe. The standard curve established with a linear correlation (R2) of 0.998 and efficiency of 99% between the Ct value and the logarithm of the plasmid copy number. The reproducibility and specificity of the real-time PCR assay were confirmed by using plasmids containing DtxR genes or DNAs extracted from well-known bacteria or viruses causing duck diseases. The real-time PCR assay was 100 times more sensitive than the conventional PCR. The results reveal that the established real-time PCR assay might be a useful method for diagnosis and quantitative detection of Riemerella anatipestifer in birds.

  9. Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

    Directory of Open Access Journals (Sweden)

    Harris Dana M

    2013-01-01

    Full Text Available Abstract Background Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens. Methods Identification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx, as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH. Results In blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5. Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6. Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9% as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI −92.4 to 267.1. Identification by PNA-FISH averaged 16.4 hours (95% CI −57.3 to 90.0. Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%. For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS, discontinuation of vancomycin could result in savings of $20.00/day. Conclusions In this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to

  10. Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

    Science.gov (United States)

    Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

    2016-02-17

    The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

  11. Specific and Rapid Detection of Camellia oleifera Anthracnose ...

    African Journals Online (AJOL)

    The internal transcribed spacer (ITS) regions and the 5.8S rRNA gene of strain C1 of the pathogenic fungus Colletetrichum gloeosporioides were sequenced in order to design specific PCR primers for pathogen detection. Alignment of the sequence data of strain C1 and the other Colletetrichum species obtained from the ...

  12. Rapid and sensitive detection of potyvirus infecting tropical tuber ...

    African Journals Online (AJOL)

    Specific cDNA probe was generated from the amplicon, and the probe was then successfully used for the diagnosis of the potyviruses infecting the major tuber crops through biotinylated nucleic acid spot hybridisation. The specific probe developed could detect the potyviruses infecting tuber crops namely SPFMV, DsMV ...

  13. Rapid response flood detection using the MSG geostationary satellite

    DEFF Research Database (Denmark)

    Proud, Simon Richard; Fensholt, Rasmus; Rasmussen, Laura Vang

    2011-01-01

    A novel technique for the detection of flooded land using satellite data is presented. This new method takes advantage of the high temporal resolution of the Spinning Enhanced Visible and InfraRed Imager (SEVIRI) aboard the Meteosat Second Generation (MSG) series of satellites to derive several p...

  14. Development of rapid, specific and sensitive detection of Cucumber ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-03-06

    Mar 6, 2009 ... antibodies are biotinylated, and biotin bound antibodies revealed by reaction with a universal streptavidin-enzyme conjugate. Recently, an assay which incorporates biotiny- lated antibodies, and conjugates comprising streptavidin coupled to homopolymers of HRP improved detection sensitivity by 12 – 25 ...

  15. Rapid detection of Ganoderma lucidum and assessment of inhibition ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... revolutionized the field of plant pathology in diagnosing various plant pathogens (Henson and French, 1993). The internal transcribed spacer (ITS) regions of ribosomal. RNA gene (rDNA) have been selected as specific targets for PCR detection of Ganoderma (Utomo and Niepold,. 2000). Secondly, the ...

  16. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

    DEFF Research Database (Denmark)

    Batchelor, Miranda; Hopkins, Katie L; Liebana, Ernesto

    2008-01-01

    We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum ......We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended...

  17. Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips.

    Science.gov (United States)

    Ma, Qinglin; Liu, Houming; Ye, Feidi; Xiang, Guangxin; Shan, Wanshui; Xing, Wanli

    2017-12-01

    To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Contrast Enhancement of Mammograms for Rapid Detection of Microcalcification Clusters

    Directory of Open Access Journals (Sweden)

    Hajar Moradmand

    2014-08-01

    Full Text Available Introduction Breast cancer is one of the most common types of cancer among women.  Early detection of breast cancer is the key to reducing the associated mortality rate. The presence of microcalcifications clusters (MCCs is one of the earliest signs of breast cancer. Due to poor imaging contrast of mammograms and noise contamination, radiologists may overlook some diagnostic signs, specially the presence of MCCs. In order to improve cancer detection, image enhancement methods are often used to aid radiologists. In this paper, a new enhancement method was presented for the accurate and early detection of MCCs in mammograms. Materials and Methods The proposed system consisted of four main steps including: 1 image scaling;2 breast region segmentation;3 noise cancellation using a filter, which is sensitive to MCCs; and 4 contrast enhancement of mammograms using Contrast-Limited Adaptive Histogram Equalization (CLAHE and wavelet transform. To evaluate this method, 120 clinical mammograms were used. Results To evaluate the performance of the image enhancement algorithm, contrast improvement index (CII was used. The proposed enhancement method in this research achieved the highest CII in comparison with other methods applied in this study. The Validity of the results was confirmed by an expert radiologist through visual inspection. Conclusion Detection of MCCs significantly improved in contrast-enhanced mammograms. The proposed method could be helpful for radiologists to easily detect MCCs; it could also decrease the number of biopsies and reduce the frequency of clinical misdiagnosis. Moreover, it could be useful prior to segmentation or classification stages.

  19. Rapid in situ toxicity testing with luminescent bacteria Photorhabdus luminescens and Vibrio fischeri adapted to a small portable luminometer.

    Science.gov (United States)

    Masner, Petr; Javůrková, Barbora; Bláha, Luděk

    2017-02-01

    The present study demonstrates development of a rapid testing protocol based on a small portable luminometer using flash kinetic assessment of bacterial bioluminescence. The laboratory comparisons based on six model organic toxicants and two metals showed significant correlations between responses of freshwater bacteria Photorhabdus luminescens and standard marine bacterial species Vibrio fisheri. While P. luminescens was less sensitive in standard arrangements, the responses of both organisms were comparable in the newly introduced portable luminometer setup. The applicability and reproducibility of the portable luminometer protocol was further demonstrated in the assessment of 43 European wastewater effluents that were simultaneously tested for toxicity and analysed for 150 organic and 20 inorganic contaminants grouped into 13 major chemical classes. Clear association between the toxic responses in both compared bacterial species and the elevated levels of inorganic compounds (toxic metals), chlorophenols and benzotriazole anticorrosives was observed. The new protocol with a portable luminometer provides a fast (30 s) response and may be used as a tool for rapid in situ toxicity evaluation of freshwater environmental samples such as effluents.

  20. Polythiophene synthesis coupled to quartz crystal microbalance and Raman spectroscopy for detecting bacteria.

    Science.gov (United States)

    Kengne-Momo, R P; Lagarde, F; Daniel, Ph; Pilard, J F; Durand, M J; Thouand, G

    2012-12-01

    A simple electrochemical procedure was used for the synthesis of a polythiophene containing para-benzenesulfonyl chloride groups. The obtained polymer was shown to be very reactive and directly able to covalently bind nucleophile biomolecules. Protein A and a specific antibody were then successively immobilized on the conductive polymer through a covalent bonding of Protein A with the as-prepared linker for bacteria trapping purpose. All reactions were controlled in situ by cyclic voltammetry, quartz crystal microbalance and Raman spectroscopy. The results were compared to those previously obtained on gold surface modified with the same chemical linker. The conductive polymer led to a very high rate of antibody recognition compared to the gold surface and to literature, probably due to a large available surface obtained after polymerization. One example of pathogenic bacteria "Salmonella enterica paratyphi" detection was successfully tested on the substrates. The presented results are promising for the future design of simple and inexpensive immunocapture-based sensors.

  1. Rapid Detection of Visually Provocative Animals by Preschool Children and Adults

    Science.gov (United States)

    Penkunas, Michael J.; Coss, Richard G.

    2013-01-01

    The ability to detect dangerous animals rapidly in complex landscapes has been historically important during human evolution. Previous research has shown that snake images are more readily detected than images of benign animals. To provide a stringent test of superior snake detection in preschool children and adults, Experiment 1 consisted of two…

  2. Survival and detection of coliforms, Enterobacteriaceae, and gram-negative bacteria in Greek yogurt.

    Science.gov (United States)

    Hervert, C J; Martin, N H; Boor, K J; Wiedmann, M

    2017-02-01

    Despite the widespread use of coliforms as indicator bacteria, increasing evidence suggests that the Enterobacteriaceae (EB) and total gram-negative groups more accurately reflect the hygienic status of high-temperature, short-time pasteurized milk and processing environments. If introduced into milk as postpasteurization contamination, these bacteria may grow to high levels and produce a wide range of sensory-related defects. However, limited information is available on the use and survival of bacterial hygiene indicators in dairy products outside of pasteurized fluid milk and cheese. The goal of this study was to (1) provide information on the survival of a diverse set of bacterial hygiene indicators in the low pH environment of Greek yogurt, (2) compare traditional and alternative detection methods for their ability to detect bacterial hygiene indicators in Greek yogurt, and (3) offer insight into optimal hygiene indicator groups for use in low-pH fermented dairy products. To this end, we screened 64 bacterial isolates, representing 24 dairy-relevant genera, for survival and detection in Greek yogurt using 5 testing methods. Before testing, isolates were inoculated into plain, 0% fat Greek yogurt (pH 4.35 to 4.65), followed by a 12-h hold period at 4 ± 1°C. Yogurts were subsequently tested using Coliform Petrifilm (3M, St. Paul, MN) to detect coliforms; Enterobacteriaceae Petrifilm (3M), violet red bile glucose agar and the D-Count (bioMérieux, Marcy-l'Étoile, France) to detect EB; and crystal violet tetrazolium agar (CVTA) to detect total gram-negative bacteria. Overall, the non-EB gram-negative isolates showed significantly larger log reductions 12 h after inoculation into Greek yogurt (based on bacterial numbers recovered on CVTA) compared with the coliform and noncoliform EB isolates tested. The methods evaluated varied in their ability to detect different microbial hygiene indicators in Greek yogurt. Crystal violet tetrazolium agar detected the highest

  3. Population coding in area V4 during rapid shape detections

    Science.gov (United States)

    Weiner, Katherine F.

    2015-01-01

    While previous studies have suggested that neuronal correlations are common in visual cortex over a range of timescales, the effect of correlations on rapid visually based decisions has received little attention. We trained Macaca mulatta to saccade to a peripherally presented shape embedded in dynamic noise as soon as the shape appeared. While the monkeys performed the task, we recorded from neuronal populations (5–29 cells) using a microelectrode array implanted in area V4, a visual area thought to be involved in form perception. While modest correlations were present between cells during visual stimulation, their magnitude did not change significantly subsequent to the appearance of a shape. We quantified the reliability and temporal precision with which neuronal populations signaled the appearance of the shape and predicted the animals' choices using mutual information analyses. To study the impact of correlations, we shuffled the activity from each cell across observations while retaining stimulus-dependent modulations in firing rate. We found that removing correlations by shuffling across trials minimally affected the reliability or timing with which pairs, or larger groups of cells, signaled the presence of a shape. To assess the downstream impact of correlations, we also studied how shuffling affected the ability of V4 populations to predict behavioral choices. Surprisingly, shuffling created a modest increase in the accuracy of such predictions, suggesting that the reliability of downstream neurons is slightly compromised by activity correlations. Our findings are consistent with neuronal correlations having a minimal effect on the reliability and timing of rapid perceptual decisions. PMID:25787961

  4. Clinical feasibility of rapid confocal melanoma feature detection

    Science.gov (United States)

    Hennessy, Ricky; Jacques, Steve; Pellacani, Giovanni; Gareau, Daniel

    2010-02-01

    In vivo reflectance confocal microscopy shows promise for the early detection of malignant melanoma. One diagnostic trait of malignancy is the presence of pagetoid melanocytes in the epidermis. For automated detection of MM, this feature must be identified quantitatively through software. Beginning with in vivo, noninvasive confocal images from 10 unequivocal MMs and benign nevi, we developed a pattern recognition algorithm that automatically identified pagetoid melanocytes in all four MMs and identified none in five benign nevi. One data set was discarded due to artifacts caused by patient movement. With future work to bring the performance of this pattern recognition technique to the level of the clinicians on difficult lesions, melanoma diagnosis could be brought to primary care facilities and save many lives by improving early diagnosis.

  5. An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue.

    Science.gov (United States)

    Becerra, Sandra C; Roy, Daniel C; Sanchez, Carlos J; Christy, Robert J; Burmeister, David M

    2016-04-12

    Bacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections should only occur after positive diagnosis. Currently, diagnosis is delayed due to lengthy culturing methods which may also fail to identify the presence of bacteria. While newer costly bacterial identification methods are being explored, a simple and inexpensive diagnostic tool would aid in immediate and accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E) and Gram stains have been employed, but are far from optimal when analyzing tissue samples due to non-specific staining. The goal of the current study was to develop a modification of the Gram stain that enhances the contrast between bacteria and host tissue. A modified Gram stain was developed and tested as an alternative to Gram stain that improves the contrast between Gram positive bacteria, Gram negative bacteria and host tissue. Initially, clinically relevant strains of Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry techniques involving bacterial strain-specific fluorescent antibodies as validation tools. H&E and Gram stain of serial biopsy sections were then compared to a modification of the Gram stain incorporating a counterstain that highlights collagen found in tissue. The modified Gram stain clearly identified both Gram positive and Gram negative bacteria, and when compared to H&E or Gram stain alone provided excellent contrast between bacteria and non-viable burn eschar. Moreover, when applied to surgical biopsies from patients that underwent burn debridement this technique was able to clearly detect bacterial morphology within host tissue. We describe a modification of the Gram stain that provides improved contrast of Gram positive and Gram negative microorganisms within host

  6. Rapid Isolation and Detection for RNA Biomarkers for TBI Diagnostics

    Science.gov (United States)

    2016-10-01

    isolation of glioblastoma exosomes from 50 µL of un -diluted plasma in fifteen to twenty minutes. We also showed tri-color fluorescent detection of the...to carryout immunofluorescence analysis of brain specific exosomal protein biomarkers. We believe this new technique is very close to achieving true...we can continue to carry out further work in order to achieve our final TBI project goals, which required use of TBI patient samples. With regard

  7. gmos: Rapid Detection of Genome Mosaicism over Short Evolutionary Distances.

    Science.gov (United States)

    Domazet-Lošo, Mirjana; Domazet-Lošo, Tomislav

    2016-01-01

    Prokaryotic and viral genomes are often altered by recombination and horizontal gene transfer. The existing methods for detecting recombination are primarily aimed at viral genomes or sets of loci, since the expensive computation of underlying statistical models often hinders the comparison of complete prokaryotic genomes. As an alternative, alignment-free solutions are more efficient, but cannot map (align) a query to subject genomes. To address this problem, we have developed gmos (Genome MOsaic Structure), a new program that determines the mosaic structure of query genomes when compared to a set of closely related subject genomes. The program first computes local alignments between query and subject genomes and then reconstructs the query mosaic structure by choosing the best local alignment for each query region. To accomplish the analysis quickly, the program mostly relies on pairwise alignments and constructs multiple sequence alignments over short overlapping subject regions only when necessary. This fine-tuned implementation achieves an efficiency comparable to an alignment-free tool. The program performs well for simulated and real data sets of closely related genomes and can be used for fast recombination detection; for instance, when a new prokaryotic pathogen is discovered. As an example, gmos was used to detect genome mosaicism in a pathogenic Enterococcus faecium strain compared to seven closely related genomes. The analysis took less than two minutes on a single 2.1 GHz processor. The output is available in fasta format and can be visualized using an accessory program, gmosDraw (freely available with gmos).

  8. Detecting rapid mass movements using electrical self-potential measurements

    Science.gov (United States)

    Heinze, Thomas; Limbrock, Jonas; Pudasaini, Shiva P.; Kemna, Andreas

    2017-04-01

    Rapid mass movements are a latent danger for lives and infrastructure in almost any part of the world. Often such mass movements are caused by increasing pore pressure, for example, landslides after heavy rainfall or dam breaking after intrusion of water in the dam. Among several other geophysical methods used to observe water movement, the electrical self-potential method has been applied to a broad range of monitoring studies, especially focusing on volcanism and dam leakage but also during hydraulic fracturing and for earthquake prediction. Electrical self-potential signals may be caused by various mechanisms. Though, the most relevant source of the self-potential field in the given context is the streaming potential, caused by a flowing electrolyte through porous media with electrically charged internal surfaces. So far, existing models focus on monitoring water flow in non-deformable porous media. However, as the self-potential is sensitive to hydraulic parameters of the soil, any change in these parameters will cause an alteration of the electric signal. Mass movement will significantly influence the hydraulic parameters of the solid as well as the pressure field, assuming that fluid movement is faster than the pressure diffusion. We will present results of laboratory experiments under drained and undrained conditions with fluid triggered as well as manually triggered mass movements, monitored with self-potential measurements. For the undrained scenarios, we observe a clear correlation between the mass movements and signals in the electric potential, which clearly differ from the underlying potential variations due to increased saturation and fluid flow. In the drained experiments, we do not observe any measurable change in the electric potential. We therefore assume that change in fluid properties and release of the load causes disturbances in flow and streaming potential. We will discuss results of numerical simulations reproducing the observed effect. Our

  9. gmos: Rapid Detection of Genome Mosaicism over Short Evolutionary Distances.

    Directory of Open Access Journals (Sweden)

    Mirjana Domazet-Lošo

    Full Text Available Prokaryotic and viral genomes are often altered by recombination and horizontal gene transfer. The existing methods for detecting recombination are primarily aimed at viral genomes or sets of loci, since the expensive computation of underlying statistical models often hinders the comparison of complete prokaryotic genomes. As an alternative, alignment-free solutions are more efficient, but cannot map (align a query to subject genomes. To address this problem, we have developed gmos (Genome MOsaic Structure, a new program that determines the mosaic structure of query genomes when compared to a set of closely related subject genomes. The program first computes local alignments between query and subject genomes and then reconstructs the query mosaic structure by choosing the best local alignment for each query region. To accomplish the analysis quickly, the program mostly relies on pairwise alignments and constructs multiple sequence alignments over short overlapping subject regions only when necessary. This fine-tuned implementation achieves an efficiency comparable to an alignment-free tool. The program performs well for simulated and real data sets of closely related genomes and can be used for fast recombination detection; for instance, when a new prokaryotic pathogen is discovered. As an example, gmos was used to detect genome mosaicism in a pathogenic Enterococcus faecium strain compared to seven closely related genomes. The analysis took less than two minutes on a single 2.1 GHz processor. The output is available in fasta format and can be visualized using an accessory program, gmosDraw (freely available with gmos.

  10. Core-shell of FePt@SiO2-Au magnetic nanoparticles for rapid SERS detection.

    Science.gov (United States)

    Hardiansyah, Andri; Chen, An-Yu; Liao, Hung-Liang; Yang, Ming-Chien; Liu, Ting-Yu; Chan, Tzu-Yi; Tsou, Hui-Ming; Kuo, Chih-Yu; Wang, Juen-Kai; Wang, Yuh-Lin

    2015-12-01

    In this study, multifunctional hybrid nanoparticles composed of iron platinum (FePt), silica (SiO2), and gold nanoparticles (AuNPs) had been developed for surface-enhanced Raman scattering (SERS) application. Core-shell structure of SiO2 and FePt nanoparticles (FePt@SiO2) was fabricated through sol-gel process and then immobilized gold nanoparticles onto the surface of FePt@SiO2, which displays huge Raman enhancement effect and magnetic separation capability. The resulting core-shell nanoparticles were subject to evaluation by transmission electron microscopy (TEM), Energy-dispersive X-ray spectroscopy (EDX), zeta potential measurement, and X-ray photoelectron spectroscopy (XPS). TEM observation revealed that the particle size of resultant nanoparticles displayed spherical structure with the size ~30 nm and further proved the successful immobilization of Au onto the surface of FePt@SiO2. Zeta potential measurement exhibited the successful reaction between FePt@SiO2 and AuNPs. The rapid SERS detection and identification of small biomolecules (adenine) and microorganisms (gram-positive bacteria, Staphylococcus aureus) was conducted through Raman spectroscopy. In summary, the novel core-shell magnetic nanoparticles could be anticipated to apply in the rapid magnetic separation under the external magnetic field due to the core of the FePt superparamagnetic nanoparticles and label-free SERS bio-sensing of biomolecules and bacteria.

  11. The potential of spectral and hyperspectral-imaging techniques for bacterial detection in food: A case study on lactic acid bacteria.

    Science.gov (United States)

    Foca, Giorgia; Ferrari, Carlotta; Ulrici, Alessandro; Sciutto, Giorgia; Prati, Silvia; Morandi, Stefano; Brasca, Milena; Lavermicocca, Paola; Lanteri, Silvia; Oliveri, Paolo

    2016-06-01

    Official methods for the detection of bacteria are based on culture techniques. These methods have limitations such as time consumption, cost, detection limits and the impossibility to analyse a large number of samples. For these reasons, the development of rapid, low-cost and non-destructive analytical methods is a task of growing interest. In the present study, the capability of spectral and hyperspectral techniques to detect bacterial surface contamination was investigated preliminarily on gel cultures, and subsequently on sliced cooked ham. In more detail, two species of lactic acid bacteria (LAB) were considered, namely Lactobacillus curvatus and Lactobacillus sakei, both of which are responsible for common alterations in sliced cooked ham. Three techniques were investigated, with different equipment, respectively: a macroscopic hyperspectral scanner operating in the NIR (10,470-5880cm(-1)) region, a FT-NIR spectrophotometer equipped with a transmission arm as the sampling tool, working in the 12,500-5800cm(-1) region, and a FT-MIR microscopy operating in the 4000-675cm(-1) region. Multivariate exploratory data analysis, in particular principal component analysis (PCA), was applied in order to extract useful information from original data and from hyperspectrograms. The results obtained demonstrate that the spectroscopic and imaging techniques investigated can represent an effective and sensitive tool to detect surface bacterial contamination in samples and, in particular, to recognise species to which bacteria belong. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Low cost and manufacturable complete microTAS for detecting bacteria.

    Science.gov (United States)

    Sauer-Budge, Alexis F; Mirer, Paul; Chatterjee, Anirban; Klapperich, Catherine M; Chargin, David; Sharon, Andre

    2009-10-07

    In this paper, we present a fully integrated lab-on-a-chip and associated instrument for the detection of bacteria from liquid samples. The system conducts bacterial lysis, nucleic acid isolation and concentration, polymerase chain reaction (PCR), and end-point fluorescent detection. To enable truly low-cost manufacture of the single-use disposable chip, we designed the plastic chip in a planar format without any active components to be amenable to injection molding and utilized a novel porous polymer monolith (PPM) embedded with silica that has been shown to lyse bacteria and isolate the nucleic acids from clinical samples (M. D. Kulinski, M. Mahalanabis, S. Gillers, J. Y. Zhang, S. Singh and C. M. Klapperich, Biomed. Microdevices, 2009, 11, 671-678).(1) The chip is made of Zeonex(R), a thermoplastic with a high melting temperature to allow PCR, good UV transmissibility for UV-curing of the PPM, and low auto-fluorescence for fluorescence detection of the amplicon. We have built a prototype instrument to automate control of the fluids, temperature cycling, and optical detection with the capability of accommodating various chip designs. To enable fluid control without including valves or pumps on the chip, we utilized a remote valve switching technique. To allow fluid flow rate changes on the valveless chip, we incorporated speed changing fluid reservoirs. The PCR thermal cycling was achieved with a ceramic heater and air cooling, while end-point fluorescence detection was accomplished with an optical spectrometer; all integrated in the instrument. The chip seamlessly and automatically is mated to the instrument through an interface block that presses against the chip. The interface block aligns and ensures good contact of the chip to the temperature controlled region and the optics. The integrated functionality of the chip was demonstrated using Bacillus subtilis as a model bacterial target. A Taqman assay was employed on-chip to detect the isolated bacterial DNA.

  13. Rapid detection of worms using ICMP-T3 analysis

    Science.gov (United States)

    Gray, Robert S.; Berk, Vincent H.

    2004-09-01

    Identification of an active Internet worm is a manual process where security analysts must observe and analyze unusual activity on multiple firewalls, intrusion-detection systems or hosts. A worm might not be positively identified until it already has spread to most of the Internet, eliminating many defensive options. In previous work, we developed an automated system that can identify active worms seconds or minutes after they first begin to spread, a necessary precursor to halting the spread of the worm rather than simply cleaning up afterward. The system collects ICMP Destination Unreachable messages from instrumented network routers, identifies those patterns of unreachable messages that indicate malicious scanning activity, and then searches for patterns of scanning activity that indicate a propagating worm. In this paper, we compare the performance of two different detection strategies, our previous threshold approach and a new line-fit approach, for different worm-propagation techniques, noise environments, and system parameters. These techniques work for worms that generate at least some of their target addresses through a random process, a feature of most recent worms. Although both being powerful methods for fast worm identification, the new line-fit approach proves to be significantly more noise resistant.

  14. Detection and Genotyping of Coxiella burnetii and Coxiella-Like Bacteria in Horses in South Korea.

    Science.gov (United States)

    Seo, Min-Goo; Lee, Seung-Hun; VanBik, Dorene; Ouh, In-Ohk; Yun, Sun-Hee; Choi, Eunsang; Park, Yong-Soo; Lee, Sang-Eun; Kim, Jong Wan; Cho, Gil-Jae; Kwon, Oh-Deog; Kwak, Dongmi

    2016-01-01

    Coxiella burnetii and Coxiella-like bacteria (CLB) are genetically and ecologically distinct despite some genetic similarities. Furthermore, CLB are exceptionally diverse and widespread in ticks, but rarely detected in domestic animals. Since Coxiella bacteria can be transmitted from infected horses by inhalation or by coming in contact with ticks during activities such as horseback riding, it is necessary to study their prevalence. To the best of our knowledge, this is the first large-scale nationwide investigation of the prevalence of C. burnetii and CLB among horses reared in South Korea. Of 816 blood samples collected between 2007 and 2013, 11 (1.3%) were identified as C. burnetii by ELISA, and six (0.7%) as CLB by 16S rRNA sequencing. While a sequence from Jeju Island was similar (97.9-100%) to those within clade B, five sequences obtained from the northern region were categorized into a new clade, indicating the sequence diversity of the genus Coxiella. Studies until date had detected CLB only in ticks; here, we describe their detection in mammals. Given their zoonotic potential, strategic monitoring and appropriate control programs for Coxiella species need to be established.

  15. Detection of bacteria from a cecal anaerobic competitive exclusion culture with an immunoassay electrochemiluminescence sensor

    Science.gov (United States)

    Beier, Ross C.; Young, Colin R.; Stanker, Larry H.

    1999-01-01

    A competitive exclusion (CE) culture of chicken cecal anaerobes has been developed and used in this laboratory for control of Salmonella typhimurium in chickens. The CE culture consists of 29 different species of micro-organisms, and is known as CF3. Detection of one of the CF3 bacteria, Eubacteria, and S. typhimurium were demonstrated using a commercial immunomagnetic (IM) electrochemiluminescence (ECL) sensor, the ORIGENR Analyzer. Analysis was achieved using a sandwich immunoassay. Bacteria were captured on antibody- conjugated 280 micron sized magnetic beads followed by binding of reporter antibodies labelled with ruthenium (II) tris(dipyridyl) chelate [Ru(bpy)32+]. The magnetic beads were then trapped on an electrode in the reaction cell of the ORIGENR Analyzer by a magnet, and the ECL was evoked from Ru(bpy)32+ on the tagged reporter antibodies by an electrical potential at the electrode. Preliminary IM-ECL assays with Eubacteria yielded a detection limit of 105 cfu/mL. Preliminary IM-ECL assays with S. typhimurium yielded a similar detection limit of 105 cfu/mL.

  16. Rapid detection of fungal alpha-amylase in the work environment with a lateral flow immunoassay

    NARCIS (Netherlands)

    Bogdanovic, J.; Koets, M.; Sander, I.; Wouters, I.; Meijster, T.; Heederik, D.J.J.; Amerongen, van A.; Doekes, G.

    2006-01-01

    Background Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with

  17. Rapid trace detection of triacetone triperoxide (TATP) by complexation reactions during desorption electrospray ionization.

    Science.gov (United States)

    Cotte-Rodríguez, Ismael; Chen, Hao; Cooks, R Graham

    2006-03-07

    Desorption electrospray ionization (DESI) mass spectrometry is used for rapid, specific and sensitive detection of trace amounts of the notorious explosive TATP present on ambient surfaces by alkali metal complexation in a simple spray technique.

  18. Effects of clonidine and scopolamine on multiple target detection in rapid serial visual presentation

    NARCIS (Netherlands)

    Brown, S.B.R.E.; Slagter, H.A.; van Noorden, M.S.; Giltay, E.J.; van der Wee, N.J.A.; Nieuwenhuis, S.

    2016-01-01

    Rationale: The specific role of neuromodulator systems in regulating rapid fluctuations of attention is still poorly understood. Objectives: In this study, we examined the effects of clonidine and scopolamine on multiple target detection in a rapid serial visual presentation task to assess the role

  19. Saponin promotes rapid identification and antimicrobial susceptibility profiling of Gram-positive and Gram-negative bacteria in blood cultures with the Vitek 2 system.

    Science.gov (United States)

    Lupetti, A; Barnini, S; Morici, P; Ghelardi, E; Nibbering, P H; Campa, M

    2013-04-01

    The rapid identification and antimicrobial susceptibility testing (AST) of bacteria in clinical blood cultures is crucial to optimise antimicrobial therapy. A previous study involving small sample numbers revealed that the addition of saponin to blood cultures, further referred to as the new method, shortened considerably the turn-around time for the identification and AST of Gram-positive cocci as compared to the current method involving an overnight subculture. Here, we extend previous results and compare the identification and AST of blood cultures containing Gram-negative bacilli by the new and current methods. The identification and AST of 121 Gram-positive and 109 Gram-negative bacteria in clinical monomicrobial blood cultures by the new and current methods and, in the case of Gram-negative bacilli, by direct (no additions) inoculation into an automated system (rapid method) was assessed using the Vitek 2 system. Discrepancies between the results obtained with the different methods were solved by manual methods. The new method correctly identified 88 % of Gram-positive and 98 % of Gram-negative bacteria, and the rapid method correctly identified 94 % of Gram-negative bacteria. The AST for all antimicrobials by the new method were concordant with the current method for 55 % and correct for an additional 9 % of Gram-positive bacteria, and concordant with the current method for 62 % and correct for an additional 21 % of Gram-negative bacilli. The AST by the rapid method was concordant with the current method for 62 % and correct for an additional 12 % of Gram-negative bacilli. Together, saponin-treated monomicrobial blood cultures allow rapid and reliable identification and AST of Gram-positive and Gram-negative bacteria.

  20. Rapid earthquake detection through GPU-Based template matching

    Science.gov (United States)

    Mu, Dawei; Lee, En-Jui; Chen, Po

    2017-12-01

    The template-matching algorithm (TMA) has been widely adopted for improving the reliability of earthquake detection. The TMA is based on calculating the normalized cross-correlation coefficient (NCC) between a collection of selected template waveforms and the continuous waveform recordings of seismic instruments. In realistic applications, the computational cost of the TMA is much higher than that of traditional techniques. In this study, we provide an analysis of the TMA and show how the GPU architecture provides an almost ideal environment for accelerating the TMA and NCC-based pattern recognition algorithms in general. So far, our best-performing GPU code has achieved a speedup factor of more than 800 with respect to a common sequential CPU code. We demonstrate the performance of our GPU code using seismic waveform recordings from the ML 6.6 Meinong earthquake sequence in Taiwan.

  1. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

    Science.gov (United States)

    Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

  2. Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations

    Directory of Open Access Journals (Sweden)

    Law eJodi Woan-Fei

    2015-01-01

    Full Text Available The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR, multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA, loop-mediated isothermal amplification (LAMP and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases.

  3. Oral bacteria in placental tissues: increased molecular detection in pregnant periodontitis patients.

    Science.gov (United States)

    Blanc, V; O'Valle, F; Pozo, E; Puertas, A; León, R; Mesa, F

    2015-10-01

    The objective of this study was to identify the DNA of oral bacteria in placental samples from women with and without periodontitis who had or had not had preterm births and/or low birthweight (PB/LBW) neonates. Data were gathered from 57 puerperal women in relation to socio-demographic, gynaecological, and periodontal variables and to placental histomorphology. Fifty-seven biopsies, 28 from mothers with periodontitis, were taken aseptically from preterm placentas (n = 36) and from full-term placentas (n = 21). Total DNA was extracted, and the presence of 15 oral bacteria was assessed using Nested-PCR. The placentas from women with periodontitis showed a higher prevalence of periodontopathogens compared to those from women without periodontitis (P = 0.009). Samples showed low prevalences of Actinomyces israelii, Parvimonas micra and Tannerella forsythia. An association was found between Eikenella corrodens in placenta and periodontitis (P = 0.002). The most ubiquitous bacterium, Fusobacterium nucleatum, was more prevalent in mothers with periodontitis and PB/LBW (P = 0.033). Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were not detected. These results, along with previous findings, show that oral bacteria may be normally present in the placenta, however, the levels of certain oral pathogens in the placenta would highly depend on the mother's periodontal state. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV)

    OpenAIRE

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M.; Sugumar, Vimal; Taju, Gani; Sahul Hameed, A. S.; Paknikar, Kishore M.

    2017-01-01

    Background White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. Metho...

  5. Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection

    OpenAIRE

    Lama, Lara

    2017-01-01

    This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins. Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate...

  6. Rapid and visual detection ofMycobacterium aviumsubsp.paratuberculosisby recombinase polymerase amplification combined with a lateral flow dipstick.

    Science.gov (United States)

    Guimin, Zhao; Hongmei, Wang; Peili, Hou; Chengqiang, He; Hongbin, He

    2017-12-28

    Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. Quick diagnosis could facilitate control; however widespread point-of-care testing is infrequently done due to the lack of robust method. Isothermal recombinase polymerase amplification (RPA) technique has emerged as a novel DNA amplify assay for use in rapid diagnosis. Here, an RPA combined with lateral flow dipstick (LFD) assay was developed to estimate DNA from M.paratuberculosis . First, the specificity and sensitivity of RPA-nfo primer and probe sets were assessed. The assay successfully detected M.paratuberculosis DNA in 30 minutes at 39°C, limit of detection up to eight copies per reaction, which was equivalent with the real-time quantitative PCR (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium and five pathogenic enteric bacteria. Then, 612 clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR and ELISA assays respectively, also the established RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR. This is the first report utilizing an RPA-LFD assay to visual and rapid detect M.paratuberculosis . Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource constrained setting.

  7. Development of detection medium for hard-to-culture beer-spoilage lactic acid bacteria.

    Science.gov (United States)

    Suzuki, K; Asano, S; Iijima, K; Kuriyama, H; Kitagawa, Y

    2008-05-01

    To develop a detection medium for hard-to-culture beer-spoilage lactic acid bacteria (LAB). Four hard-to-culture beer-spoilage strains of LAB, belonging to Lactobacillus paracollinoides and Lactobacillus lindneri, have been obtained by repeatedly subculturing the wild-type strains in beer. To develop a countermeasure against these hard-to-culture beer-spoilage LAB, a beer-based medium was modified. As a consequence, the supplementation of a small amount of de Man Rogosa Sharpe medium was found to enhance the growth of hard-to-culture beer-spoilage LAB strains obtained in this study. In addition, sodium acetate was shown to improve the selectivity of this beer-based medium. Further comparative study was performed with five other media widely used for the detection of beer-spoilage LAB in the brewing industry. This study revealed that the newly developed medium, designated advanced beer-spoiler detection (ABD) medium, possessed superior sensitivity for hard-to-culture beer-spoilage LAB and comparable sensitivity with easy-to-culture beer-spoilage LAB. Moreover, ABD medium was found to suppress the growth of nonspoilage micro-organisms, and thereby allow the selective growth of beer-spoilage LAB. Advanced beer-spoiler detection medium is considered as an effective tool for comprehensive detection of beer-spoilage LAB in breweries. The detection by ABD medium can be used as an indicator for differentiating the beer-spoilage ability of LAB without further confirmatory tests in breweries.

  8. Detection of viruses and atypical bacteria associated with acute respiratory infection of children in Hubei, China.

    Science.gov (United States)

    Wu, Zegang; Li, Yan; Gu, Jian; Zheng, Hongyun; Tong, Yongqing; Wu, Qing

    2014-02-01

    Acute respiratory infection is the major cause of disease and death in children, particularly in developing countries. However, the spectrum of pathogenic viruses and atypical bacteria that exist in many of these countries remains incompletely characterized. The aim of this study was to examine the spectrum of pathogenic viruses and atypical bacteria associated with acute respiratory infection in children under the age of 16. A total of 10 435 serum sera specimens were collected from hospitalized children presenting with acute respiratory infection symptoms. Indirect immunofluorescence assays were performed to detect immunoglobulin M antibodies against nine common pathogens: mycoplasma pneumonia, influenza virus B, respiratory syncytial virus, parainfluenza virus, adenovirus, influenza virus A, legionella pneumophila, coxiella burnetii and chamydophila pneumonia. Of the 10 435 specimens examined, 7046 tested positive for at least one pathogen. Among all of the tested pathogens, mycoplasma pneumonia had the highest detection rate (56.9%). Influenza virus A and influenza virus B epidemics occurred during both winter and summer. The detection rate of respiratory syncytial virus and adenovirus was higher in spring. Cases of mixed infection were more complex: 4136 specimens (39.6%) tested positive for ≥2 pathogens. There were statistically significant difference in detection rates of mycoplasma pneumonia, influenza virus B, respiratory syncytial virus, parainfluenza virus, adenovirus, influenza virus A, legionella pneumophila and chamydophila pneumonia among different age groups (P acute respiratory infection among children in Hubei of China were mycoplasma pneumonia, influenza virus B and respiratory syncytial virus. The detection rates for each pathogen displayed specific seasonal and age group variations. © 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.

  9. A novel, multiplex, real-time PCR–based approach for the detection of the commonly occurring pathogenic fungi and bacteria

    Science.gov (United States)

    2013-01-01

    Background Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified. The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. Results A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons. With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. Conclusions This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice. PMID:24364823

  10. A novel, multiplex, real-time PCR-based approach for the detection of the commonly occurring pathogenic fungi and bacteria.

    Science.gov (United States)

    Horváth, Ádám; Pető, Zoltán; Urbán, Edit; Vágvölgyi, Csaba; Somogyvári, Ferenc

    2013-12-23

    Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified.The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons.With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice.

  11. Wheat germ agglutinin and Lens culinaris agglutinin sensitized anisotropic silver nanoparticles in detection of bacteria: A simple photometric assay.

    Science.gov (United States)

    Mikaelyan, Mariam V; Poghosyan, Gayane G; Hendrickson, Olga D; Dzantiev, Boris B; Gasparyan, Vardan K

    2017-08-15

    Efficacy of anisotropic silver nanoparticles sensitized with wheat germ agglutinin (WGA) and Lens culinaris agglutinin (LCA) was studied for detection of Staphylococcus aureus and Escherichia coli. It was demonstrated that interaction of these nanoparticles with bacteria stabilizes them and prevents their aggregation upon addition of sodium chloride; such stabilization depends on bacteria concentration. High concentration of bacteria results in higher stabilization whereas low concentration leads to aggregation of nanoparticles. Optical changes as a result of aggregation correlate with bacteria concentration. The developed approach allows the detection of Gram-positive bacteria (S.aureus) with the lowest detectable concentration of 103 cells/mL and Gram-negative bacteria (E. coli) with the lowest detectable concentration of 3 × 103 cells/mL using WGA-sensitized nanoparticles. In the case of LCA-sensitized nanoparticles the lowest detection was 5 × 103 cells/mL for S. aureus and 5 × 104 cells/mL for E. coli. Copyright © 2017. Published by Elsevier B.V.

  12. Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

    Science.gov (United States)

    Kuhnert, P; Heyberger-Meyer, B; Burnens, A P; Nicolet, J; Frey, J

    1997-06-01

    The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.

  13. A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase.

    Science.gov (United States)

    Li, Z; Chang, S; Lin, L; Li, Y; An, Q

    2011-08-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC-utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Determination of bacterial ACC consumption by the PCR-plate ninhydrin-ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. The PCR-plate ninhydrin-ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  14. An evaluation of a genotoxicity assay with liver s9 for activation and luminescent bacteria for detection

    Science.gov (United States)

    Johnson, B. Thomas

    1992-01-01

    A new short-term in vitro genotoxicity assay with marine bioluminescent bacteria was evaluated for sensitivity and cost. Known under the trade name of Mutatox™, this assay is a simple and rapid screening tool that detects DNA-damaging substances (genotoxins) by measuring light output from an isolated dark mutant strain of the luminescent bacterium Photobacterium phosphoreum. A positive response indicates the ability of the test chemical to restore the luminescent state in the dark mutant strain; the degree of light increase indicates the relative genotoxicity of the sample. In this study, the Mutatox assay with rat hepatic fractions (S9) as an exogenous metabolic activation system detected genotoxic activity with known progenotoxins: 2-acetamidofluorene, aflatoxin B1, 2-aminoanthracene, 2-aminofluorene, 2-aminonaphthalene, benzo[a]pyrene, 3-methyl-cholanthrene, and pyrene. Each chemical clearly demonstrated a dose response between 5.0 and 0.6 μg per tube. Known nongenotoxic controls carbofuran, di-2-ethylhexyl phthalate, malathion, simazine, and permethrin showed no genotoxic responses. The optimum assay conditions were determined to be rat S9 concentration of 0.4 mg/ml, preincubation at 37°C for 30 min, and 18 h incubation at 23°C. Genotoxicity data were obtained in <24 h. The Mutatox assay compared favorably in sensitivity with the Ames test; it was easier and more rapid to perform and, as a result, cost less. The sensitivity, specificity, and predictive value suggested that the Mutatox assay could be a valuable screening tool to monitor complex environmental samples for genotoxins.

  15. Detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations.

    Science.gov (United States)

    Kloepfer, Kirsten M; Lee, Wai Ming; Pappas, Tressa E; Kang, Theresa J; Vrtis, Rose F; Evans, Michael D; Gangnon, Ronald E; Bochkov, Yury A; Jackson, Daniel J; Lemanske, Robert F; Gern, James E

    2014-05-01

    Detection of either viral or bacterial pathogens is associated with wheezing in children; however, the influence of both bacteria and viruses on illness symptoms has not been described. We evaluated bacterial detection during the peak rhinovirus season in children with and without asthma to determine whether an association exists between bacterial infection and the severity of rhinovirus-induced illnesses. Three hundred eight children (166 with asthma and 142 without asthma) aged 4 to 12 years provided 5 consecutive weekly nasal samples during September and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were performed on all nasal samples. Detection rates were 53%, 17%, and 11% for H influenzae, S pneumoniae, and M catarrhalis, respectively, with detection of rhinovirus increasing the risk of detecting bacteria within the same sample (odds ratio [OR], 2.0; 95% CI, 1.4-2.7; P rhinovirus, S pneumoniae was associated with increased cold symptoms (mean, 2.7 [95% CI, 2.0-3.5] vs 1.8 [95% CI, 1.5-2.2]; P = .006) and moderate asthma exacerbations (18% [95% CI, 12% to 27%] vs 9.2% [95% CI, 6.7% to 12%]; P = .006). In the presence of rhinovirus, S pneumoniae was associated with increased moderate asthma exacerbations (22% [95% CI, 16% to 29%] vs 15% [95% CI, 11% to 20%]; P = .01). Furthermore, M catarrhalis detected alongside rhinovirus increased the likelihood of experiencing cold symptoms, asthma symptoms, or both compared with isolated detection of rhinovirus (OR, 2.0 [95% CI, 1.0-4.1]; P = .04). Regardless of rhinovirus status, H influenzae was not associated with respiratory symptoms. Rhinovirus infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M catarrhalis and S pneumoniae contribute to the severity of respiratory tract illnesses, including asthma exacerbations

  16. Biosynthesis of CdS nanoparticles: A fluorescent sensor for sulfate-reducing bacteria detection.

    Science.gov (United States)

    Qi, Peng; Zhang, Dun; Zeng, Yan; Wan, Yi

    2016-01-15

    CdS nanoparticles were synthesized with an environmentally friendly method by taking advantage of the characteristic metabolic process of sulfate-reducing bacteria (SRB), and used as fluorescence labels for SRB detection. The presence of CdS nanoparticles was observed within and immediately surrounded bacterial cells, indicating CdS nanoparticles were synthesized both intracellularly and extracellularly. Moreover, fluorescent properties of microbial synthesized CdS nanoparticles were evaluated for SRB detection, and a linear relationship between fluorescence intensity and the logarithm of bacterial concentration was obtained in the range of from 1.0×10(2) to 1.0×10(7)cfu mL(-1). The proposed SRB detection method avoided the use of biological bio-recognition elements which are easy to lose their specific recognizing abilities, and the bacterial detection time was greatly shortened compared with the widely used MPN method which would take up to 15 days to accomplish the detection process. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Rapid identification of bacteria from positive blood culture bottles by MALDI-TOF MS following short-term incubation on solid media.

    Science.gov (United States)

    Altun, Osman; Botero-Kleiven, Silvia; Carlsson, Sarah; Ullberg, Måns; Özenci, Volkan

    2015-11-01

    Rapid identification of bacteria from blood cultures enables early initiation of appropriate antibiotic treatment in patients with bloodstream infections (BSI). The objective of the present study was to evaluate the use of matrix-associated laser desorption ionization-time of flight (MALDI-TOF) MS after a short incubation on solid media for rapid identification of bacteria from positive blood culture bottles. MALDI-TOF MS was performed after 2.5 and 5.5 h plate incubation of samples from positive blood cultures. Identification scores with values ≥ 1.7 were accepted as successful identification if the results were confirmed by conventional methods. Conventional methods included MALDI-TOF MS, Vitek 2, and diverse biochemical and agglutination tests after overnight culture. In total, 515 positive blood cultures with monomicrobial bacterial growth representing one blood culture per patient were included in the study. There were 229/515 (44.5%) and 286/515 (55.5%) blood culture bottles with Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB), respectively. MALDI-TOF MS following short-term culture could accurately identify 300/515 (58.3%) isolates at 2.5 h, GNB being identified in greater proportion (180/229; 78.6%) than GPB (120/286; 42.0%). In an additional 124/515 bottles (24.1%), identification was successful at 5.5 h, leading to accurate identification of bacteria from 424/515 (82.3%) blood cultures after short-term culture. Interestingly, 11/24 of the isolated anaerobic bacteria could be identified after 5.5 h. The present study demonstrates, in a large number of clinical samples, that MALDI-TOF MS following short-term culture on solid medium is a reliable and rapid method for identification of bacteria from blood culture bottles with monomicrobial bacterial growth.

  18. Detection of emerging antibiotic resistance in bacteria isolated from subclinical mastitis in cattle in West Bengal.

    Science.gov (United States)

    Das, Arnab; Guha, Chanchal; Biswas, Ujjwal; Jana, Partha Sarathi; Chatterjee, Amaresh; Samanta, Indranil

    2017-05-01

    The aim of this work was to detect antibiotic resistance in Gram-negative bacteria isolated from subclinical mastitis in cattle in West Bengal. The milk samples were collected from the cattle suffering with subclinical mastitis in West Bengal. The milk samples were inoculated into the nutrient broth and incubated at 37°C. On the next day, the growth was transferred into nutrient agar and MacConkey agar. All the pure cultures obtained from nutrient agar slant were subjected to Gram-staining and standard biochemical tests. All the bacterial isolates were tested in vitro for their sensitivity to different antibiotics commonly used in veterinary practices. All Gram-negative isolates including positive control were subjected to polymerase chain reaction (PCR) for detection of bla CTX-M , bla TEM , bla SHV , bla VIM , tetA, tetB, tetC, and tetM genes considered for extended-spectrum β-lactamase (ESBL), metallo-β-lactamase, and tetracycline resistance. In total, 50 Gram-negative organisms (Escherichia coli, Proteus, Pseudomonas, Klebsiella, and Enterobacter) were isolated from milk samples of subclinical mastitis infected cattle. Among these Gram-negative isolates, 48% (24/50) were found either ESBL producing or tetracycline resistant. Out of total 50 Gram-negative isolates, bla CTX-M was detected in 18 (36%) isolates, and 6 (12%) harbored bla TEM genes in PCR. None of the isolates carried bla SHV genes. Further, in this study, 5 (10%) isolates harbored tet(A) gene, and 8 (16%) isolates carried tet(B) gene. No tet(C) gene was detected from the isolates. This study showed emerging trend of antibiotic-resistant Gram-negative bacteria associated with subclinical mastitis in cattle in West Bengal, India.

  19. Detection of emerging antibiotic resistance in bacteria isolated from subclinical mastitis in cattle in West Bengal

    Directory of Open Access Journals (Sweden)

    Arnab Das

    2017-05-01

    Full Text Available Aim: The aim of this work was to detect antibiotic resistance in Gram-negative bacteria isolated from subclinical mastitis in cattle in West Bengal. Materials and Methods: The milk samples were collected from the cattle suffering with subclinical mastitis in West Bengal. The milk samples were inoculated into the nutrient broth and incubated at 37°C. On the next day, the growth was transferred into nutrient agar and MacConkey agar. All the pure cultures obtained from nutrient agar slant were subjected to Gram-staining and standard biochemical tests. All the bacterial isolates were tested in vitro for their sensitivity to different antibiotics commonly used in veterinary practices. All Gram-negative isolates including positive control were subjected to polymerase chain reaction (PCR for detection of blaCTX-M, blaTEM, blaSHV, blaVIM, tetA, tetB, tetC, and tetM genes considered for extended-spectrum β-lactamase (ESBL, metallo-β-lactamase, and tetracycline resistance. Results: In total, 50 Gram-negative organisms (Escherichia coli, Proteus, Pseudomonas, Klebsiella, and Enterobacter were isolated from milk samples of subclinical mastitis infected cattle. Among these Gram-negative isolates, 48% (24/50 were found either ESBL producing or tetracycline resistant. Out of total 50 Gram-negative isolates, blaCTX-M was detected in 18 (36% isolates, and 6 (12% harbored blaTEM genes in PCR. None of the isolates carried blaSHV genes. Further, in this study, 5 (10% isolates harbored tet(A gene, and 8 (16% isolates carried tet(B gene. No tet(C gene was detected from the isolates. Conclusion: This study showed emerging trend of antibiotic-resistant Gram-negative bacteria associated with subclinical mastitis in cattle in West Bengal, India.

  20. Miniaturized extinction culturing is the preferred strategy for rapid isolation of fast‐growing methane‐oxidizing bacteria

    Science.gov (United States)

    Hoefman, Sven; van der Ha, David; De Vos, Paul; Boon, Nico; Heylen, Kim

    2012-01-01

    Summary Methane‐oxidizing bacteria (MOB) have a large potential as a microbial sink for the greenhouse gas methane as well as for biotechnological purposes. However, their application in biotechnology has so far been hampered, in part due to the relative slow growth rate of the available strains. To enable the availability of novel strains, this study compares the isolation of MOB by conventional dilution plating with miniaturized extinction culturing, both performed after an initial enrichment step. The extinction approach rendered 22 MOB isolates from four environmental samples, while no MOB could be isolated by plating. In most cases, extinction culturing immediately yielded MOB monocultures making laborious purification redundant. Both type I (Methylomonas spp.) and type II (Methylosinus sp.) MOB were isolated. The isolated methanotrophic diversity represented at least 11 different strains and several novel species based on 16S rRNA gene sequence dissimilarity. These strains possessed the particulate (100%) and soluble (64%) methane monooxygenase gene. Also, 73% of the strains could be linked to a highly active fast‐growing mixed MOB community. In conclusion, miniaturized extinction culturing was more efficient in rapidly isolating numerous MOB requiring little effort and fewer materials, compared with the more widely applied plating procedure. This miniaturized approach allowed straightforward isolation and could be very useful for subsequent screening of desired characteristics, in view of their future biotechnological potential. PMID:22070783

  1. Rapid identification of mycobacteria and rapid detection of drug resistance in Mycobacterium tuberculosis in cultured isolates and in respiratory specimens.

    Science.gov (United States)

    Yam, Wing-Cheong; Siu, Kit-Hang Gilman

    2013-01-01

    Recent advances in molecular biology and better understanding of the genetic basis of drug resistance have allowed rapid identification of mycobacteria and rapid detection of drug resistance of Mycobacterium tuberculosis present in cultured isolates or in respiratory specimens. In this chapter, several simple nucleic acid amplification-based techniques are introduced as molecular approach for clinical diagnosis of tuberculosis. A one-tube nested IS6110-based polymerase chain reaction (PCR) is used for M. tuberculosis complex identification; the use of a multiplex allele-specific PCR is demonstrated to detect the isoniazid resistance; PCR-sequencing assays are applied for rifampicin and ofloxacin resistance detection and 16S rDNA sequencing is utilized for identification of mycobacterial species from cultures of acid fast bacilli (AFB). Despite the high specificity and sensitivity of the molecular techniques, mycobacterial culture remains the "Gold Standard" for tuberculosis diagnosis. Negative results of molecular tests never preclude the infection or the presence of drug resistance. These technological advancements are, therefore, not intended to replace the conventional tests, but rather have major complementary roles in tuberculosis diagnosis.

  2. Electrochemical Genosensor To Detect Pathogenic Bacteria (Escherichia coli O157:H7) As Applied in Real Food Samples (Fresh Beef) To Improve Food Safety and Quality Control.

    Science.gov (United States)

    Abdalhai, Mandour H; Fernandes, António Maximiano; Xia, Xiaofeng; Musa, Abubakr; Ji, Jian; Sun, Xiulan

    2015-05-27

    The electrochemical genosensor is one of the most promising methods for the rapid and reliable detection of pathogenic bacteria. In a previous work, we performed an efficient electrochemical genosensor detection of Staphylococcus aureus by using lead sulfide nanoparticles (PbSNPs). As a continuation of this study, in the present work, the electrochemical genosensor was used to detect Escherichia coli O157:H7. The primer and probes were designed using NCBI database and Sigma-Aldrich primer and probe software. The capture and signalizing probes were modified by thiol (SH) and amine (NH2), respectively. Then, the signalizing probe was connected using cadmium sulfide nanoparticles (CdSNPs), which showed well-defined peaks after electrochemical detection. The genosensor was prepared by immobilization of complementary DNA on the gold electrode surface, which hybridizes with a specific fragment gene from pathogenic to make a sandwich structure. The conductivity and sensitivity of the sensor were increased by using multiwalled carbon nanotubes (MWCNT) that had been modified using chitosan deposited as a thin layer on the glass carbon electrode (GCE) surface, followed by a deposit of bismuth. The peak currents of E. coli O157:H7 correlated in a linear fashion with the concentration of tDNA. The detection limit was 1.97 × 10(-14) M, and the correlation coefficient was 0.989. A poorly defined current response was observed as the negative control and baseline. Our results showed high sensitivity and selectivity of the electrochemical DNA biosensor to the pathogenic bacteria E. coli O157:H7. The biosensor was also used to evaluate the detection of pathogen in real beef samples contaminated artificially. Compared with other electrochemical DNA biosensors, we conclude that this genosensor provides for very efficient detection of pathogenic bacteria. Therefore, this method may have potential application in food safety and related fields.

  3. A parylene-based dual channel microelectrophoresis system for rapid mutation detection via heteroduplex analysis

    NARCIS (Netherlands)

    Sukas, S.; Erson, Ayse Elif; Sert, Cuneyt; Kulah, Haluk

    2008-01-01

    A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 μm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing

  4. Are early warning scores the only way to rapidly detect and manage deterioration?

    Science.gov (United States)

    Odell, Mandy

    A systematic literature review recently highlighted the complexity of nursing practice in terms of detecting and managing deteriorating ward patients (Odell et al, 2009). The findings suggest that rapid response systems, including early warning scores, may not be the only solution to the problems of detecting and managing signs of deterioration. This article summarises the findings of this review.

  5. Early Detection Rapid Response Program Targets New Noxious Weed Species in Washington State

    Science.gov (United States)

    Andreas, Jennifer E.; Halpern, Alison D.; DesCamp, Wendy C.; Miller, Timothy W.

    2015-01-01

    Early detection, rapid response is a critical component of invasive plant management. It can be challenging, however, to detect new invaders before they become established if landowners cannot identify species of concern. In order to increase awareness, eye-catching postcards were developed in Washington State as part of a noxious weed educational…

  6. A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

    Science.gov (United States)

    Benacer, Douadi; Zain, Siti Nursheena Mohd; Lewis, John W; Khalid, Mohd Khairul Nizam Mohd; Thong, Kwai Lin

    2017-01-01

    This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

  7. CONCEPTUAL APPROACHES TO THE RAPID DETECTION OF CAMPYLOBACTER SPP. IN MEAT OF SLAUGHTER ANIMALS

    Directory of Open Access Journals (Sweden)

    D. S. Bataeva

    2017-01-01

    Full Text Available The modern approach to quality assurance of food products based on the ISO 9000 standards indicates the need for the implementation of quality management systems in processing plants. According to the analysis of scientific publication databases (Science Direct and Web of Science, it is established that only 0.5–1.7% of publications are related to studying meat of slaughter animals (except for birds concerning the presence of Campylobacter. The priority method of investigation is PCR. Ready-to-use PCR test system was developed for the detection of Campylobacter spp. on the basis of selected gene-specific primers to bacteria of Campylobacter genus. Specificity of the test system is established for Gram-negative bacteria of Salmonella, Escherichia, and Proteus genera, and for oxidase-positive Aeromonas. Gene-specific primers for Campylobacter were selected and ready-to-use PCR test system was developed on their basis. It was found that the selected primers have 100% convergence to the genome of Campylobacter genus bacteria, the PCR efficiency is not less than 95%, and the detection limit is not more than 1× 104 CFU/g. When estimating the specificity of the primers, it was taken into account that the bacteria of Campylobacter genus may be incorporated in a consortium with intestine microbiome, mainly with Enterobacteriaceae and lactic acid bacteria. However, Bolton’s enrichment medium is selective and, during the cultivation process, suppresses the growth of Gram-positive lactic acid bacteria. It was found that the selected primers were 100% specific and did not give false positive reactions with this group of microorganisms. The developed test system was successfully validated in a cycle of qualitative tests in the FEPAS system and implemented into laboratory practice. It was proved that the developed test system may be used both in screening at the stages of Campylobacter enrichment and in identification of pure culture of the

  8. 9 CFR 113.27 - Detection of extraneous viable bacteria and fungi in live vaccines.

    Science.gov (United States)

    2010-01-01

    ... bacteria and fungi in live vaccines. 113.27 Section 113.27 Animals and Animal Products ANIMAL AND PLANT... bacteria and fungi in live vaccines. Unless otherwise specified by the Administrator or elsewhere exempted... Seed Bacteria shall be tested for extraneous viable bacteria and fungi as prescribed in this section. A...

  9. Molecular Detection of Extended-Spectrum Beta-Lactamase in Isolated Bacteria from Blood Cultures

    Directory of Open Access Journals (Sweden)

    Rashid Ramazanzadeh

    2015-11-01

    Full Text Available Introduction: ESBLs are a B -lactamases which had ability to hydrolyse third-generation cephalosporins and  aztreonam.  ESBLs producer bacteria are resistant to  a wide variety of antimicrobials and they made a serious global health concern for treatment strategies. So, aim of this study as to  molecular detection of ESBLs in  bacteria isolated from blood  cultures in hospitals from Kurdistan Province, Iran.Methods: Biochemical test, antimicrobial susceptibility test by disc method, ESBL detection by NCCLs Phenotypic and PCR method for ESBL detection were applied. Results were analyzed by using SPSS 11.5 (p < 0.05.Results: 96 S. epidermidis isolated from blood cultures, E. coli, Enterobacter spp., Klebsiella spp.,  P.  aeruginosa,  Salmonella  spp.,  C.  freundii,  S.  maltophilia,  also  S.  aureus,  and S.epidermis. Maximum resistance was 75% for CP and minimum resistance was 25% for GM. Of the 96 isolates, 20 (20.83% produced ESBLs. Also 11.46%, 20.83%, 12.5%, 9.38% and 2.08%  were  positive  for  TEM, CTX-M, SHV, OXA-1  and  OXA-2  ensymes,  respectively.Conclusion: Inappropriate therapy for infections with ESBL producers is cause of prolongs hospital stay and mortality.  So, more research on drug resistance with ESBL is necessary.

  10. Rapid and selective detection of E. coli O157:H7 combining phagomagnetic separation with enzymatic colorimetry.

    Science.gov (United States)

    Zhang, Yun; Yan, Chenghui; Yang, Hang; Yu, Junping; Wei, Hongping

    2017-11-01

    Mammal IgG antibodies are normally used in conventional immunoassays for E. coli O157:H7, which could lead to false positive results from the presence of protein A producing S. aureus. In this study, a natural specific bacteriophage was isolated and then conjugated with magnetic beads as a capture element in a sandwich format for the rapid and selective detection of E. coli O157:H7. To the best of our knowledge, it was the first time to utilize a natural bacteriophage to develop a phagomagnetic separation combined with colorimetric assay for E. coli O157:H7. The method has an overall time less than 2h with a detection limit of 4.9×10 4 CFU/mL. No interference from S. aureus was observed. Furthermore, the proposed method was successfully applied to detect E. coli O157:H7 in spiked skim milk. The proposed detection system provided a potential method for E. coli O157:H7 and other pathogenic bacteria in food samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

    Science.gov (United States)

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2015-09-01

    Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2-3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also

  12. Extended-spectrum beta-lactamase-producing bacteria are not detected in supragingival plaque samples from human fecal carriers of ESBL-producing Enterobacteriaceae

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    Arne Søraas

    2014-08-01

    Full Text Available Background: The prevalence of infections caused by Cefotaximase-Munich (CTX-M-type extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E has rapidly increased during the past 15 years. Enterobacteriaceae are commonly found in the gastrointestinal tract and long-term intestinal carriage is considered important for the spread of ESBL and as a source of clinical infections. Oral biofilm such as supragingival plaque is known to contain numerous antibiotic resistance determinants and may also represent a poorly investigated site for ESBL carriage and further spread. Objective: To investigate possible carriage of ESBL-producing bacteria in supragingival plaque of known fecal carriers of these bacteria. Design: We screened for the presence of aerobic and anaerobic ESBL-producing bacteria and blaCTX-M in supragingival plaque samples from healthy human adults with culture-verified fecal carriage of CTX-M-producing Escherichia coli. The presence or absence of Enterobacteriaceae and ESBL-producing bacteria in plaque samples was evaluated using culture-based methods and consensus CTX-M PCR. Results: Oral samples were obtained from 17 participants with known previous carriage of ESBL-producing E. coli. No ESBL-producing bacteria or ESBL genes were detected using culture-based and molecular methods. One colony of Rahnella aquatilis harboring the class A ESBL gene bla RAHN-1/2 was identified in an oral sample from one of the participants. Conclusion: This pilot study supports the notion that the presence of CTX-M-producing bacteria is uncommon in oral plaque of healthy human adult fecal carriers. Due to the limited number of persons tested, a low prevalence of oral ESBL-carriage in healthy adults or carriage in selected groups of patients cannot be excluded. To our knowledge, this is the first description of an R. aquatilis with the RAHN-1/2 gene in the oral cavity.

  13. [Detection of causative bacteria for bovine mastitis and their susceptibility to beta-lactam antibacterial agents].

    Science.gov (United States)

    Kamata, S; Matsunaga, T; Uchida, K; Uchida, K

    1990-10-01

    During the period from November 1988 to May 1989, causative bacteria in a total of 172 clinical mastitis cases observed in 66 farms in 5 districts with 6 areas in Japan were examined and frequencies of their occurrences were determined. Susceptibilities (in MICs) of the isolates to 6 beta-lactam antibacterial agents were also determined. As a result, coagulase-negative staphylococci (CNS) was identified in 94 out of 172 cases (54.7%) and were the most prevalent. Corynebacterium spp., Gram-negative bacteria and Staphylococcus aureus were found in 52 (30.2%), 49 (28.5%) and 43 (25.0%) cases, respectively. Four species of Streptococcus family (S. agalactiae, S. dysgalactiae, S. uberis and S. bovis) were identified in a total of 58 cases (33.7%). Susceptibility testing of CNS to cefoperazone (CPZ), cefazolin (CEZ), benzylpenicillin (PCG), ampicillin (ABPC), methicillin (DMPPC) and cloxacillin (MCIPC) showed that all MIC80's (inhibiting bacterial growth of 80% of all isolates) were within a range from 0.10 to 3.13 micrograms/ml and that there was no marked differences in antibacterial effects among the antibiotics used. The highest antibacterial effect on S. aureus was exhibited by MCIPC, which inhibited the growth of all isolates at 0.39 microgram/ml. The MICs of DMPPC against all isolates of S. aureus were 3.13 micrograms/ml or less and no methicillin-resistant S. aureus (MRSA) was detected. There was no difference in antibacterial activities against Streptococcus family between penicillin antibiotics (DMPPC, MCIPC, ABPC and PCG) and cephem antibiotics (CPZ and CEZ), both of which showed excellent antibacterial activities. Cephem antibiotics exhibited higher activities against Gram-negative bacteria than penicillin antibiotics. Especially CPZ, the third generation cephem, showed excellent antibacterial activity against Escherichia coli, Klebsiella spp., Enterobacter spp., as well as against other Enterobacteriaceae.

  14. Development of a novel genetically modified bioluminescent-bacteria-based assay for detection of fluoroquinolones in animal-derived foods.

    Science.gov (United States)

    Cheng, Guyue; Dong, Xiaobing; Wang, Yulian; Peng, Dapeng; Wang, Xu; Hao, Haihong; Xie, Shuyu; Qu, Wei; Liu, Zhenli; Yuan, Zonghui

    2014-12-01

    Fluoroquinolones (FQNs) are broad-spectrum antibacterial agents widely used in animal husbandry and aquaculture. The residues and antimicrobial resistance of such antibiotics are a major public health concern. To realize multianalyte detection of FQN residues, a genetically modified bacterium, Escherichia coli pK12 harboring plasmid pRecAlux3, was constructed in this study to develop a bioluminescent-bacteria-based assay for the detection of FQNs in animal-derived foods. This assay was based on the principle of induction of an SOS response by FQNs via inducing the recA-promoter-fused luciferase reporter gene existing on the plasmid pRecAlux3. E. coli pK12 was able to recognize 11 FQNs: difloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, norfloxacin, danofloxacin, ofloxacin, pefloxacin, lomefloxacin, marbofloxacin, and orbifloxacin. This method could be applied to 11 edible tissues, including milk, fish muscle, and the muscles, livers, and kidneys of cattle, chickens, and pigs, with a very simple and rapid sample extraction procedure using only phosphate-buffered saline. The limits of detection of the FQNs were between 12.5 and 100 μg kg(-1), all of which were lower than the maximum residue limits. Most of the recoveries of the FQNs were in the range from 60 to 120 %, and the interassay coefficients of variation were less than 30 %. This method, confirmed by high-performance liquid chromatography, is reliable and can be used as both a screening test and a semiquantitative assay, when the identity of a single type of FQN is known.

  15. "Salvage microbiology": detection of bacteria directly from clinical specimens following initiation of antimicrobial treatment.

    Directory of Open Access Journals (Sweden)

    John J Farrell

    Full Text Available PCR coupled with electrospray ionization mass spectrometry (ESI-MS is a diagnostic approach that has demonstrated the capacity to detect pathogenic organisms from culture negative clinical samples after antibiotic treatment has been initiated. [1] We describe the application of PCR/ESI-MS for detection of bacteria in original patient specimens that were obtained after administration of antibiotic treatment in an open investigation analysis.We prospectively identified cases of suspected bacterial infection in which cultures were not obtained until after the initiation of antimicrobial treatment. PCR/ESI-MS was performed on 76 clinical specimens that were submitted for conventional microbiology testing from 47 patients receiving antimicrobial treatment.In our series, 72% (55/76 of cultures obtained following initiation of antimicrobial treatment were non-diagnostic (45 negative cultures; and 10 respiratory specimens with normal flora (5, yeast (4, or coagulase-negative staphylococcus (1. PCR/ESR-MS detected organisms in 83% (39/47 of cases and 76% (58/76 of the specimens. Bacterial pathogens were detected by PCR/ESI-MS in 60% (27/45 of the specimens in which cultures were negative. Notably, in two cases of relapse of prosthetic knee infections in patients on chronic suppressive antibiotics, the previous organism was not recovered in tissue cultures taken during extraction of the infected knee prostheses, but was detected by PCR/ESI-MS.Molecular methods that rely on nucleic acid amplification may offer a unique advantage in the detection of pathogens collected after initiation of antimicrobial treatment and may provide an opportunity to target antimicrobial therapy and "salvage" both individual treatment regimens as well as, in select cases, institutional antimicrobial stewardship efforts.

  16. Paper-based Biosensor for Rapid Colorimetric Detection of Pathogenic Bacteria Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The ability to monitor microbial contamination remains a critical technique in the mitigation of risk to crew health and vehicle systems during human spaceflight...

  17. Development of a Methodology for the Rapid Detection of Coliform Bacteria.

    Science.gov (United States)

    1981-02-27

    and 563Y) using an eyepiece micrometer . Surfactants were also judged visibly in terms of desree of settling, flocculation, coalescence, and bulk phase...Independent control of individual flow rates was accomplished by means of flow loops, outfitted with vernier handled metering valves, and connected by T...settings were determined for each input stream using both deionized water and silicone oil to maintain a constant flow equilibrium. Vernier settings

  18. Detection of airborne psychrotrophic bacteria and fungi in food storage refrigerators.

    Science.gov (United States)

    Altunatmaz, Sema Sandikci; Issa, Ghassan; Aydin, Ali

    2012-10-01

    The purpose of this study was to determine the microbiological air quality (psychrotrophic bacteria and airborne fungi) and distribution of fungi in different types of ready-to-eat (RTE) food-storage refrigerators (n=48) at selected retail stores in the city of Edirne, Turkey. Refrigerators were categorized according to the type of RTE food-storage: meat products, vegetables, desserts, or a mix of food types. Microbiological quality of air samples was evaluated by using a Mas-100 Eco Air Sampler. Four refrigerators (all containing meat products, 8.3%) produced air samples with undetectable microorganisms. The highest detected mean value of airborne psychrotrophic bacteria and fungi was 82.3 CFU/m(3) and 54.6 CFU/m(3), respectively and were found in mixed-food refrigerators. The dominant airborne fungal genera found were Penicillium (29.0%), Aspergillus (12.0%), Mucor (9%), Cladosporium (8%), Botyrtis (7%), and Acremonium (6%). By definition, RTE food does not undergo a final treatment to ensure its safety prior to consumption. Therefore, ensuring a clean storage environment for these foods is important to prevent food-borne disease and other health risks.

  19. Detection of airborne psychrotrophic bacteria and fungi in food storage refrigerators

    Directory of Open Access Journals (Sweden)

    Sema Sandikci Altunatmaz

    2012-12-01

    Full Text Available The purpose of this study was to determine the microbiological air quality (psychrotrophic bacteria and airborne fungi and distribution of fungi in different types of ready-to-eat (RTE food-storage refrigerators (n=48 at selected retail stores in the city of Edirne, Turkey. Refrigerators were categorized according to the type of RTE food-storage: meat products, vegetables, desserts, or a mix of food types. Microbiological quality of air samples was evaluated by using a Mas-100 Eco Air Sampler. Four refrigerators (all containing meat products, 8.3% produced air samples with undetectable microorganisms. The highest detected mean value of airborne psychrotrophic bacteria and fungi was 82.3 CFU/m³ and 54.6 CFU/m³, respectively and were found in mixed-food refrigerators. The dominant airborne fungal genera found were Penicillium (29.0%, Aspergillus (12.0%, Mucor (9%, Cladosporium (8%, Botyrtis (7%, and Acremonium (6%. By definition, RTE food does not undergo a final treatment to ensure its safety prior to consumption. Therefore, ensuring a clean storage environment for these foods is important to prevent food-borne disease and other health risks.

  20. Detection of putative periodontopathic bacteria in type 1 diabetic and healthy children: A comparative study

    Directory of Open Access Journals (Sweden)

    Ponnudurai Arangannal

    2013-01-01

    Full Text Available Aim: The aim of this study was to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans in type 1 diabetic and healthy children. Materials and Methods: Fifty type 1 diabetic and 50 healthy children in the age group of 7-14 years were recruited for the study. Subgingival plaque samples collected from permanent first molars were subjected to polymerase chain reaction assay to detect 16S rRNA gene of P. gingivalis, T. forsythia, T. denticola and A. actinomycetemcomitans. The data were analyzed using Fisher exact test. The P < 0.05 was considered statistically significant. Results: The prevalence of subgingival periodontal pathogens in diabetic and healthy children was 2% and 4% for P. gingivalis, 34% and 34% for T. denticola, 20% and 18% for A. actinomycetemcomitans and for T. forsythia, 4% and 34%, respectively. Significant statistical difference was not observed with regard to the prevalence of P. gingivalis, T. denticola, and A. actinomycetemcomitans among type 1 diabetic and healthy children (P = 1.00. Conversely, T. forsythia was less prevalent in diabetic children compared to healthy children. Conclusion: Statistical significance was not observed for the prevalence of periodontopathic bacteria in type 1 diabetic subjects. The results of the present study thus reveal the absence of risk of periodontitis by these bacterial species in type 1 diabetic subjects.

  1. Rapid Salmonella detection using an acoustic wave device combined with the RCA isothermal DNA amplification method

    Directory of Open Access Journals (Sweden)

    Antonis Kordas

    2016-12-01

    Full Text Available Salmonella enterica serovar Typhimurium is a major foodborne pathogen that causes Salmonellosis, posing a serious threat for public health and economy; thus, the development of fast and sensitive methods is of paramount importance for food quality control and safety management. In the current work, we are presenting a new approach where an isothermal amplification method is combined with an acoustic wave device for the development of a label free assay for bacteria detection. Specifically, our method utilizes a Love wave biosensor based on a Surface Acoustic Wave (SAW device combined with the isothermal Rolling Circle Amplification (RCA method; various protocols were tested regarding the DNA amplification and detection, including off-chip amplification at two different temperatures (30 °C and room temperature followed by acoustic detection and on-chip amplification and detection at room temperature, with the current detection limit being as little as 100 Bacteria Cell Equivalents (BCE/sample. Our acoustic results showed that the acoustic ratio, i.e., the amplitude over phase change observed during DNA binding, provided the only sensitive means for product detection while the measurement of amplitude or phase alone could not discriminate positive from negative samples. The method's fast analysis time together with other inherent advantages i.e., portability, potential for multi-analysis, lower sample volumes and reduced power consumption, hold great promise for employing the developed assay in a Lab on Chip (LoC platform for the integrated analysis of Salmonella in food samples.

  2. Rapid Impingement Detection System with Uniform Sampling for Ball-and-Socket Joint

    Science.gov (United States)

    Cai, Ding; Lee, Won-Sook; Joslin, Chris; Beaulé, Paul

    Detecting the position and the level of joint impingement Femoroacetabular impingement is often a key to computer-aided surgical plan to normalize joint kinematics. So far most of the current impingement detection methods for ball-and-socket joint are not efficient or only report a few collided points as the detection results. In this chapter, we present a novel real-time impingement detection system with rapid memory-efficient uniform sampling and surface-to-surface distance measurement feature to estimate the overall impingement. Our system describes near-spherical objects in spherical coordinate system, which reduces the space complexity and the computation costs. The sampling design further reduces the memory cost by generating uniform sampling orientations. The rapid and accurate impingement detection with surface-to-surface distance measurement can provide more realistic detailed information to estimate the overall impingement on the ball-and-socket joint, which is particularly useful for computer-aided surgical plan.

  3. Rapid detection of Mycobacterium avium in stool samples from AIDS patients by immunomagnetic PCR.

    OpenAIRE

    Li, Z; Bai, G H; von Reyn, C. F.; Marino, P.; Brennan, M J; Gine, N; Morris, S. L.

    1996-01-01

    Direct PCR detection of bacteria in clinical samples is often hindered by the presence of compounds that inhibit the PCR. To improve and accelerate the diagnosis of Mycobacterium avium-M. intracellulare complex infections, an immunomagnetic PCR (IM-PCR) assay was developed. This IM-PCR procedure combines the separation of mycobacteria by antimycobacterial monoclonal antibody coupled to magnetic beads with an M. avium-M. intracellulare complex-specific PCR protocol based on 16S rRNA gene seque...

  4. Random amplified ribosomal DNA restriction analysis for rapid identification of thermophilic Actinomycete-like bacteria involved in hypersensitivity pneumonitis.

    Science.gov (United States)

    Harvey, I; Cormier, Y; Beaulieu, C; Akimov, V N; Mériaux, A; Duchaine, C

    2001-07-01

    Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 degrees C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taql and HhaI restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.

  5. Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection.

    Science.gov (United States)

    Hu, Hongmei; Li, Zhenhua; Zhang, Xiaoning; Xu, Chunxiu; Guo, Yuanming

    2013-10-01

    A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis (NAMCE) with LIF detection, A homemade pump-free negative pressure sampling device was used for rapid bias-free sampling in NAMCE, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in catecholamines in urine samples. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Reduced ability to detect surface-related biofilm bacteria after antibiotic exposure under in vitro conditions

    DEFF Research Database (Denmark)

    Ravn, Christen; Furustrand Tafin, Ulrika; Bétrisey, Bertrand

    2016-01-01

    Background and purpose - Antibiotic treatment of patients before specimen collection reduces the ability to detect organisms by culture. We investigated the suppressive effect of antibiotics on the growth of non-adherent, planktonic, and surface-related biofilm bacteria in vitro by using sonication...... and microcalorimetry methods. Patients and methods - Biofilms of Staphylococcus aureus, S. epidermidis, Escherichia coli, and Propionibacterium acnes were formed on porous glass beads and exposed for 24 h to antibiotic concentrations from 1 to 1,024 times the minimal inhibitory concentration (MIC) of vancomycin......, daptomycin, rifampin, flucloxacillin, or ciprofloxacin. The beads were then sonicated to dislodge biofilm, followed by culture and measurement of growth-related heat flow by microcalorimetry of the resulting sonication fluid. Results - Vancomycin did not inhibit the heat flow of staphylococci and P. acnes...

  7. Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR.

    Science.gov (United States)

    O'Grady, Justin; Ruttledge, Margaret; Sedano-Balbás, Sara; Smith, Terry J; Barry, Thomas; Maher, Majella

    2009-02-01

    A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24h incubation in half-Fraser broth, 4h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1-5CFU/25g food sample and can be performed in 2 working days compared to up to 7days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n=175) and controls (n=31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.

  8. Rapid detection of Naegleria fowleri in water distribution pipeline biofilms and drinking water samples.

    Science.gov (United States)

    Puzon, Geoffrey J; Lancaster, James A; Wylie, Jason T; Plumb, Iason J

    2009-09-01

    Rapid detection of pathogenic Naegleria fowler in water distribution networks is critical for water utilities. Current detection methods rely on sampling drinking water followed by culturing and molecular identification of purified strains. This culture-based method takes an extended amount of time (days), detects both nonpathogenic and pathogenic species, and does not account for N. fowleri cells associated with pipe wall biofilms. In this study, a total DNA extraction technique coupled with a real-time PCR method using primers specific for N. fowleri was developed and validated. The method readily detected N. fowleri without preculturing with the lowest detection limit for N. fowleri cells spiked in biofilm being one cell (66% detection rate) and five cells (100% detection rate). For drinking water, the detection limit was five cells (66% detection rate) and 10 cells (100% detection rate). By comparison, culture-based methods were less sensitive for detection of cells spiked into both biofilm (66% detection for pipe wall biofilm samples obtained from a distribution network enabled the detection of N. fowleri in under 6 h, versus 3+ daysforthe culture based method. Further, comparison of the real-time PCR data from the field samples and the standard curves enabled an approximation of N. fowleri cells in the biofilm and drinking water. The use of such a method will further aid water utilities in detecting and managing the persistence of N. fowleri in water distribution networks.

  9. Assessment of three rapid methods for the detection of methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Soares, Maria João; Soares, Carlos; Mendes, Ana Constança; Guimarães, Maria Luís; Cabeda, José Manuel; Amorim, José Manuel

    2004-01-01

    We evaluated three rapid methods to detect methicillin-resistant Staphylococcus aureus (MRSA) and compared them with PCR amplification of mecA. A total of 103 S. aureus strains were studied by MRSA-Screen, BBL Crystal, Velogene Genomic and mecA PCR. All the methods detected the 61 MRSA strains having the mecA gene, showing 100% sensitivity and specificity. Despite the correlation between all the rapid methods and PCR, the ease of use and shorter turnaround time of MRSA-Screen were important factors leading to the selection of this method as the routine screening technique for MRSA.

  10. A rapid, simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples.

    Science.gov (United States)

    Wang, Jinhong; Zhang, Yan; Cui, Yanyan; Yan, Yaqun; Wang, Xiaoxing; Wang, Rongjun; Jian, Fuchun; Zhang, Longxian; Ning, Changshen

    2017-03-27

    A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62°C for 60min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5×10(0)copies/μL, 100 times more than that of conventional PCR (5×10(2)copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Rapid Detection and Identification of Yersinia pestis from Food Using Immunomagnetic Separation and Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Kingsley K. Amoako

    2012-01-01

    Full Text Available Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through eating infected animals that were not adequately cooked or handling of meat from infected animals makes the possible use of Y. pestis in a foodborne bioterrorism attack a reality. Rapid, efficient food sample preparation and detection systems that will help overcome the problem associated with the complexity of the different matrices and also remove any ambiguity in results will enable rapid informed decisions to be made regarding contamination of food with biothreat agents. We have developed a rapid detection assay that combines the use of immunomagnetic separation and pyrosequencing in generating results for the unambiguous identification of Y. pestis from milk (0.9 CFU/mL, bagged salad (1.6 CFU/g, and processed meat (10 CFU/g. The low detection limits demonstrated in this assay provide a novel tool for the rapid detection and confirmation of Y. pestis in food without the need for enrichment. The combined use of the iCropTheBug system and pyrosequencing for efficient capture and detection of Y. pestis is novel and has potential applications in food biodefence.

  12. Current and emerging technologies for rapid detection and characterization of Salmonella in poultry and poultry products.

    Science.gov (United States)

    Park, Si Hong; Aydin, Muhsin; Khatiwara, Anita; Dolan, Maureen C; Gilmore, David F; Bouldin, Jennifer L; Ahn, Soohyoun; Ricke, Steven C

    2014-04-01

    Salmonella is the leading cause of foodborne illnesses in the United States, and one of the main contributors to salmonellosis is the consumption of contaminated poultry and poultry products. Since deleterious effects of Salmonella on public health and the economy continue to occur, there is an ongoing need to develop more advanced detection methods that can identify Salmonella accurately and rapidly in foods before they reach consumers. Rapid detection and identification methods for Salmonella are considered to be an important component of strategies designed to prevent poultry and poultry product-associated illnesses. In the past three decades, there have been increasing efforts towards developing and improving rapid pathogen detection and characterization methodologies for application to poultry and poultry products. In this review, we discuss molecular methods for detection, identification and genetic characterization of Salmonella associated with poultry and poultry products. In addition, the advantages and disadvantages of the established and emerging rapid detection and characterization methods are addressed for Salmonella in poultry and poultry products. The methods with potential application to the industry are highlighted in this review. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  14. Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration.

    Science.gov (United States)

    Stern, Daniel; Olson, Victoria A; Smith, Scott K; Pietraszczyk, Marko; Miller, Lilija; Miethe, Peter; Dorner, Brigitte G; Nitsche, Andreas

    2016-12-09

    The rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce. In this work we present a point-of-care test for the detection of Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 104 plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus. Given the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples.

  15. Rapid Preclinical Detection of Sheeppox Virus by a Real-Time PCR Assay▿

    Science.gov (United States)

    Balinsky, C. A.; Delhon, G.; Smoliga, G.; Prarat, M.; French, R. A.; Geary, S. J.; Rock, D. L.; Rodriguez, L. L.

    2008-01-01

    Sheeppox virus (SPPV) is a member of the Capripoxvirus (CaPV) genus of the Poxviridae family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples. PMID:18032617

  16. Rapid preclinical detection of sheeppox virus by a real-time PCR assay.

    Science.gov (United States)

    Balinsky, C A; Delhon, G; Smoliga, G; Prarat, M; French, R A; Geary, S J; Rock, D L; Rodriguez, L L

    2008-02-01

    Sheeppox virus (SPPV) is a member of the Capripoxvirus (CaPV) genus of the Poxviridae family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples.

  17. Rapid and specific detection of Asian- and African-lineage Zika viruses

    Science.gov (United States)

    Chotiwan, Nunya; Brewster, Connie D.; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K.; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M.; Fauver, Joseph R.; Andre, Barb; Gray, Meg; Black, William C.; Kading, Rebekah C.; Ebel, Gregory D.; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T. A.; Brault, Aaron C.; Harris, Eva; Foy, Brian D.; Quackenbush, Sandra L.; Perera, Rushika; Rovnak, Joel

    2017-01-01

    Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. PMID:28469032

  18. Piezoelectric immunosensor for the direct and rapid detection of Francisella tularensis.

    Science.gov (United States)

    Pohanka, M; Skládal, P

    2007-01-01

    A novel immunosensing device based on a piezoelectric sensor for direct detection of the biological warfare agent Francisella tularensis was developed. This sensor includes mouse polyclonal antibody immobilized in a layer of protein A covalently linked to the gold electrode of the sensor. The immunosensor is able to detect F. tularensis with the limit of detection 10(5) CFU/mL with a typical measuring cycle > 5 min. The sensor was successfully evaluated for rapid detection of F. tularensis spikes in drinking water and milk; no deterioration of sensitivity in comparison with buffer solutions was observed. The proposed concept of a rapid measurement of microbial agents seems to be promising for evaluation of samples after short pre-cultivation enrichment.

  19. Electrochemical biosensors for rapid detection of Escherichia coli O157:H7.

    Science.gov (United States)

    Xu, Meng; Wang, Ronghui; Li, Yanbin

    2017-01-01

    Electrochemical biosensors have shown great promise in the development of rapid methods for the detection of foodborne pathogens and have been intensively studied over the past two decades. The scope of this review is to summarize the advancements made in the development of electrochemical biosensors for the rapid detection of one of the most common foodborne pathogens, Escherichia coli O157:H7. The article is intended to include different configurations of electrochemical biosensors based on the sensing principles and measured electrical parameters, as well as the latest improvements of technology in the progress of electrochemical biosensor development to detect E. coli O157:H7. By discussing the current and future trend based on some of excellent published literatures and reviews, this survey is hoped to illustrate a broad and comprehensive understanding of electrochemical biosensors for the detection of foodborne pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. PRODUCCIÓN DE ANTISUEROS PARA LA DETECCIÓN DE ÁCIDO INDOLACÉTICO EN CULTIVOS DE BACTERIAS PROMOTORAS DEL CRECIMIENTO VEGETAL Antisera Production to Detect Indoleacetic Acid in Cultures of Plant-Growth Promoting Bacteria

    Directory of Open Access Journals (Sweden)

    MARCIA M ROJAS

    2012-05-01

    Full Text Available Se obtuvieron antisueros en conejo utilizando como antígeno el AIA adherido a membranas de nitrocelulosa que mostraron un elevado título y especificidad. Mediante la técnica de inmunoadsorción por manchas marcadas con oro coloidal se detectó la producción de esta auxina por cepas de los géneros Gluconacetobacter, Herbaspirillum, Azospirillum, Pseudomonas, Burkholderia y Bacillus empleando como antígenos los sobrenadantes de los cultivos. Para cuantificar la producción de AIA y corroborar los datos obtenidos se empleó la técnica colorimétrica derivada de Salkowski. Los resultados muestran que todos los géneros bacterianos estudiados tienen la capacidad de producir AIA y se demuestra la factibilidad del uso de este antisuero policlonal para la detección de este metabolito. Teniendo en cuenta las potencialidades de estas bacterias, resulta de gran importancia la utilización de antisueros y técnicas serológicas para la detección rápida y sencilla de este tipo de metabolitos en bacterias asociadas a cultivos de interés económico.Rabbit polyclonal antisera against indoleacetic acid (IAA bound to nitrocellulose membrane were obtained, which exhibited a high titer and specificity. The dot immunobinding technique with colloidal gold was used to detect auxin production by several strains belonging to Gluconacetobacter, Herbaspirillum, Azospirillum, Pseudomonas, Burkholderia and Bacillus genera, using culture supernatants as antigens. Moreover, auxin production was quantified by the Salkowski's method to corroborate the previous results. It was found that that all the studied microorganisms produce IAA and the feasibility of using these antisera to detect the metabolite was confirmed. Taking into account the potentialities of plant growth promoting bacteria as biofertilizers, the use of these antisera for a rapid and easy detection of IAA in bacteria associated with important crops is thus recommended.

  1. Rapid whole-genome sequencing for detection and characterization of microorganisms directly from clinical samples.

    Science.gov (United States)

    Hasman, Henrik; Saputra, Dhany; Sicheritz-Ponten, Thomas; Lund, Ole; Svendsen, Christina Aaby; Frimodt-Møller, Niels; Aarestrup, Frank M

    2014-01-01

    Whole-genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples, this could further reduce diagnostic times and thereby improve control and treatment. A major bottleneck is the availability of fast and reliable bioinformatic tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatic tools for the analysis of sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria, and direct sequencing on pellets from the urine samples. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples but in pure cultures from only 17 samples. WGS improved the identification of the cultivated bacteria, and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibilities. Complete agreement was observed between species identification, multilocus sequence typing, and phylogenetic relationships for Escherichia coli and Enterococcus faecalis isolates when the results of WGS of cultured isolates and urine samples were directly compared. Sequencing directly from the urine enabled bacterial identification in polymicrobial samples. Additional putative pathogenic strains were observed in some culture-negative samples. WGS directly on clinical samples can provide clinically relevant information and drastically reduce diagnostic times. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem, a publicly available bioinformatic tool was developed in this study.

  2. PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes.

    Science.gov (United States)

    Janczarek, Monika; Palusińska-Szysz, Marta

    2016-05-01

    Legionella bacteria are organisms of public health interest due to their ability to cause pneumonia (Legionnaires' disease) in susceptible humans and their ubiquitous presence in water supply systems. Rapid diagnosis of Legionnaires' disease allows the use of therapy specific for the disease. L. pneumophila serogroup 1 is the most common cause of infection acquired in community and hospital environments. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this work, simplex and duplex PCR assays with the use of new molecular markers pcs and pmtA involved in phosphatidylcholine synthesis were specified for rapid and cost-efficient identification and distinguishing Legionella species. The sets of primers developed were found to be sensitive and specific for reliable detection of Legionella belonging to the eight most clinically relevant species. Among these, four primer sets I, II, VI, and VII used for duplex-PCRs proved to have the highest identification power and reliability in the detection of the bacteria. Application of this PCR-based method should improve detection of Legionella spp. in both clinical and environmental settings and facilitate molecular typing of these organisms.

  3. Real-time in situ detection and quantification of bacteria in the Arctic environment

    Directory of Open Access Journals (Sweden)

    Linda Powers

    2014-03-01

    Full Text Available At present, there are no methods that determine the total microbial load on an abiotic substrate in real time. The utility of such a capability ranges from sterilization and medical diagnostics to the search for new microorganisms in the environment and study of their ecological niches. We report the development of a hand-held, fluorescence detection device and demonstrate its applicability to the field detection of Arctic bacteria. This technology is based on the early pioneering work of Britton Chance which elucidated the intrinsic fluorescence of a number of metabolites and protein cofactors in cells, including reduced pyridine nucleotides, cytochromes and flavins. A PDA controls the device (fluorescence excitation and data collection and processes the multiwavelength signals to yield bacterial cell counts, including estimates of live cells, dead cells and endospores. Unlike existing methods for cell counting, this method requires no sample contact or addition of reagents. The use of this technology is demonstrated with in situ measurements of two sub-glacial microbial communities at sites in Palander and colonized surface rocks in the Bockfjord Volcanic Complex during AMASE 2008 (Arctic Mars Analog Svalbard Expedition. The total bacterial load on the interrogated sample surfaces ranged from 109 cells/cm2.

  4. Detection, identification and characterization of bacteriocin-producing lactic acid bacteria from retail food products.

    Science.gov (United States)

    Garver, K I; Muriana, P M

    1993-09-01

    Forty bacteriocin-producing (Bac+) lactic acid bacteria (LAB) were isolated from food samples purchased from retail supermarkets and local farms. Of the 40 Bac+ isolates, 18 were isolated from 85 food samples by enrichment (21% isolation rate) whereas eight were obtained from 63 samples by direct plating (13% isolation rate). By direct plating, Bac+ LAB were detected at levels up to 2.4 x 10(5) cfu/g in ready-to-eat meats. The Bac+ isolates were identified by carbohydrate fermentation patterns, SDS-PAGE protein patterns, and other biochemical characteristics; SDS-PAGE proved invaluable in identifying strains that could not be identified by other means. Differential inhibitory spectra against indicator microorganisms assisted in the identification of 19 unique Bac+ isolates. Bac+ LAB included Enterococcus faecalis, Lactobacillus curvatus, Lb. delbrueckii, Lb. plantarum, Lactococcus lactis, and Pediococcus acidilactici. Lb. curvatus (four strains) and Lc. lactis (nine strains) were the only isolates inhibitory to foodborne pathogens including Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus. Some Lc. lactis isolates inhibited as many as nine Gram-positive genera. Lb. curvatus FS47 and FS65 grew to high cell densities and produced bacteriocin at 6 degrees C; however, Lc. lactis FS56 produced greater levels of bacteriocin at lower cell densities. The high incidence of Bac+ LAB detected in retail foods indicates that the public is consuming a wide variety of Bac+ LAB that occur as natural contaminants. These data suggest a greater role for bacteriocins as biopreservatives in food.

  5. GES-5 among the β-lactamases detected in ubiquitous bacteria isolated from aquatic environment samples.

    Science.gov (United States)

    Manageiro, Vera; Ferreira, Eugénia; Caniça, Manuela; Manaia, Célia M

    2014-02-01

    In this study, we investigated the β-lactamase-encoding genes responsible for β-lactam resistance phenotypes detected among 56 Gram-negative isolates (Gamma- and Alpha-proteobacteria) recovered from wastewater, urban streams, and drinking water. The β-lactam resistance mechanisms detected in 36 isolates comprised the presence of class A (blaTEM-1 , blaSHV-1 , blaSHV-11 , blaGES-5 ), class B (ImiS, L1), class C (blaCMY-2 , blaCMY-34 , blaCMY-65 , blaCMY-89 , blaCMY-90 , blaACC-5 , blaACT-13 ), and class D (blaOXA-309)β-lactamase-encoding genes, some variants described for the first time here. Notably, the results showed antimicrobial resistance genes related not only to commonly used antibiotics, but also to carbapenems, providing the first description of a GES-5-producing Enterobacteriaceae. The importance of ubiquitous bacteria thriving in aquatic environments as reservoirs or carriers of clinically relevant resistance determinants was confirmed, and the need to monitor water habitats as potential sources for the emergence and/or spread of antibiotic resistance in the environment was highlighted. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  6. A nationwide web-based automated system for early outbreak detection and rapid response in China

    Directory of Open Access Journals (Sweden)

    Yilan Liao

    2011-03-01

    Full Text Available Timely reporting, effective analyses and rapid distribution of surveillance data can assist in detecting the aberration of disease occurrence and further facilitate a timely response. In China, a new nationwide web-based automated system for outbreak detection and rapid response was developed in 2008. The China Infectious Disease Automated-alert and Response System (CIDARS was developed by the Chinese Center for Disease Control and Prevention based on the surveillance data from the existing electronic National Notifiable Infectious Diseases Reporting Information System (NIDRIS started in 2004. NIDRIS greatly improved the timeliness and completeness of data reporting with real time reporting information via the Internet. CIDARS further facilitates the data analysis, aberration detection, signal dissemination, signal response and information communication needed by public health departments across the country. In CIDARS, three aberration detection methods are used to detect the unusual occurrence of 28 notifiable infectious diseases at the county level and to transmit that information either in real-time or on a daily basis. The Internet, computers and mobile phones are used to accomplish rapid signal generation and dissemination, timely reporting and reviewing of the signal response results. CIDARS has been used nationwide since 2008; all Centers for Disease Control and Prevention (CDC in China at the county, prefecture, provincial and national levels are involved in the system. It assists with early outbreak detection at the local level and prompts reporting of unusual disease occurrences or potential outbreaks to CDCs throughout the country.

  7. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    Science.gov (United States)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  8. Novel touchdown-PCR method for the detection of putrescine producing gram-negative bacteria in food products.

    Science.gov (United States)

    Wunderlichová, Leona; Buňková, Leona; Koutný, Marek; Valenta, Tomáš; Buňka, František

    2013-06-01

    Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99-91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. A microdevice for rapid, monoplex and colorimetric detection of foodborne pathogens using a centrifugal microfluidic platform.

    Science.gov (United States)

    Sayad, Abkar; Ibrahim, Fatimah; Mukim Uddin, Shah; Cho, Jongman; Madou, Marc; Thong, Kwai Lin

    2018-02-15

    Outbreaks of foodborne diseases have become a global health concern; hence, many improvements and developments have been made to reduce the risk of food contamination. We developed a centrifugal microfluidic automatic wireless endpoint detection system integrated with loop mediated isothermal amplification (LAMP) for monoplex pathogen detection. Six identical sets were designed on the microfluidic compact disc (CD) to perform 30 genetic analyses of three different species of foodborne pathogens. The consecutive loading, mixing, and aliquoting of the LAMP primers/reagents and DNA sample solutions were accomplished using an optimized square-wave microchannel, metering chambers and revulsion per minute (RPM) control. We tested 24 strains of pathogenic bacteria (Escherichia coli, Salmonella spp and Vibrio cholerae), with 8 strains of each bacterium, and performed DNA amplification on the microfluidic CD for 60min. Then, the amplicons of the LAMP reaction were detected using the calcein colorimetric method and further analysed via the developed electronic system interfaced with Bluetooth wireless technology to transmit the results to a smartphone. The system showed a limit of detection (LOD) of 3 × 10-5ngμL-1 DNA by analysing the colour change when tested with chicken meat spiked with the three pathogenic bacteria. Since the entire process was performed in a fully automated way and was easy to use, our microdevice is suitable for point-of-care (POC) testing with high simplicity, providing affordability and accessibility even to poor, resource-limited settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Virus detection using Viro-Adembeads, a rapid capture system for viruses, and plaque assay in intentionally virus-contaminated beverages.

    Science.gov (United States)

    Hatano, Ben; Kojima, Asato; Sata, Tetsutaro; Katano, Harutaka

    2010-01-01

    Intentional contamination of beverages with microbes is one type of bioterrorist threat. While bacteria and fungus can be easily collected by a centrifuge, viruses are difficult to collect from virus-contaminated beverages. In this study, we demonstrated that Viro-Adembeads, a rapid-capture system for viruses using anionic polymer-coated magnetic beads, collected viruses from beverages contaminated intentionally with vaccinia virus and human herpesvirus 8. Real-time PCR showed that the recovery rates of the contaminated viruses in green tea and orange juice were lower than those in milk and water. Plaque assay showed that green tea and orange juice cut the efficiency of vaccinia virus infection in CV-1 cells. These results suggest that the efficiency of virus detection depends on the kind of beverage being tested. Viro-Adembeads would be a useful tool for detecting viruses rapidly in virus-contaminated beverages used in a bioterrorist attack.

  11. HIV-Selectest enzyme immunoassay and rapid test: ability to detect seroconversion following HIV-1 infection.

    Science.gov (United States)

    Khurana, Surender; Norris, Philip J; Busch, Michael P; Haynes, Barton F; Park, Susan; Sasono, Pretty; Mlisana, Koleka; Salim, Abdool Karim; Hecht, Frederick M; Mulenga, Joseph; Chomba, Elwyn; Hunter, Eric; Allen, Susan; Nemo, George; Rodriguez-Chavez, Isaac R; Margolick, Joseph B; Golding, Hana

    2010-01-01

    HIV-Selectest is a serodiagnostic enzyme immunoassay (EIA), containing p6 and gp41 peptides, designed to differentiate between vaccine-induced antibodies and true infections. A rapid test version of the HIV-Selectest was developed. Both assays detected HIV antibodies in men and women within 2 to 4 weeks of infection, with sensitivity similar to third-generation EIAs.

  12. Rapid and early detection of salmonella serotypes with hyperspectral microscope and multivariate data analysis

    Science.gov (United States)

    This study was designed to evaluate hyperspectral microscope images for early and rapid detection of Salmonella serotypes: S. Enteritidis, S. Heidelberg, S. Infantis, S. Kentucky, and S. Typhimurium at incubation times of 6, 8, 10, 12, and 24 hours. Images were collected by an acousto-optical tunab...

  13. Rapid and real-time detection technologies for emerging viruses of ...

    Indian Academy of Sciences (India)

    2008-10-17

    Oct 17, 2008 ... The development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number threats posed by emerging and re-emerging viruses in the recent past. The conventional identification methods require time-consuming culturing ...

  14. A miniaturized optoelectronic system for rapid quantitative label-free detection of harmful species in food

    NARCIS (Netherlands)

    Raptis, Ioannis; Misiakos, Konstantinos; Makarona, Eleni; Salapatas, Alexandros; Petrou, Panagiota; Kakabakos, Sotirios; Botsialas, Athanasios; Jobst, Gerhard; Haasnoot, Willem; Fernandez-Alba, Amadeo; Lees, Michelle; Valamontes, Evangelos

    2016-01-01

    Optical biosensors have emerged in the past decade as the most promising candidates for portable, highly-sensitive bioanalytical systems that can be employed for in-situ measurements. In this work, a miniaturized optoelectronic system for rapid, quantitative, label-free detection of harmful

  15. Evaluation of accuracy of OraQuick ® rapid test in detecting HIV ...

    African Journals Online (AJOL)

    Objective: The accuracy of OraQuick® rapid test in detecting HIV 1 & 2 antibodies in saliva is evaluated against the blood EIA benchmark tests with confirmatory testing, against which OraQuick® accuracy is determined. Method: Paired samples of saliva and blood from 281 Nigerians were tested for HIV antibodies, and ...

  16. Lateral flow immunoassay for the rapid detection of citrus tristeza virus

    Science.gov (United States)

    A lateral flow methodology was developed using gold nanoparticles for rapid detection of Citrus tristeza virus (CTV). The test strip was based on a sandwich immunoassay and could be accomplished within 10 minutes. A sample was considered negative for CTV when only the control line appeared; whereas,...

  17. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    National Research Council Canada - National Science Library

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    .... The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  18. Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

    Science.gov (United States)

    Tsai, Shinn Shyong; Chang, Yeng Ling; Huang, Yen Li; Liu, Hung Jen; Ke, Guan Ming; Chiou, Chwei Jang; Hsieh, Yao Ching; Chang, Tsung Chou; Cheng, Li Ting; Chuang, Kuo Pin

    2014-05-01

    There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity.

  19. Rapid Newcastle Disease Virus Detection Based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout

    DEFF Research Database (Denmark)

    Tian, Bo; Ma, Jing; Zardán Gómez de la Torre, Teresa

    2016-01-01

    efficiency of loop-mediated isothermal amplification (LAMP) with an optomagnetic nanoparticle-based readout system, we demonstrate ultrasensitive and rapid detection of Newcastle disease virus RNA. Biotinylated amplicons of LAMP and reverse transcription LAMP (RT-LAMP) bind to streptavidin-coated magnetic...

  20. Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei

    OpenAIRE

    Bauernfeind, Adolf; Roller, Carsten; Meyer, Detlef; Jungwirth, Renate; Schneider, Ines

    1998-01-01

    A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.

  1. Rapid Anomaly Detection and Tracking via Compressive Time-Spectra Measurement

    Science.gov (United States)

    2016-02-12

    0002AM Title: Rapid anomaly detection and tracking via compressive time- spectra measurement Contract performance period: 05 Nov 2013 - 04... ground truth signal broadening technique...and tracking has direct applications in lower- cost, higher- performance sensors particularly in the shortwave infrared where focal plane array

  2. Rapid detection of single nucleotide mutation in p53 gene based on ...

    Indian Academy of Sciences (India)

    ... for the rapid detection of a specific DNA sequence related to the p53 gene is described. The structure and morphology of the synthesized graphene nanosheets and Au nanoparticles were characterized through transmission electron microscopy, UV–Vis spectroscopyand energy dispersion X-ray spectroscopy techniques.

  3. Human Plasmodium knowlesi infection detected by rapid diagnostic tests for malaria

    NARCIS (Netherlands)

    J.J. van Hellemond (Jaap); M. Rutten (Martine); R. Koelewijn (Rob); A.M. Zeeman (Anne Marie); J. Verweij (Jaap); P.J. Wismans (Pieter); C.H. Kocken (Clemens); P.J.J. van Genderen (Perry)

    2009-01-01

    textabstractWe describe a PCR-confirmed case of Plasmodium knowlesi infection with a high parasitemia level and clinical signs of severe malaria in a migrant worker from Malaysian Borneo in the Netherlands. Investigations showed that commercially available rapid antigen tests for detection of human

  4. Evaluation of Various Culture Media for Detection of Rapidly Growing Mycobacteria from Patients with Cystic Fibrosis.

    Science.gov (United States)

    Preece, Clair L; Wichelhaus, Thomas A; Perry, Audrey; Jones, Amanda L; Cummings, Stephen P; Perry, John D; Hogardt, Michael

    2016-07-01

    Isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly growing species that colonize the lungs of patients with CF. Extended incubation on Burkholderia cepacia selective agar (BCSA) has been recommended as an expedient culture method for the isolation of rapidly growing NTM in this setting. The aim of this study was to assess five selective media designed for the isolation of Burkholderia cepacia complex, along with two media designed for the isolation of mycobacteria (rapidly growing mycobacteria [RGM] medium and Middlebrook 7H11 agar), for their abilities to isolate NTM. All seven media were challenged with 147 isolates of rapidly growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for the isolation of B. cepacia complex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium, compared with only 7 samples using the most selective brand of BCSA (P = 0.023). RGM medium offers a superior option, compared to other selective agars, for the isolation of rapidly growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly growing NTM in all submitted sputum samples from patients with CF. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Rapid and Highly Sensitive Detection of Lead Ions in Drinking Water Based on a Strip Immunosensor

    Directory of Open Access Journals (Sweden)

    Chuanlai Xu

    2013-03-01

    Full Text Available In this study, we have first developed a rapid and sensitive strip immunosensor based on two heterogeneously-sized gold nanoparticles (Au NPs probes for the detection of trace lead ions in drinking water. The sensitivity was 4-fold higher than that of the conventional LFA under the optimized conditions. The visual limit of detection (LOD of the amplified method for qualitative detection lead ions was 2 ng/mL and the LOD for semi-quantitative detection could go down to 0.19 ng/mL using a scanning reader. The method suffered from no interference from other metal ions and could be used to detect trace lead ions in drinking water without sample enrichment. The recovery of the test samples ranged from 96% to 103%. As the detection method could be accomplished within 15 min, this method could be used as a potential tool for preliminary monitoring of lead contamination in drinking water.

  6. Rapid and highly sensitive detection of lead ions in drinking water based on a strip immunosensor.

    Science.gov (United States)

    Kuang, Hua; Xing, Changrui; Hao, Changlong; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

    2013-03-28

    In this study, we have first developed a rapid and sensitive strip immunosensor based on two heterogeneously-sized gold nanoparticles (Au NPs) probes for the detection of trace lead ions in drinking water. The sensitivity was 4-fold higher than that of the conventional LFA under the optimized conditions. The visual limit of detection (LOD) of the amplified method for qualitative detection lead ions was 2 ng/mL and the LOD for semi-quantitative detection could go down to 0.19 ng/mL using a scanning reader. The method suffered from no interference from other metal ions and could be used to detect trace lead ions in drinking water without sample enrichment. The recovery of the test samples ranged from 96% to 103%. As the detection method could be accomplished within 15 min, this method could be used as a potential tool for preliminary monitoring of lead contamination in drinking water.

  7. Rapid amperometric detection of coliforms based on MWNTs/Nafion composite film modified glass carbon electrode.

    Science.gov (United States)

    Cheng, Yuxiao; Liu, Yajun; Huang, Jingjing; Xian, Yuezhong; Zhang, Wen; Zhang, Zhonghai; Jin, Litong

    2008-03-15

    A multi-wall carbon nanotubes (MWNTs)/Nafion modified glassy carbon electrode (GCE) was fabricated for the rapid amperometric detection of coliforms, represented by Escherichia coli (E. coli). In the bacterial solution, beta-galactosidase which was used as an indicator of coliforms reacted with substrate, p-aminophenol-beta-galactopyranoside (PAPG), and produced p-aminophenol (PAP). PAP was detected by MWNTs/Nafion modified GCE. Due to the cation-exchange capacity of Nafion and the electrocatalytic ability of MWNTs, the detection sensitivity of PAP was improved and the detection time of coliforms was shortened. The bacterial can be detected within 5h ranging from 10 to 10(4)cfu/mL. The MWNTs/Nafion modified GCE was easy to be constructed and regenerated. To our best knowledge, it was the first time to use MWNTs/Nafion modified GCE to detect the concentration of coliforms.

  8. Screen-printed fluorescent sensors for rapid and sensitive anthrax biomarker detection

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Inkyu; Oh, Wan-Kyu; Jang, Jyongsik, E-mail: jsjang@plaza.snu.ac.kr

    2013-05-15

    Highlights: •We fabricated flexible anthrax sensors with a simple screen-printing method. •The sensors selectively detected B. anthracis biomarker. •The sensors provide the visible alarm against anthrax attack. -- Abstract: Since the 2001 anthrax attacks, efforts have focused on the development of an anthrax detector with rapid response and high selectivity and sensitivity. Here, we demonstrate a fluorescence sensor for detecting anthrax biomarker with high sensitivity and selectivity using a screen-printing method. A lanthanide–ethylenediamine tetraacetic acid complex was printed on a flexible polyethersulfone film. Screen-printing deposition of fluorescent detecting moieties produced fluorescent patterns that acted as a visual alarm against anthrax.

  9. Rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectroscopy with heterodyne detection.

    Science.gov (United States)

    Hiramatsu, Kotaro; Luo, Yizhi; Ideguchi, Takuro; Goda, Keisuke

    2017-11-01

    High-speed Raman spectroscopy has become increasingly important for analyzing chemical dynamics in real time. To address the need, rapid-scan Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectroscopy has been developed to realize broadband CARS measurements at a scan rate of more than 20,000 scans/s. However, the detection sensitivity of FT-CARS spectroscopy is inherently low due to the limited number of photons detected during each scan. In this Letter, we show our experimental demonstration of enhanced sensitivity in rapid-scan FT-CARS spectroscopy by heterodyne detection. Specifically, we implemented heterodyne detection by superposing the CARS electric field with an external local oscillator (LO) for their interference. The CARS signal was amplified by simply increasing the power of the LO without the need for increasing the incident power onto the sample. Consequently, we achieved enhancement in signal intensity and the signal-to-noise ratio by factors of 39 and 5, respectively, compared to FT-CARS spectroscopy with homodyne detection. The sensitivity-improved rapid-scan FT-CARS spectroscopy is expected to enable the sensitive real-time observation of chemical dynamics in a broad range of settings, such as combustion engines and live biological cells.

  10. Development of a double-antibody sandwich ELISA for rapid detection of Bacillus Cereus in food

    Science.gov (United States)

    Zhu, Longjiao; He, Jing; Cao, Xiaohan; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-01-01

    Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 104–2.8 × 106 cells/mL with a detection limit (LOD) of 0.9 × 103 cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens. PMID:26976753

  11. Commercially Available Rapid Methods for Detection of Selected Food-borne Pathogens.

    Science.gov (United States)

    Valderrama, Wladir B; Dudley, Edward G; Doores, Stephanie; Cutter, Catherine N

    2016-07-03

    Generally, the enumeration and isolation of food-borne pathogens is performed using culture-dependent methods. These methods are sensitive, inexpensive, and provide both qualitative and quantitative assessment of the microorganisms present in a sample, but these are time-consuming. For this reason, researchers are developing new techniques that allow detection of food pathogens in shorter period of time. This review identifies commercially available methods for rapid detection and quantification of Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and Shiga toxin-producing Escherichia coli in food samples. Three categories are discussed: immunologically based methods, nucleic acid-based assays, and biosensors. This review describes the basic mechanism and capabilities of each method, discusses the difficulties of choosing the most convenient method, and provides an overview of the future challenges for the technology for rapid detection of microorganisms.

  12. Portable microfluidic raman system for rapid, label-free early disease signature detection

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Meiye [Sandia National Laboratories (SNL-CA), Livermore, CA (United States); Davis, Ryan Wesley [Sandia National Laboratories (SNL-CA), Livermore, CA (United States); Hatch, Anson [Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

    2015-09-01

    In the early stages of infection, patients develop non-specific or no symptoms at all. While waiting for identification of the infectious agent, precious window of opportunity for early intervention is lost. The standard diagnostics require affinity reagents and sufficient pathogen titers to reach the limit of detection. In the event of a disease outbreak, triaging the at-risk population rapidly and reliably for quarantine and countermeasure is more important than the identification of the pathogen by name. To expand Sandia's portfolio of Biological threat management capabilities, we will utilize Raman spectrometry to analyze immune subsets in whole blood to rapidly distinguish infected from non-infected, and bacterial from viral infection, for the purpose of triage during an emergency outbreak. The goal of this one year LDRD is to determine whether Raman spectroscopy can provide label-free detection of early disease signatures, and define a miniaturized Raman detection system meeting requirements for low- resource settings.

  13. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    NARCIS (Netherlands)

    Quevedo, Beatrice; Giertsen, Elin; Zijnge, Vincent; Luethi-Schaller, Helga; Guggenheim, Bernhard; Thurnheer, Thomas; Gmuer, Rudolf

    2011-01-01

    Background: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to

  14. Single walled carbon nanotube-based electrical biosensor for the label-free detection of pathogenic bacteria

    DEFF Research Database (Denmark)

    Yoo, S. M.; Baek, Y. K.; Shin, S.

    2016-01-01

    We herein describe the development of a single-walled carbon nanotube (SWNT)-based electrical biosensor consisting of a two-terminal resistor, and report its use for the specific, label-free detection of pathogenic bacteria via changes in conductance. The ability of this biosensor to recognize...

  15. Label-free electrical sensing of bacteria in eye wash samples: A step towards point-of-care detection of pathogens in patients with infectious keratitis.

    Science.gov (United States)

    Pandya, Hardik J; Kanakasabapathy, Manoj Kumar; Verma, Saloni; Chug, Manjyot Kaur; Memic, Adnan; Gadjeva, Mihaela; Shafiee, Hadi

    2017-05-15

    The diagnosis of keratitis is based on visual exam, tissue cytology, and standard microbial culturing to determine the type of the infectious pathogen. To prescribe appropriate therapy, it is important to distinguish between bacterial, fungal, and viral keratitis, as the treatments are quite different. Diagnosis of the causative organism has a substantial prognostic importance. Further, timely knowledge of the nature of the pathogen is also critical to adapt therapy in patients unresponsive to empiric treatment options, which occurs in 10% of all cases. Currently, the identification of the nature of the pathogen that causes keratitis is achieved via microbial culture screening, which is laboratory-based, expensive, and time-consuming. The most frequent pathogens that cause the corneal ulcers are P. aeruginosa and S. aureus. Here, we report a microchip for rapid (<1h) detection of P. aeruginosa (6294), S. aureus(LAC), through on-chip electrical sensing of bacterial lysate. We evaluated the microchip with spiked samples of PBS with bacteria concentration between 101 to 108 CFU/mL. The least diluted bacteria concentration in bacteria-spiked samples with statistically significant impedance change was 10 CFU/mL. We further validated our assay by comparing our microchip results with the standard culture-based methods using eye washes obtained from 13 infected mice. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).

    Science.gov (United States)

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sugumar, Vimal; Taju, Gani; Sahul Hameed, A S; Paknikar, Kishore M

    2017-01-01

    White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR. The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.

  17. [Rapid-tests detection evaluation of Clostridium difficile toxins and microbiological investigation].

    Science.gov (United States)

    Nakagawa, Risa; Iinuma, Yoshitsugu; Yamamoto, Masaki; Matsumura, Yasufumi; Shirano, Michinori; Matsushima, Aki; Nagao, Miki; Saito, Takashi; Takakura, Shunji; Ito, Yutaka; Higuchi, Takeshi; Tanaka, Michio; Ichiyama, Satoshi

    2010-03-01

    We evaluated two rapid toxin tests for C. difficile combined with stool specimen cultures used from January 2006 to March 2009. Stool specimens numbered 877, 102 among which were from the cases of diagnosed clinical C. difficile-associated diarrhea (CDAD). Rapid toxin A 'Uniquick' detection kits were used until October 2007 and toxin A&B 'TOX A/B' detection kits thereafter. Clinical CDAD was considered the detection gold standard. Uniquick sensitivity, specificity, and positive and negative predictive values were 54.3%, 99.1%, 90.5%, and 93.2% while those for TOX A/B were 46.2%, 97.6%, 65.2%, and 95.0% and for culture 42.2%, 95.5%, 55.1%, and 92.6%. Rapid toxin tests tended to have better sensitivity than culture results although not significantly so, and Uniquick showed significantly better positive predictive value than TOX A/B or culture results. Among clinical CDAD cases, concordance with culture was 24.3% for Uniquick and 53.1% for TOX A/B. For stored strains, 27 were typed toxin A+B+ (48.1%), toxin A-B+ (37.0%) and toxin A-B- (14.8%) with toxin gene detection by PCR. Eight of the 10 toxin A-B+ strains were classified into two cluster by ribotyping, and 7 of those were detected in two hospital wards, indicated the possibility of nosocomial toxin A-B+ strain spread. The rapid toxin test for both toxins A and B should be used if toxin A-B+ predominate. Simultaneous culture testing may be useful for detecting clinical CDAD more accurately, however.

  18. Biocontrol and Rapid Detection of Food-Borne Pathogens Using Bacteriophages and Endolysins.

    Science.gov (United States)

    Bai, Jaewoo; Kim, You-Tae; Ryu, Sangryeol; Lee, Ju-Hoon

    2016-01-01

    Bacteriophages have been suggested as natural food preservatives as well as rapid detection materials for food-borne pathogens in various foods. Since Listeria monocytogenes-targeting phage cocktail (ListShield) was approved for applications in foods, numerous phages have been screened and experimentally characterized for phage applications in foods. A single phage and phage cocktail treatments to various foods contaminated with food-borne pathogens including E. coli O157:H7, Salmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Cronobacter sakazakii, and Vibrio spp. revealed that they have great potential to control various food-borne pathogens and may be alternative for conventional food preservatives. In addition, phage-derived endolysins with high host specificity and host lysis activities may be preferred to food applications rather than phages. For rapid detection of food-borne pathogens, cell-wall binding domains (CBDs) from endolysins have been suggested due to their high host-specific binding. Fluorescence-tagged CBDs have been successfully evaluated and suggested to be alternative materials of expensive antibodies for various detection applications. Most recently, reporter phage systems have been developed and tested to confirm their usability and accuracy for specific detection. These systems revealed some advantages like rapid detection of only viable pathogenic cells without interference by food components in a very short reaction time, suggesting that these systems may be suitable for monitoring of pathogens in foods. Consequently, phage is the next-generation biocontrol agent as well as rapid detection tool to confirm and even identify the food-borne pathogens present in various foods.

  19. Biocontrol and Rapid Detection of Food-borne Pathogens Using Bacteriophages and Endolysins

    Directory of Open Access Journals (Sweden)

    Jaewoo eBai

    2016-04-01

    Full Text Available Bacteriophages have been suggested as natural food preservatives as well as rapid detection materials for food-borne pathogens in various foods. Since Listeria monocytogenes-targeting phage cocktail (ListShield was approved for applications in foods, numerous phages have been screened and experimentally characterized for phage applications in foods. A single phage and phage cocktail treatments to various foods contaminated with food-borne pathogens including E. coli O157:H7, Salmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Cronobacter sakazakii, and Vibrio spp. revealed that they have great potential to control various food-borne pathogens and may be alternative for conventional food preservatives. In addition, phage-derived endolysins with high host specificity and host lysis activities may be preferred to food applications rather than phages. For rapid detection of food-borne pathogens, cell-wall binding domains (CBDs from endolysins have been suggested due to their high host-specific binding. Fluorescence-tagged CBDs have been successfully evaluated and suggested to be alternative materials of expensive antibodies for various detection applications. Most recently, reporter phage systems have been developed and tested to confirm their usability and accuracy for specific detection. These systems revealed some advantages like rapid detection of only viable pathogenic cells without interference by food components in a very short reaction time, suggesting that these systems may be suitable for monitoring of pathogens in foods. Consequently, phage is the next-generation biocontrol agent as well as rapid detection tool to confirm and even identify the food-borne pathogens present in various foods.

  20. Detection probability models for bacteria, and how to obtain them from heterogeneous spiking data. An application to Bacillus anthracis.

    Science.gov (United States)

    Hedell, Ronny; Stephansson, Olga; Mostad, Petter; Andersson, Mats Gunnar

    2017-01-16

    Efficient and correct evaluation of sampling results with respect to hypotheses about the concentration or distribution of bacteria generally requires knowledge about the performance of the detection method. To assess the sensitivity of the detection method an experiment is usually performed where the target matrix is spiked (i.e. artificially contaminated) with different concentrations of the bacteria, followed by analyses of the samples using the pre-enrichment method and the analytical detection method of interest. For safety reasons or because of economic or time limits it is not always possible to perform exactly such an experiment, with the desired number of samples. In this paper, we show how heterogeneous data from diverse sources may be combined within a single model to obtain not only estimates of detection probabilities, but also, crucially, uncertainty estimates. We indicate how such results can then be used to obtain optimal conclusions about presence of bacteria, and illustrate how strongly the sampling results speak in favour of or against contamination. In our example, we consider the case when B. cereus is used as surrogate for B. anthracis, for safety reasons. The statistical modelling of the detection probabilities and of the growth characteristics of the bacteria types is based on data from four experiments where different matrices of food were spiked with B. anthracis or B. cereus and analysed using plate counts and qPCR. We show how flexible and complex Bayesian models, together with inference tools such as OpenBUGS, can be used to merge information about detection probability curves. Two different modelling approaches, differing in whether the pre-enrichment step and the PCR detection step are modelled separately or together, are applied. The relative importance on the detection curves for various existing data sets are evaluated and illustrated. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Rapid detection of avian influenza A virus by immunochromatographic test using a novel fluorescent dye.

    Science.gov (United States)

    Yeo, Seon-Ju; Cuc, Bui Thi; Kim, Soon-Ai; Kim, Do Thi Hoang; Bao, Duong Tuan; Tien, Trinh Thi Thuy; Anh, Nguyen Thi Viet; Choi, Do-Young; Chong, Chom-Kyu; Kim, Hak Sung; Park, Hyun

    2017-08-15

    Sensitive and rapid diagnostic systems for avian influenza (AI) virus are required to screen large numbers of samples during a disease outbreak and to prevent the spread of infection. In this study, we employed a novel fluorescent dye for the rapid and sensitive recognition of AI virus. The styrylpyridine phosphor derivative was synthesized by adding allyl bromide as a stable linker and covalently immobilizing it on latex beads with antibodies generating the unique Red dye 53-based fluorescent probe. The performance of the innovative rapid fluorescent immnunochromatographic test (FICT) employing Red dye 53 in detecting the AI virus (A/H5N3) was 4-fold and 16-fold higher than that of Europium-based FICT and the rapid diagnostic test (RDT), respectively. In clinical studies, the presence of human nasopharyngeal specimens did not alter the performance of Red dye 53-linked FICT for the detection of H7N1 virus. Furthermore, in influenza A virus-infected human nasopharyngeal specimens, the sensitivity of the Red dye 53-based assay and RDT was 88.89% (8/9) and 55.56% (5/9) relative to rRT-PCR, respectively. The photostability of Red dye 53 was higher than that of fluorescein isothiocyanate (FITC), showing a stronger fluorescent signal persisting up to 8min under UV. The Red dye 53 could therefore be a potential probe for rapid fluorescent diagnostic systems that can recognize AI virus in clinical specimens. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A portable cell-based optical detection device for rapid detection of Listeria and Bacillus toxins

    Science.gov (United States)

    Banerjee, Pratik; Banada, Padmapriya P.; Rickus, Jenna L.; Morgan, Mark T.; Bhunia, Arun K.

    2005-11-01

    A mammalian cell-based optical biosensor was built to detect pathogenic Listeria and Bacillus species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic L. monocytogenes strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from B. cereus strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic L. innocua (F4247) and a B. substilis induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic Listeria and Bacillus species and can potentially be used onsite for food safety or in biosecurity application.

  3. Molecular methods routinely used to detect Coxiella burnetii in ticks cross-react with Coxiella-like bacteria

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    Jourdain Elsa

    2015-11-01

    Full Text Available Background: Q fever is a widespread zoonotic disease caused by Coxiella burnetii. Ticks may act as vectors, and many epidemiological studies aim to assess C. burnetii prevalence in ticks. Because ticks may also be infected with Coxiella-like bacteria, screening tools that differentiate between C. burnetii and Coxiella-like bacteria are essential. Methods: In this study, we screened tick specimens from 10 species (Ornithodoros rostratus, O. peruvianus, O. capensis, Ixodes ricinus, Rhipicephalus annulatus, R. decoloratus, R. geigy, O. sonrai, O. occidentalis, and Amblyomma cajennense known to harbor specific Coxiella-like bacteria, by using quantitative PCR primers usually considered to be specific for C. burnetii and targeting, respectively, the IS1111, icd, scvA, p1, and GroEL/htpB genes. Results: We found that some Coxiella-like bacteria, belonging to clades A and C, yield positive PCR results when screened with primers initially believed to be C. burnetii-specific. Conclusions: These results suggest that PCR-based surveys that aim to detect C. burnetii in ticks by using currently available methods must be interpreted with caution if the amplified products cannot be sequenced. Future molecular methods that aim at detecting C. burnetii need to take into account the possibility that cross-reactions may exist with Coxiella-like bacteria.

  4. Rapid and accurate detection of Escherichia coli growth by fluorescent pH-sensitive organic nanoparticles for high-throughput screening applications.

    Science.gov (United States)

    Si, Yang; Grazon, Chloé; Clavier, Gilles; Rieger, Jutta; Audibert, Jean-Frédéric; Sclavi, Bianca; Méallet-Renault, Rachel

    2016-01-15

    Rapid detection of bacterial growth is an important issue in the food industry and for medical research. Here we present a novel kind of pH-sensitive fluorescent nanoparticles (FANPs) that can be used for the rapid and accurate real-time detection of Escherichia coli growth. These organic particles are designed to be non-toxic and highly water-soluble. Here we show that the coupling of pH sensitive fluoresceinamine to the nanoparticles results in an increased sensitivity to changes in pH within a physiologically relevant range that can be used to monitor the presence of live bacteria. In addition, these FANPs do not influence bacterial growth and are stable over several hours in a complex medium and in the presence of bacteria. The use of these FANPs allows for continuous monitoring of bacterial growth via real-time detection over long time scales in small volumes and can thus be used for the screening of a large number of samples for high-throughput applications such as screening for the presence of antibiotic resistant strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Cellulose production and cellulose synthase gene detection in acetic acid bacteria.

    Science.gov (United States)

    Valera, Maria José; Torija, Maria Jesús; Mas, Albert; Mateo, Estibaliz

    2015-02-01

    The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

  6. Nanomotion Detection Method for Testing Antibiotic Resistance and Susceptibility of Slow-Growing Bacteria.

    Science.gov (United States)

    Villalba, María Ines; Stupar, Petar; Chomicki, Wojciech; Bertacchi, Massimiliano; Dietler, Giovanni; Arnal, Laura; Vela, María Elena; Yantorno, Osvaldo; Kasas, Sandor

    2017-12-04

    Infectious diseases are caused by pathogenic microorganisms and are often severe. Time to fully characterize an infectious agent after sampling and to find the right antibiotic and dose are important factors in the overall success of a patient's treatment. Previous results suggest that a nanomotion detection method could be a convenient tool for reducing antibiotic sensitivity characterization time to several hours. Here, the application of the method for slow-growing bacteria is demonstrated, taking Bordetella pertussis strains as a model. A low-cost nanomotion device is able to characterize B. pertussis sensitivity against specific antibiotics within several hours, instead of days, as it is still the case with conventional growth-based techniques. It can discriminate between resistant and susceptible B. pertussis strains, based on the changes of the sensor's signal before and after the antibiotic addition. Furthermore, minimum inhibitory and bactericidal concentrations of clinically applied antibiotics are compared using both techniques and the suggested similarity is discussed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Detection of relapsing fever Borrelia spp., Bartonella spp. and Anaplasmataceae bacteria in argasid ticks in Algeria.

    Science.gov (United States)

    Lafri, Ismail; El Hamzaoui, Basma; Bitam, Idir; Leulmi, Hamza; Lalout, Reda; Mediannikov, Oleg; Chergui, Mohamed; Karakellah, Mohamed; Raoult, Didier; Parola, Philippe

    2017-11-01

    Argasid ticks (soft ticks) are blood-feeding arthropods that can parasitize rodents, birds, humans, livestock and companion animals. Ticks of the Ornithodoros genus are known to be vectors of relapsing fever borreliosis in humans. In Algeria, little is known about relapsing fever borreliosis and other bacterial pathogens transmitted by argasid ticks. Between May 2013 and October 2015, we investigated the presence of soft ticks in 20 rodent burrows, 10 yellow-legged gull (Larus michahellis) nests and animal shelters in six locations in two different bioclimatic zones in Algeria. Six species of argasid ticks were identified morphologically and through 16S rRNA gene sequencing. The presence and prevalence of Borrelia spp., Bartonella spp., Rickettsia spp. and Anaplasmataceae was assessed by qPCR template assays in each specimen. All qPCR-positive samples were confirmed by standard PCR, followed by sequencing the amplified fragments. Two Borrelia species were identified: Borrelia hispanica in Ornithodoros occidentalis in Mostaganem, and Borrelia cf. turicatae in Carios capensis in Algiers. One new Bartonella genotype and one new Anaplasmataceae genotype were also identified in Argas persicus. The present study highlights the presence of relapsing fever borreliosis agents, although this disease is rarely diagnosed in Algeria. Other bacteria of unknown pathogenicity detected in argasid ticks which may bite humans deserve further investigation.

  8. Detection of relapsing fever Borrelia spp., Bartonella spp. and Anaplasmataceae bacteria in argasid ticks in Algeria.

    Directory of Open Access Journals (Sweden)

    Ismail Lafri

    2017-11-01

    Full Text Available Argasid ticks (soft ticks are blood-feeding arthropods that can parasitize rodents, birds, humans, livestock and companion animals. Ticks of the Ornithodoros genus are known to be vectors of relapsing fever borreliosis in humans. In Algeria, little is known about relapsing fever borreliosis and other bacterial pathogens transmitted by argasid ticks.Between May 2013 and October 2015, we investigated the presence of soft ticks in 20 rodent burrows, 10 yellow-legged gull (Larus michahellis nests and animal shelters in six locations in two different bioclimatic zones in Algeria. Six species of argasid ticks were identified morphologically and through 16S rRNA gene sequencing. The presence and prevalence of Borrelia spp., Bartonella spp., Rickettsia spp. and Anaplasmataceae was assessed by qPCR template assays in each specimen. All qPCR-positive samples were confirmed by standard PCR, followed by sequencing the amplified fragments. Two Borrelia species were identified: Borrelia hispanica in Ornithodoros occidentalis in Mostaganem, and Borrelia cf. turicatae in Carios capensis in Algiers. One new Bartonella genotype and one new Anaplasmataceae genotype were also identified in Argas persicus.The present study highlights the presence of relapsing fever borreliosis agents, although this disease is rarely diagnosed in Algeria. Other bacteria of unknown pathogenicity detected in argasid ticks which may bite humans deserve further investigation.

  9. An investigation of indirect conductimetry for detection of some food-borne bacteria.

    Science.gov (United States)

    Bolton, F J

    1990-11-01

    Indirect conductimetry using a rapid automated bacterial impedance technique was investigated. Strains of Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Salmonella spp. grown in Whitley Impedance broth all elicited indirect conductimetric changes. These indirect conductance responses were improved by the addition of 2 g/l glucose to the medium and resulted in maximum changes of 2340-4300 microS with associated maximum rates of change of 520-1210 microS/h. Furthermore, the indirect conductimetric assay detected growth of staphylococci, listeria and salmonella in media containing high concentrations of salts used as selective agents in culture media for the isolation of these organisms.

  10. One-step cell lysis suitable for quantitative bacteria detection in inhibitor-laden sands

    Science.gov (United States)

    Lim, Hyun Jeong; Choi, Jung-Hyun; Son, Ahjeong

    2015-04-01

    Complexity and heterogeneity of soils often hinder effective DNA extraction from the soil matrix. In particular, conventional DNA extraction techniques require extensive purification which makes DNA extraction time-consuming and labor-intensive. Other drawbacks include lower recovery yield, degradation, and damage of DNA, which are also caused by intensive purifications during DNA extraction. Therefore a rapid and simple and yet effective DNA pretreatment method is preferred for environmental monitoring and screening. This study has evaluated the feasibility of simple physical pretreatment for effective cell lysis of bacteria in sands. Bead beating method was selected as an effective physical cell lysis method in this study. We examined the capability of this physical lysis for Pseudomonas putida seeded sands without additional chemical purification steps. The lysate from the method was analysed by the quantitative polymerase chain reaction (qPCR) assay and subsequently compared to that by commercial DNA extraction kit. The best lysis condition (treatment with 0.1 mm glass beads at 3000 rpm for 3 minutes) was selected. The qPCR results of bead beating treated samples showed the better performance than that of conventional DNA extraction kit. Moreover, the qPCR assay was performed to the sands laden with qPCR inhibitors (humic acids, clay, and magnesium), which generally present in environmental samples. Further experiments with the sands containing less than 10 μg/g of humic acids and 70% of clay showed successful quantification results of qPCR assay. In conclusion, the bead beating method is useful for simplified DNA extraction prior to qPCR analysis for sand samples of particular composition. It is expected that this approach will be beneficial for environmental in-situ analysis or immediate pre-screening. It also provides the groundwork for future studies with real soil samples that have various physico-chemical properties.

  11. ENTEROPATHOGENS DETECTED IN A DAYCARE CENTER, SOUTHEASTERN BRAZIL: BACTERIA, VIRUS, AND PARASITE RESEARCH

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    Edna Donizetti Rossi Castro

    2015-02-01

    Full Text Available Introduction: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. Methods: From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. Results: A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. Conclusions: For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens.

  12. Enteropathogens detected in a daycare center, Southeastern Brazil: bacteria, virus, and parasite research.

    Science.gov (United States)

    Castro, Edna Donizetti Rossi; Germini, Marcela Cristina Braga Yassaka; Mascarenhas, Joana D'Arc Pereira; Gabbay, Yvone Benchimol; de Lima, Ian Carlos Gomes; Lobo, Patrícia dos Santos; Fraga, Valéria Daltibari; Conceição, Luciana Moran; Machado, Ricardo Luiz Dantas; Rossit, Andréa Regina Baptista

    2015-01-01

    The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens.

  13. Sensitivity of a rapid immuno-chromatographic test for hepatitis C antibodies detection.

    Science.gov (United States)

    Desbois, Delphine; Vaghefi, Parissa; Savary, Jeanine; Dussaix, Elisabeth; Roque-Afonso, Anne-Marie

    2008-02-01

    Enzyme-linked immunoassays (ELISA) are the most widely used anti-hepatitis C virus (HCV) screening tests but simple, instrument and electricity-free screening tests have been developed with results available in a few minutes. The sensitivity of a rapid immuno-chromatographic assay for the detection of anti-HCV antibodies was evaluated on 421 HCV RNA-positive samples from chronic carriers and compared with ELISA method. The sensitivity of the ELISA method was 99.3% and the sensitivity of the rapid test was 95.5%. False negative results were independent of HCV genotype, but were associated with human immunodeficiency virus (HIV)-positive status. Among HIV-negative people, sensitivities of the rapid test and the EIA assay were 99.2% and 100%, respectively. Whereas among HIV-positive people, sensitivities were 77.5% and 96.3%. The immuno-chromatographic test is rapid and simple, and could be used along with rapid anti-HIV determination, in settings with limited facilities or when rapid results are required.

  14. Survey and Rapid detection of Bordetella pertussis in clinical samples targeting the BP485 in China

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    Wei eLiu

    2015-03-01

    Full Text Available Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg/µl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 Bordetella pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing (MLST groups with different alleles of the virulence-related genes including 4 alleles of ptxA, 6 of prn, 4 of tcfA, 2 of fim2 and 3 of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.

  15. Rapid Detection of Land Cover Changes Using Crowdsourced Geographic Information: A Case Study of Beijing, China

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    Yuan Meng

    2017-08-01

    Full Text Available Land cover change (LCC detection is a significant component of sustainability research including ecological economics and climate change. Due to the rapid variability of natural environment, effective LCC detection is required to capture sufficient change-related information. Although such information has been available through remotely sensed images, the complicated image processing and classification make it time consuming and labour intensive. In contrast, the freely available crowdsourced geographic information (CGI contains easily interpreted textual information, and thus has the potential to be applied for capturing effective change-related information. Therefore, this paper presents and evaluates a method using CGI for rapid LCC detection. As a case study, Beijing is chosen as the study area, and CGI is applied to monitor LCC information. As one kind of CGI which is generated from commercial Internet maps, points of interest (POIs with detailed textual information are utilised to detect land cover in 2016. Those POIs are first classified into land cover nomenclature based on their textual information. Then, a kernel density approach is proposed to effectively generate land cover regions in 2016. Finally, with GlobeLand30 in 2010 as baseline map, LCC is detected using the post-classification method in the period of 2010–2016 in Beijing. The result shows that an accuracy of 89.20% is achieved with land cover regions generated by POIs, indicating that POIs are reliable for rapid LCC detection. Additionally, an LCC detection comparison is proposed between remotely sensed images and CGI, revealing the advantages of POIs in terms of LCC efficiency. However, due to the uneven distribution, remotely sensed images are still required in areas with few POIs.

  16. Integrated Circuits for Rapid Sample Processing and Electrochemical Detection of Biomarkers

    Science.gov (United States)

    Besant, Justin

    The trade-off between speed and sensitivity of detection is a fundamental challenge in the design of point-of-care diagnostics. As the relevant molecules in many diseases exist natively at extremely low levels, many gold-standard diagnostic tests are designed with high sensitivity at the expense of long incubations needed to amplify the target analytes. The central aim of this thesis is to design new strategies to detect biologically relevant analytes with both high speed and sensitivity. The response time of a biosensor is limited by the ability of the target analyte to accumulate to detectable levels at the sensor surface. We overcome this limitation by designing a range of integrated devices to optimize the flux of the analyte to the sensor by increasing the effective analyte concentration, shortening the required diffusion distance, and confining the analyte in close proximity to the sensor. We couple these devices with novel ultrasensitive electrochemical transduction strategies to convert rare analytes into a detectable signal. We showcase the clinical utility of these approaches with several applications including cancer diagnosis, bacterial identification, and antibiotic susceptibility profiling. We design and optimize a device to isolate rare cancer cells from the bloodstream with near 100% efficiency and 10 000-fold specificity. We analyse pathogen specific nucleic acids by lysing bacteria in close proximity to an electrochemical sensor and find that this approach has 10-fold higher sensitivity than standard lysis in bulk solution. We design an electronic chip to readout the antibiotic susceptibility profile with an hour-long incubation by concentrating bacteria into nanoliter chambers with integrated electrodes. Finally, we report a strategy for ultrasensitive visual readout of nucleic acids as low as 100 fM within 10 minutes using an amplification cascade. The strategies presented could guide the development of fast, sensitive and low-cost diagnostics

  17. DEVELOPMENT OF A RAPID DIAGNOSTIC KIT FOR DETECTION OF SALMONELLA ENTERITIDIS IN FOOD USING INDIRECT COAGGLUTINATION TECHNIQUE

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    Wyanda Arnafia

    2017-12-01

    Full Text Available The objective of this study was to develop a rapid, simple, cheap, sensitive, and specific assay for detection of Salmonella Enteritidis in food. The kit-prototype was developed by using indirect coagglutination technique with three main components, namely Staphylococcus aureus Cowan I, rabbit IgG anti-chicken Fc IgY and chicken IgY anti-S. Enteritidis. Isa Brown layer chickens were used to produce specific antibodies against S. Enteritidis. Monospecific antisera were prepared by absorption method. Staphylococcus aureus Cowan I was coupled with rabbit IgG anti-chicken Fc IgY and monospecific antisera anti-S. Enteritidis. Kit-prototype was compared with multiplex polymerase chain reaction to determine sensitivity and specificity of kit-prototype. Artificially inoculated food sample was used to determine the limit of detection of kit-prototype in a food sample. Indirect coagglutination kit-prototype was able to differentiate positive control from negative control without self-agglutination reaction. This assay has a high specificity to S. Enteritidis without significant cross-reactivity towards other bacteria. Kit-prototype was able to detect 108 CFU/mL of S. Enteritidis in the buffer and 1 CFU/mL of S. Enteritidis in a food sample after selective enrichment procedure. The application of this kit was able to give a fast result (reaction can be observed in 10 sec, to be applied in a sample without extraction in the preparation of antigen and to reduce detection time of S. Enteritidis in food until 4 days.

  18. A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

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    Laura C Bonney

    2017-10-01

    Full Text Available Crimean-Congo Haemorrhagic fever Virus (CCHFV is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia.An isothermal recombinase polymerase amplification (RPA assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan.The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

  19. A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

    Science.gov (United States)

    Bonney, Laura C; Watson, Robert J; Afrough, Babak; Mullojonova, Manija; Dzhuraeva, Viktoriya; Tishkova, Farida; Hewson, Roger

    2017-10-01

    Crimean-Congo Haemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia. An isothermal recombinase polymerase amplification (RPA) assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes) detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan. The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

  20. Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis.

    Directory of Open Access Journals (Sweden)

    Sören Hansen

    Full Text Available The detection of Mycobacterium avium subsp. paratuberculosis (MAP infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure.In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%.The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.

  1. Selective cultivation and rapid detection of Staphylococcus aureus by computer vision.

    Science.gov (United States)

    Wang, Yong; Yin, Yongguang; Zhang, Chaonan

    2014-03-01

    In this paper, we developed a selective growth medium and a more rapid detection method based on computer vision for selective isolation and identification of Staphylococcus aureus from foods. The selective medium consisted of tryptic soy broth basal medium, 3 inhibitors (NaCl, K2 TeO3 , and phenethyl alcohol), and 2 accelerators (sodium pyruvate and glycine). After 4 h of selective cultivation, bacterial detection was accomplished using computer vision. The total analysis time was 5 h. Compared to the Baird-Parker plate count method, which requires 4 to 5 d, this new detection method offers great time savings. Moreover, our novel method had a correlation coefficient of greater than 0.998 when compared with the Baird-Parker plate count method. The detection range for S. aureus was 10 to 10(7) CFU/mL. Our new, rapid detection method for microorganisms in foods has great potential for routine food safety control and microbiological detection applications. © 2014 Institute of Food Technologists®

  2. Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool,

    Directory of Open Access Journals (Sweden)

    Flávio da Silva Mesquita

    Full Text Available Abstract Objective: The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. Methods: The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. Results: From 313 positive samples by immunofluorescence assays, 282 (90% were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. Conclusions: This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics.

  3. Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool.

    Science.gov (United States)

    Mesquita, Flávio da Silva; Oliveira, Danielle Bruna Leal de; Crema, Daniela; Pinez, Célia Miranda Nunes; Colmanetti, Thaís Cristina; Thomazelli, Luciano Matsumia; Gilio, Alfredo Elias; Vieira, Sandra Elisabeth; Martinez, Marina Baquerizo; Botosso, Viviane Fongaro; Durigon, Edison Luiz

    The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  4. [Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

    Science.gov (United States)

    Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

    2015-01-01

    To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance

  5. Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection.

    Directory of Open Access Journals (Sweden)

    Roy Cohen

    Full Text Available Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs. As proof of principle, we use oriented immobilization of pyruvate kinase (PK and luciferase (Luc on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE, a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815 with the current gold standard for biomarker detection, ELISA-with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active

  6. Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

    Science.gov (United States)

    Cohen, Roy; Lata, James P.; Lee, Yurim; Hernández, Jean C. Cruz; Nishimura, Nozomi; Schaffer, Chris B.; Mukai, Chinatsu; Nelson, Jacquelyn L.; Brangman, Sharon A.; Agrawal, Yash; Travis, Alexander J.

    2015-01-01

    Background Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices. Methods and findings We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA. Conclusions Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using

  7. In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition

    DEFF Research Database (Denmark)

    Kofoed, Michael Vedel; Stief, Peter; Poulsen, Morten

    reduction of nitrate to dinitrogen gas, is essential for the removal of fixed nitrogen from natural and engineered ecosystems. However, community structure and activity dynamics of denitrifying bacteria in most systems are poorly understood, partially due to difficulties in identifying and quantifying...... (active) denitrifiers. The goal of this study was therefore to develop a protocol for the in situ detection of denitrifying bacterial cells by targeting the mRNA of denitrification genes, hereby linking denitrification activity directly to the single-cell level. Target genes were narG (encoding nitrate...... reductase) and nosZ (encoding nitrous oxide reductase), to detect nitrate-reducing and completely denitrifying bacteria, respectively. Enzyme-labelled oligonucleotide probes and digoxygenin-labelled polynucleotide probes were evaluated for in situ hybridization in combination with immunochemical detection...

  8. Evaluation of BacLite Rapid MRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistant S. aureus (MRSA from screening swabs

    Directory of Open Access Journals (Sweden)

    Skyrme Margaret

    2006-09-01

    Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA is a major nosocomial pathogen worldwide. The need for accurate and rapid screening methods to detect MRSA carriers has been clearly established. The performance of a novel assay, BacLite Rapid MRSA (Acolyte Biomedica, UK for the rapid detection (5 h and identification of hospital associated ciprofloxacin resistant strains of MRSA directly from nasal swab specimens was compared to that obtained by culture on Mannitol salt agar containing Oxacillin (MSAO after 48 h incubation. Results A total of 1382 nasal screening swabs were tested by multiple operators. The BacLite Rapid MRSA test detected 142 out of the 157 confirmed MRSA that were detected on MSAO giving a diagnostic sensitivity of 90.4, diagnostic specificity of 95.7% and a negative predictive value of 98.7%. Of the 15 false negatives obtained by the BacLite Rapid MRSA test, seven grew small amounts ( Conclusion The Baclite MRSA test is easy to use and provides a similar level of sensitivity to conventional culture for the detection of nasal carriage of MRSA with the advantage that the results are obtained much more rapidly.

  9. Comparison between the Traditional and a Rapid Screening Test for Cryoimmunoglobulins Detection

    Directory of Open Access Journals (Sweden)

    Federica Romitelli

    2015-01-01

    Full Text Available Objectives. A new rapid, automatic, and sensitive screening test useful to detect cryoglobulins in serum samples is proposed. Design and Methods. The increase of turbidity during the cryoglobulin aggregation was monitored spectrophotometrically in sera from 400 patients with clinical evidence of cryoglobulinemia related disorders and 100 controls. Results were correlated to those obtained by the traditional method. Results. Kinetics of the aggregation curves were described by their maximum turbidity increase, lag time, and slope. Despite a partial correspondence between the traditional and the rapid test, patients with symptomatic cryoglobulinemia showed turbidity values significantly higher than the determined cutoff. Moreover, a functional classification of cryoglobulins is proposed. Conclusions. Due to its high reproducibility, operator independence, low cost, and results obtained within 2 hours, the rapid test can be used as a “real time” monitoring of cryoglobulinemia related diseases and for the evaluation of plasmapheresis efficacy.

  10. The rapid identification of lactic acid bacteria present in Chilean winemaking processes using culture-independent analysis.

    Science.gov (United States)

    Ilabaca, Carolina; Jara, Carla; Romero, Jaime

    2014-01-01

    A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S ribosomal RNA (rRNA) genes was developed to identify lactic acid bacteria (LAB) that are commonly present in winemaking processes (Oenococcus, Pediococcus, Lactobacillus, and Leuconostoc). This culture-independent approach revealed the presence of Oenococcus in the spontaneous malolactic fermentation in industrial Chilean wines.

  11. Substrate-independent luminescent phage-based biosensor to specifically detect enteric bacteria such as E. coli.

    Science.gov (United States)

    Franche, Nathalie; Vinay, Manon; Ansaldi, Mireille

    2017-01-01

    Water quality is a major safety consideration in environments that are impacted by human activity. The key challenge of the COMBITOX project is to develop a unique instrument that can accommodate several biodetector systems (see the accompanying COMBITOX papers) able to detect different pollutants such as bacteria, toxins, and heavy metals. The output signal chosen by our consortium is based on luminescence detection. Our group recently developed phage-based biosensors using gfp as a reporter gene to detect enteric bacteria in complex environments such as sea water, and the main challenge we faced was to adapt our biodetector to a luminescent signal that could fit the COMBITOX project requirements. Another key point was to use a substrate-independent reporter system in order to avoid substrate addition in the detection prototype. This paper describes the development of a phage-based biodetector using a luminescent and substrate-independent output to detect some enteric bacteria, such as Escherichia coli, in water samples. We have successfully engineered various prototypes using the HK620 and HK97 bacteriophages that use different packaging systems, and both proved functional for the integration of the full luxCDABE operon controlled by two different bacterial promoters. We show that the luxCDABE operon controlled by the PrplU bacterial promoter is the most efficient in terms of signal emission. The emission of luminescence is specific and allows the detection of 10(4) bacteria per milliliter in 1.5 h post-infection with neither a concentration nor enrichment step.

  12. Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium.

    Science.gov (United States)

    Vinay, Manon; Franche, Nathalie; Grégori, Gérald; Fantino, Jean-Raphaël; Pouillot, Flavie; Ansaldi, Mireille

    2015-01-01

    Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water.

  13. Rapid detection of malto-oligosaccharide-forming bacterial amylases by high performance anion-exchange chromatography

    DEFF Research Database (Denmark)

    Duedahl-Olesen, Lene; Larsen, K. L.; Zimmermann, W.

    2000-01-01

    High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples. Malto-oligosaccharide-formi......High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples. Malto......-oligosaccharide-forming amylases, indicated by a predominant formation of maltohexaose from starch, were produced by enzyme preparations from four of the isolates growing at pH 7.0 and 10....

  14. Rapid culture-based methods for drug-resistance detection in Mycobacterium tuberculosis.

    Science.gov (United States)

    Palomino, Juan Carlos; Martin, Anandi; Von Groll, Andrea; Portaels, Francoise

    2008-10-01

    Tuberculosis still represents a major public health problem, especially in low-resource countries where the burden of the disease is more important. Multidrug-resistant and extensively drug drug-resistant tuberculosis constitute serious problems for the efficient control of the disease stressing the need to investigate resistance to first- and second-line drugs. Conventional methods for detecting drug-resistance in Mycobacterium tuberculosis are slow and cumbersome. The most commonly used proportion method on Löwenstein-Jensen medium or Middlebrook agar requires a minimum of 3-4 weeks to produce results. Several new approaches have been proposed in the last years for the rapid and timely detection of drug-resistance in tuberculosis. This review will address phenotypic culture-based methods for rapid drug susceptibility testing in M. tuberculosis.

  15. Rapid Detection of miRNA Using Nucleic Acids-templated AgNCs

    DEFF Research Database (Denmark)

    Shah, Pratik

    MicroRNAs (miRNAs) are small ubiquitous RNA molecules (20-24nt) that negatively regulate target gene expression at the post-transcriptional level. Due to their roles in a variety of biological processes, the levels of miRNAs are dynamically changed in response to cellular and environmental signals....../AgNCs). I have showed that rapid, simple, sensitive and specific miRNA detection is possible. Two aspects of my research are 1) the implication of DNA secondary structure on the photoluminescence properties of DNA/AgNCs, 2) the development of a novel tool for miRNA detection in complex biological samples....... In the former, I revealed that the mismatched secondary structures of DNA-templates are important for the rapid formation of bright red fluorescence. Further, I suggest that the chromatic properties of DNA/AgNCs are modulated not only by sequence but also by secondary structure of DNA-templates. Moreover...

  16. Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.

    Science.gov (United States)

    Mirzai, S; Safi, S; Mossavari, N; Afshar, D; Bolourchian, M

    2016-08-31

    The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders.

  17. Rapid detection of Avian Influenza Virus - Towards point of care diagnosis

    DEFF Research Database (Denmark)

    Dhumpa, Raghuram

    Bird flu or Avian flu is an infectious disease caused by an influenza A virus of the Orthomyxoviridae family. Avian influenza virus (AIV) causes significant economic losses to the poultry industry worldwide and threatens human life with a pandemic. Pandemic of AIV is the human infection caused...... by the appearance of a “new” influenza virus as a result of antigenic shift or antigenic drift. Several outbreaks of AIV caused by the rapid spread of infection have been identified. Therefore, there is an urgent need for rapid diagnostic methods that would enable early detection and improve measurements to control...... and specificity of detecting AIV but are still cumbersome, expensive and time-consuming (1-2 days). In both classical and molecular diagnosis, the transportation of sample to the near-by reference or diagnostic laboratory is needed, and this will increases the time for diagnostic result. A simple approach would...

  18. Fluorescence-based lateral flow assays for rapid oral fluid roadside detection of cannabis use.

    Science.gov (United States)

    Plouffe, Brian D; Murthy, Shashi K

    2017-02-01

    With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral-fluid testing for Δ 9 -tetrahydrocannabinol (THC). This proof-of-concept assay uses a fluorescent-based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling-thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Rapid detection of Hendra virus antibodies: an integrated device with nanoparticle assay and chaotic micromixing.

    Science.gov (United States)

    Petkovic, K; Metcalfe, G; Chen, H; Gao, Y; Best, M; Lester, D; Zhu, Y

    2016-12-20

    Current diagnosis of infectious diseases such as Hendra virus (HeV) relies mostly on laboratory-based tests. There is an urgent demand for rapid diagnosis technology to detect and identify these diseases in humans and animals so that disease spread can be controlled. In this study, an integrated lab-on-a-chip device using a magnetic nanoparticle immunoassay is developed. The key features of the device are the chaotic fluid mixing, achieved by magnetically driven motion of nanoparticles with the optimal mixing protocol developed using chaotic transport theory, and the automatic liquid handling system for loading reagents and samples. The device has been demonstrated to detect Hendra virus antibodies in dilute horse serum samples within a short time of 15 minutes and the limit of detection is about 0.48 ng ml -1 . The device platform can potentially be used for field detection of viruses and other biological and chemical substances.

  20. An integrated micro-chip for rapid detection of magnetic particles

    KAUST Repository

    Gooneratne, Chinthaka P.

    2012-03-09

    This paper proposes an integrated micro-chip for the manipulation and detection of magnetic particles (MPs). A conducting ring structure is used to manipulate MPs toward giant magnetoresistance(GMR) sensing elements for rapid detection. The GMRsensor is fabricated in a horseshoe shape in order to detect the majority of MPs that are trapped around the conducting structure. The GMR sensing elements are connected in a Wheatstone bridge circuit topology for optimum noise suppression. Full fabrication details of the micro-chip, characterization of the GMRsensors, and experimental results with MPs are presented in this paper. Experimental results showed that the micro-chip can detect MPs from low concentration samples after they were guided toward the GMRsensors by applying current to the conducting ring structure.

  1. Rapid quantitative detection of glucose content in glucose injection by reaction headspace gas chromatography.

    Science.gov (United States)

    Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

    2017-10-20

    This work investigates an automated technique for rapid detecting the glucose content in glucose injection by reaction headspace gas chromatography (HS-GC). This method is based on the oxidation reaction of glucose in glucose injection with potassium dichromate. The carbon dioxide (CO 2 ) formed from the oxidation reaction can be quantitatively detected by GC. The results show that the relative standard deviation (RSD) of the present method was within 2.91%, and the measured glucose contents in glucose injection closely match those quantified by the reference method (relative differences glucose content in glucose injection related applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test.

    Science.gov (United States)

    Bruning, A H L; Susi, P; Toivola, H; Christensen, A; Söderlund-Venermo, M; Hedman, K; Aatola, H; Zvirbliene, A; Koskinen, J O

    2016-05-01

    Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days.

  3. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test

    Directory of Open Access Journals (Sweden)

    A.H.L. Bruning

    2016-05-01

    Full Text Available Clinically relevant diagnosis of human bocavirus 1 (HBoV1 is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days.

  4. Rapid detection of milk adulteration using intact protein flow injection mass spectrometric fingerprints combined with chemometrics.

    Science.gov (United States)

    Du, Lijuan; Lu, Weiying; Cai, Zhenzhen Julia; Bao, Lei; Hartmann, Christoph; Gao, Boyan; Yu, Liangli Lucy

    2018-02-01

    Flow injection mass spectrometry (FIMS) combined with chemometrics was evaluated for rapidly detecting economically motivated adulteration (EMA) of milk. Twenty-two pure milk and thirty-five counterparts adulterated with soybean, pea, and whey protein isolates at 0.5, 1, 3, 5, and 10% (w/w) levels were analyzed. The principal component analysis (PCA), partial least-squares-discriminant analysis (PLS-DA), and support vector machine (SVM) classification models indicated that the adulterated milks could successfully be classified from the pure milks. FIMS combined with chemometrics might be an effective method to detect possible EMA in milk. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Rapid lateral-flow immunoassay for the quantum dot-based detection of puerarin.

    Science.gov (United States)

    Qu, Huihua; Zhang, Yue; Qu, Baoping; Kong, Hui; Qin, Gaofeng; Liu, Shuchen; Cheng, Jinjun; Wang, Qingguo; Zhao, Yan

    2016-07-15

    In this study, a rapid (within 10min) quantitative lateral-flow immunoassay using a quantum dots (QDs)-antibody probe was developed for the analysis of puerarin (PUE) in water and biological samples. The competitive immunoassay was based on anti-PUE monoclonal antibody conjugated with QDs (detection reagent). Secondary antibody was immobilized on one end of a nitrocellulose membrane (control line) and PUE-bovine serum albumin conjugate was immobilized on the other end (test line). In the quantitative experiment, the detection results were scanned using a membrane strip reader and a detection curve (regression equation: y=-0.11ln(x)+0.979, R(2)=0.9816) representing the averages of the scanned data was obtained. This curve was linear from 1 to 10μg/mL. The IC50 value was 75.58ng/mL and the qualitative detection limit of PUE was 5.8ng/mL. The recovery of PUE added to phosphate-buffered saline and biological samples was in the range of 97.38-116.56%. To our knowledge, this is the first report of the quantitative detection of a natural product by QDs-based immunochromatography, which represents a powerful tool for rapidly screening PUE in plant materials and other biological samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Simple and rapid methods for detecting Salmonella enteritidis in raw eggs.

    Science.gov (United States)

    Seo, Kun-Ho; Holt, Peter S; Stone, Henry D; Gast, Richard K

    2003-10-15

    The Centers for Disease Control and Prevention estimates there were 300,000 cases of Salmonella enteritidis (SE) in 1997. Egg products were associated with many of the cases. To address this problem, many producers implemented flock surveillance of the SE situation at their facilities. A rapid and simple method for detecting SE from poultry samples is critical for the effective implementation of such testing strategies. A lateral flow device for the detection of SE utilized in this study was manufactured by Neogen, Lansing, MI. The test panel is a presumptive qualitative test system that detects only members of Group D1 Salmonella species. A series of studies were conducted to optimize the test procedure for raw eggs with different sample preparations. A novel antigen extraction method was developed for use with the test panel kit. The detection limit of the test panel kit was increased approximately tenfold when the extraction method was used. Detection of SE was 100% in raw egg pools inoculated with 10 SE cells per ml of egg and incubated at a 1:10 ratio in buffered peptone water (BPW) or tetrathionate brilliant green broth (TBG) for 24 h at 37 degrees C. The developed lateral flow test kit could provide a simple, rapid, and inexpensive method for egg producers and processors to test specifically for Salmonella group D1 serovars, such as SE, in egg samples.

  7. Rapid detection of coronary artery stenoses with real-time perfusion echocardiography during regadenoson stress.

    Science.gov (United States)

    Porter, Thomas R; Adolphson, Mary; High, Robin R; Smith, Lynette M; Olson, Joan; Erdkamp, Michelle; Xie, Feng; O'Leary, Edward; Wong, Benjamin F; Eifert-Rain, Susan; Hagen, Mary E; Abdelmoneim, Sahar S; Mulvagh, Sharon L

    2011-11-01

    Real-time myocardial contrast echocardiography permits the detection of myocardial perfusion abnormalities during stress echocardiography, which may improve the accuracy of the test in detecting coronary artery stenoses. We hypothesized that this technique could be used after a bolus injection of the selective A2A receptor agonist regadenoson to rapidly and safely detect coronary artery stenoses. In 100 patients referred for quantitative coronary angiography, real-time myocardial contrast echocardiography was performed during a continuous intravenous infusion of 3% Definity at baseline and at 2-minute intervals for up to 6 minutes after a regadenoson bolus injection (400 μg). Myocardial perfusion was assessed by examination of myocardial contrast replenishment after brief high mechanical index impulses. A perfusion defect was defined as a delay (>2 seconds) in myocardial contrast replenishment in 2 contiguous segments. Wall motion was also analyzed. The overall sensitivity/specificity/accuracy for myocardial perfusion analysis in detecting a >50% diameter stenosis was 80%/74%/78%, whereas for wall motion analysis it was 60%/72%/66% (Pregadenoson bolus (Pregadenoson bolus injection. Regadenoson real-time myocardial contrast echocardiography appears to be a feasible, safe, and rapid noninvasive method for the detection of significant coronary artery stenoses.

  8. Practical biophysics: Sensors for rapid detection of biological targets utilizing target-induced oligonucleotide annealing.

    Science.gov (United States)

    Heyduk, Tomasz

    2010-10-01

    Detection and quantitation of biomolecules is one of the most commonly performed measurements in biomedical research and clinical diagnostics. There is high demand for convenient, rapid and sensitive biomolecule detection methodologies. In this review we discuss a family of sensors that have been developed in our laboratory that share a common simple biophysical mechanism of action and that are capable of rapid detection of a diverse range of biological targets. The sensors generate fluorescence signal in the presence of the target molecule through target-induced association of short fluorochrome-labeled complementary oligonucleotides that are attached to target recognition elements of the sensors (antibodies, aptamers, etc.) via nanometer scale flexible linkers. This sensor design can be used for detecting proteins, antibodies, nucleic acids and whole cells. The assays using these sensors require only adding a sample to the sensor mix followed by simple fluorescence intensity readout. The simplicity, the speed of detection and the potential for miniaturization are the main assets of these sensors. 2010 Elsevier B.V. All rights reserved.

  9. Rapid Detection Technology for Pesticides Residues Based on Microelectrodes Impedance Immunosensor

    Directory of Open Access Journals (Sweden)

    Wen Ping Zhao

    2014-09-01

    Full Text Available Compared with conventional methods, electrochemical immunosensors have many advantages, such as low cost, high sensitivity, and rapid detection, and has certain prospects for realizing real-time-monitoring. In this paper, a design of portable pesticide residues detection instrument was presented based on an electrochemical impedance immunosensor. Firstly, we studied on an impedance immunosensor based on interdigitated array microelectrode (IDAM coupled with magnetic nanobeads-antibody conjugates (MNAC for the pesticide detection. Magnetic nanobeads (diameter 150 nm coated with anti-carbofuran antibodies were used for further amplification of the binding reaction between antibody and hapten (carbofuran. Secondly, in order to develop a portable pesticide residue apparatus, we designed the impedance detection electric circuit. Main work included designing and constructing of the system circuit, designing and debugging of the system software and so on. Thirdly, the apparatus was used for the standard pesticides solutions testing combined with immunosensor to test the reliability and stability. The pesticide added standard recovery was more than 70 % and the impedance test error was less than 5 %. The results showed that the proposed instrument had a good consistence compared with the traditional analytical methods. Thus, it would be a promising rapid detection instrument for pesticide residues in agricultural products.

  10. Rapid and label-free bioanalytical method of alpha fetoprotein detection using LSPR chip

    Science.gov (United States)

    Kim, Dongjoo; Kim, Jinwoon; Kwak, Cheol Hwan; Heo, Nam Su; Oh, Seo Yeong; Lee, Hoomin; Lee, Go-Woon; Vilian, A. T. Ezhil; Han, Young-Kyu; Kim, Woo-Sik; Kim, Gi-bum; Kwon, Soonjo; Huh, Yun Suk

    2017-07-01

    Alpha fetoprotein (AFP) is a cancer marker, particularly for hepatocellular carcinoma. Normal levels of AFP are less than 20 ng/mL; however, its levels can reach more than 400 ng/mL in patients with HCC. Enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been employed for clinical diagnosis of AFP; however, these methods are time consuming and labor intensive. In this study, we developed a localized surface plasmon resonance (LSPR) based biosensor for simple and rapid detection of AFP. This biosensor consists of a UV-Vis spectrometer, a cuvette cell, and a biosensor chip nanopatterned with gold nanoparticles (AuNPs). In our LSPR biosensor, binding of AFP to the surface of the sensor chip led to an increasing magnitude of the LSPR signals, which was measured by an ultraviolet-visible (UV-Vis) spectrometer. Our LSPR biosensor showed sufficient detectability of AFP at concentrations of 1 ng/mL to 1 μg/mL. Moreover, the overall procedure for detection of AFP was completed within 20 min. This biosensor could also be utilized for a point of care test (POCT) by employing a portable UV-Vis spectrometer. Owing to the simplicity and rapidity of the detection process, our LSPR biosensor is expected to replace traditional diagnostic methods for the early detection of diseases.

  11. Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to Duchenne muscula dystrophy carrier detection

    Energy Technology Data Exchange (ETDEWEB)

    Prior, T.W.; Wenger, G.D.; Moore, J. [Ohio State Univ., Columbus, OH (United States)] [and others

    1994-09-01

    A new strategy has been developed for rapid haplotype analysis. It is based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. The method is simple, rapid, does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using 12 commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. As a result of expanding the number of detectable polymorphisms throughout the dystrophin gene, we show how the method can easily be combined with dinucleotide analysis to improve the accuracy of carrier detection in the nondeletion cases. The technique is also shown to be used as an effective screen for improving carrier detection in several families with deletions. The finding of heterozygosity within the deletion identifies the at-risk female as a noncarrier. Using this method, we have identified and incorporated 3 new dystrophin polymorphisms (one of which in exon 16 is unique to African Americans). The method may be used other genetic diseases when mutations are unknown, or there are few dinucleotide markers in the gene proximity, or for the identification of haplotype backgrounds of mutant alleles.

  12. Simple and rapid detection of Tilletia horrida causing rice kernel smut in rice seeds.

    Science.gov (United States)

    Chen, Yu; Yang, Xue; Yao, Jian; Kyaw, Ei Phyu; Zhang, Ai-Fang; Li, Yun-Fei; Gu, Chun-Yan; Zang, Hao-Yu; Gao, Tong-Chun

    2016-09-14

    A simple and rapid method for the detection of Tilletia horrida, the causal agent of rice kernel smut, in rice seeds is developed based on specific polymerase chain reaction (PCR). To design the specific primers for the detection of T. horrida, partial sequences of internal transcribed spacer (ITS) DNA region of T. horrida, T. controversa, T. walkeri, T. ehrhartae, T. indica and T. caries were analyzed and compared. A 503-bp fragment was amplified with the designed primers from the T. horrida genomic DNA. However, no PCR product was obtained from the DNA of other five Tilletia species and 22 fungal plant pathogens tested in the present work indicating the specificity of the primers for the detection of T. horrida. The PCR was performed by directly using the spores, isolated from the 21 different rice seed samples, as template DNA. The T. horrida was detected in 6 of the samples, indicating that 28.6% of the rice samples were contaminated with the kernel smut pathogen. This simple PCR based diagnostic assay can be applied for the direct and rapid detection and identification of T. horrida to screen large numbers of rice seed samples.

  13. Apparatus and method for rapid separation and detection of hydrocarbon fractions in a fluid stream

    Science.gov (United States)

    Sluder, Charles S.; Storey, John M.; Lewis, Sr., Samuel A.

    2013-01-22

    An apparatus and method for rapid fractionation of hydrocarbon phases in a sample fluid stream are disclosed. Examples of the disclosed apparatus and method include an assembly of elements in fluid communication with one another including one or more valves and at least one sorbent chamber for removing certain classifications of hydrocarbons and detecting the remaining fractions using a detector. The respective ratios of hydrocarbons are determined by comparison with a non separated fluid stream.

  14. Biocontrol and Rapid Detection of Food-Borne Pathogens Using Bacteriophages and Endolysins

    OpenAIRE

    Bai, Jaewoo; Kim, You-Tae; Ryu, Sangryeol; Lee, Ju-Hoon

    2016-01-01

    Bacteriophages have been suggested as natural food preservatives as well as rapid detection materials for food-borne pathogens in various foods. Since Listeria monocytogenes-targeting phage cocktail (ListShield) was approved for applications in foods, numerous phages have been screened and experimentally characterized for phage applications in foods. A single phage and phage cocktail treatments to various foods contaminated with food-borne pathogens including E. coli O157:H7, Salmonella enter...

  15. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    OpenAIRE

    Hoseok Choi; Bomi Choi; Ju Tae Seo; Kyung Jin Lee; Myung Chan Gye; Young-Pil Kim

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) p...

  16. Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei

    Science.gov (United States)

    Bauernfeind, Adolf; Roller, Carsten; Meyer, Detlef; Jungwirth, Renate; Schneider, Ines

    1998-01-01

    A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel. PMID:9705426

  17. Analytical and clinical sensitivity of the 3M rapid detection influenza A+B assay.

    Science.gov (United States)

    Dale, Suzanne E; Mayer, Christine; Mayer, Marie C; Menegus, Marilyn A

    2008-11-01

    The performance of the 3M rapid detection influenza A+B (3M flu) assay was compared to the performance of other immunochromatographic assays. The clinical and analytical performance of the 3M flu assay was superior to that of BinaxNOW and Directigen EZ assays and equivalent to that of the QuickVue assay. The 3M flu assay offers an objective output and direct linkage to laboratory information systems.

  18. Analytical and Clinical Sensitivity of the 3M Rapid Detection Influenza A+B Assay ▿

    Science.gov (United States)

    Dale, Suzanne E.; Mayer, Christine; Mayer, Marie C.; Menegus, Marilyn A.

    2008-01-01

    The performance of the 3M rapid detection influenza A+B (3M flu) assay was compared to the performance of other immunochromatographic assays. The clinical and analytical performance of the 3M flu assay was superior to that of BinaxNOW and Directigen EZ assays and equivalent to that of the QuickVue assay. The 3M flu assay offers an objective output and direct linkage to laboratory information systems. PMID:18832133

  19. A Rapid Detection of Meat Spoilage using an Electronic Nose and Fuzzy-Wavelet systems

    OpenAIRE

    Kodogiannis, V.

    2018-01-01

    Freshness and safety of muscle foods are generally considered as the most important parameters for the food industry. To address the rapid detection of meat spoilage microorganisms during aerobic or modified atmosphere storage, an electronic nose with the aid of fuzzy wavelet network has been considered in this research. The proposed model incorporates a clustering pre-processing stage for the definition of fuzzy rules. The dual purpose of the proposed modelling approach is not only to classi...

  20. Clinical usefulness of multiplex PCR lateral flow in MRSA detection: a novel, rapid genetic testing method.

    Science.gov (United States)

    Nihonyanagi, Shin; Kanoh, Yuhsaku; Okada, Kiyomi; Uozumi, Toshiki; Kazuyama, Yukumasa; Yamaguchi, Tokiko; Nakazaki, Nobuhiko; Sakurai, Keizou; Hirata, Yasuyoshi; Munekata, Shinichi; Ohtani, Shinichi; Takemoto, Tsuyoshi; Bandoh, Yuki; Akahoshi, Tohru

    2012-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I-V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3 h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.

  1. Rapid detection and identification of viroids in the genus Coleviroid using a universal probe.

    Science.gov (United States)

    Jiang, Dongmei; Hou, Wanying; Sano, Teruo; Kang, Ni; Qin, Lü; Wu, Zujian; Li, Shifang; Xie, Lianhui

    2013-02-01

    A simple, low-cost hybridization assay using a universal DIG-labeled riboprobe for the rapid detection and identification of coleus viroids is presented. An octamer of 32-nucleotide sequence derived from the central conserved region (CCR) of viroids in the genus Coleviroid was used to develop a universal cRNA probe (8-central-conserved-region probe, 8CCR probe) for coleus viroids. Dot-blot hybridization assays demonstrated that the sensitivity of this probe was similar to specific probes for each CbVd, and Northern hybridization results revealed that at least four coleus viroids could be distinguished readily and simultaneously using the 8CCR probe. Batch detection assay showed that hybridization using the 8CCR probe can identify coleus viroids rapidly and effectively. This rapid and low-cost molecular hybridization technique is an effective way to survey the occurrence of coleus viroids, and has reference for the detection of other viroids and possibly viruses. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus.

    Science.gov (United States)

    Liu, Xinfeng; Guan, Yuyao; Cheng, Shiliang; Huang, Yidan; Yan, Qin; Zhang, Jun; Huang, Guanjun; Zheng, Jian; Liu, Tianqiang

    2016-12-01

    Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2×103CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment. Copyright © 2016. Published by Elsevier B.V.

  3. Comparison of rapid diagnostic tests to detect Mycobacterium avium subsp. paratuberculosis disseminated infection in bovine liver.

    Science.gov (United States)

    Zarei, Mehdi; Ghorbanpour, Masoud; Tajbakhsh, Samaneh; Mosavari, Nader

    2017-08-01

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a chronic enteritis in cattle and other domestic and wild ruminants. The presence of MAP in tissues other than intestines and associated lymph nodes, such as meat and liver, is a potential public health concern. In the present study, the relationship between the results of rapid diagnostic tests of the Johne's disease, such as serum ELISA, rectal scraping PCR, and acid-fast staining, and the presence of MAP in liver was evaluated. Blood, liver, and rectal scraping samples were collected from 200 slaughtered cattle with unknown Johne's disease status. ELISA was performed to determine the MAP antibody activity in the serum. Acid-fast staining was performed on rectal scraping samples, and PCR was performed on rectal scraping and liver samples. PCR-positive liver samples were used for mycobacterial culture. Overall, the results of this study demonstrated that MAP can be detected and cultured from liver of slaughtered cattle and rapid diagnostic tests of Johne's disease have limited value in detecting cattle with MAP infection in liver. These findings show that the presence of MAP in liver tissue may occur in cows with negative results for rapid diagnostic tests and vice versa. Hence, liver might represent another possible risk of human exposure to MAP. Given concerns about a potential zoonotic role for MAP, these results show the necessity to find new methods for detecting cattle with MAP disseminated infection.

  4. Label-free optical detection of bacteria on a 1-D photonic crystal of porous silicon

    Science.gov (United States)

    Wu, Chia-Chen; Alvarez, Sara D.; Rang, Camilla U.; Chao, Lin; Sailor, Michael J.

    2009-02-01

    The construction of a specific, label-free, bacteria biosensor using porous silicon 1-D photonic crystals will be described. Bacteria resident on the surface of porous silicon act as scattering centers for light resonant with the photonic crystal; the diffusely scattered light possesses the optical spectrum of the underlying photonic crystal. Using a spectrometer fitted to a light microscope, the bacteria are imaged without using exogenous dyes or labels and are quantified by measuring the intensity of scattered light. In order to selectively bind and identify bacteria using porous Si, we use surface modifications to reduce nonspecific binding to the surface and to engineer bacteria specificity onto the surface. Bovine serum albumin (BSA) was adsorbed to the porous Si surface to reduce nonspecific binding of bacteria. The coatings were then chemically activated to immobilize polyclonal antibodies specific to Escherichia coli. Two E. coli strains were used in our study, E. coli DH5α and non-pathogenic enterohemorrhagic Escherichia coli (EHEC) strain. The nonpathogenic Vibrio cholerae O1 strain was used to test for antibody specificity. Successful attachment of antibodies was measured using fluorescence microscopy and the scattering method was used to test for bacteria binding specificity.

  5. Rapid Discrimination of Gram-Positive and Gram-Negative Bacteria in Liquid Samples by Using NaOH-Sodium Dodecyl Sulfate Solution and Flow Cytometry

    Science.gov (United States)

    Wada, Atsushi; Kono, Mari; Kawauchi, Sawako; Takagi, Yuri; Morikawa, Takashi; Funakoshi, Kunihiro

    2012-01-01

    Background For precise diagnosis of urinary tract infections (UTI), and selection of the appropriate prescriptions for their treatment, we explored a simple and rapid method of discriminating gram-positive and gram-negative bacteria in liquid samples. Methodology/Principal Findings We employed the NaOH-sodium dodecyl sulfate (SDS) solution conventionally used for plasmid extraction from Escherichia coli and the automated urine particle analyzer UF-1000i (Sysmex Corporation) for our novel method. The NaOH-SDS solution was used to determine differences in the cell wall structures between gram-positive and gram-negative bacteria, since the tolerance to such chemicals reflects the thickness and structural differences of bacterial cell walls. The UF-1000i instrument was used as a quantitative bacterial counter. We found that gram-negative bacteria, including E. coli, in liquid culture could easily be lysed by direct addition of equal volumes of NaOH-SDS solution. In contrast, Enterococcus faecalis, which is a gram-positive bacterium, could not be completely lysed by the solution. We then optimized the reaction time of the NaOH-SDS treatment at room temperature by using 3 gram-positive and 4 gram-negative bacterial strains and determined that the optimum reaction time was 5 min. Finally, in order to evaluate the generalizability of this method, we treated 8 gram-positive strains and 8 gram-negative strains, or 4 gram-positive and 4 gram-negative strains incubated in voluntary urine from healthy volunteers in the same way and demonstrated that all the gram-positive bacteria were discriminated quantitatively from gram negative bacteria using this method. Conclusions/Significance Using our new method, we could easily discriminate gram-positive and gram-negative bacteria in liquid culture media within 10 min. This simple and rapid method may be useful for determining the treatment course of patients with UTIs, especially for those without a prior history of UTIs. The method

  6. Rapid discrimination of Gram-positive and Gram-negative bacteria in liquid samples by using NaOH-sodium dodecyl sulfate solution and flow cytometry.

    Directory of Open Access Journals (Sweden)

    Atsushi Wada

    Full Text Available BACKGROUND: For precise diagnosis of urinary tract infections (UTI, and selection of the appropriate prescriptions for their treatment, we explored a simple and rapid method of discriminating gram-positive and gram-negative bacteria in liquid samples. METHODOLOGY/PRINCIPAL FINDINGS: We employed the NaOH-sodium dodecyl sulfate (SDS solution conventionally used for plasmid extraction from Escherichia coli and the automated urine particle analyzer UF-1000i (Sysmex Corporation for our novel method. The NaOH-SDS solution was used to determine differences in the cell wall structures between gram-positive and gram-negative bacteria, since the tolerance to such chemicals reflects the thickness and structural differences of bacterial cell walls. The UF-1000i instrument was used as a quantitative bacterial counter. We found that gram-negative bacteria, including E. coli, in liquid culture could easily be lysed by direct addition of equal volumes of NaOH-SDS solution. In contrast, Enterococcus faecalis, which is a gram-positive bacterium, could not be completely lysed by the solution. We then optimized the reaction time of the NaOH-SDS treatment at room temperature by using 3 gram-positive and 4 gram-negative bacterial strains and determined that the optimum reaction time was 5 min. Finally, in order to evaluate the generalizability of this method, we treated 8 gram-positive strains and 8 gram-negative strains, or 4 gram-positive and 4 gram-negative strains incubated in voluntary urine from healthy volunteers in the same way and demonstrated that all the gram-positive bacteria were discriminated quantitatively from gram negative bacteria using this method. CONCLUSIONS/SIGNIFICANCE: Using our new method, we could easily discriminate gram-positive and gram-negative bacteria in liquid culture media within 10 min. This simple and rapid method may be useful for determining the treatment course of patients with UTIs, especially for those without a prior history

  7. Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

    Directory of Open Access Journals (Sweden)

    Camille Escadafal

    2014-03-01

    Full Text Available BACKGROUND: Yellow fever (YF is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV, is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. METHODOLOGY: The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. CONCLUSION/SIGNIFICANCE: The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction and rapid processing time (<20 min. Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for

  8. Computational modeling and experimental characterization of bacterial microcolonies for rapid detection using light scattering

    Science.gov (United States)

    Bai, Nan

    A label-free and nondestructive optical elastic forward light scattering method has been extended for the analysis of microcolonies for food-borne bacteria detection and identification. To understand the forward light scattering phenomenon, a model based on the scalar diffraction theory has been employed: a bacterial colony is considered as a biological spatial light modulator with amplitude and phase modulation to the incoming light, which continues to propagate to the far-field to form a distinct scattering 'fingerprint'. Numerical implementation via angular spectrum method (ASM) and Fresnel approximation have been carried out through Fast Fourier Transform (FFT) to simulate this optical model. Sampling criteria to achieve unbiased and un-aliased simulation results have been derived and the effects of violating these conditions have been studied. Diffraction patterns predicted by these two methods (ASM and Fresnel) have been compared to show their applicability to different simulation settings. Through the simulation work, the correlation between the colony morphology and its forward scattering pattern has been established to link the number of diffraction rings and the half cone angle with the diameter and the central height of the Gaussian-shaped colonies. In order to experimentally prove the correlation, a colony morphology analyzer has been built and used to characterize the morphology of different bacteria genera and investigate their growth dynamics. The experimental measurements have demonstrated the possibility of differentiating bacteria Salmonella, Listeria, Escherichia in their early growth stage (100˜500 µm) based on their phenotypic characteristics. This conclusion has important implications in microcolony detection, as most bacteria of our interest need much less incubation time (8˜12 hours) to grow into this size range. The original forward light scatterometer has been updated to capture scattering patterns from microcolonies. Experiments have

  9. Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection.

    Science.gov (United States)

    Balachandran, Priya; Friberg, Maria; Vanlandingham, V; Kozak, K; Manolis, Amanda; Brevnov, Maxim; Crowley, Erin; Bird, Patrick; Goins, David; Furtado, Manohar R; Petrauskene, Olga V; Tebbs, Robert S; Charbonneau, Duane

    2012-02-01

    Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

  10. EEG indices of reward motivation and target detectability in a rapid visual detection task.

    Science.gov (United States)

    Hughes, Gethin; Mathan, Santosh; Yeung, Nick

    2013-01-01

    A large corpus of data has demonstrated the sensitivity of behavioral and neural measures to variation in the availability of reward. The present study aimed to extend this work by exploring reward motivation in an RSVP task using complex satellite imagery. We found that reward motivation significantly influenced neural activity both in the preparatory period and in response to target images. Pre-stimulus alpha activity and, to a lesser degree, P3 and CNV amplitude were found to be significantly predictive of reward condition on single trials. Target-locked P3 amplitude was modulated both by reward condition and by variation in target detectability inherent to our task. We further quantified this exogenous influence, showing that P3 differences reflected single-trial variation in P3 amplitude for different targets. These findings provide theoretical insight into the neural indices of reward in an RSVP task, and have important applications in the field of satellite imagery analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays

    Directory of Open Access Journals (Sweden)

    Jinliang Wang

    2016-03-01

    Full Text Available Shiga toxin-producing Escherichia coli O157:H7 (STEC cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2 that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA; one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

  12. Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays.

    Science.gov (United States)

    Wang, Jinliang; Katani, Robab; Li, Lingling; Hegde, Narasimha; Roberts, Elisabeth L; Kapur, Vivek; DebRoy, Chitrita

    2016-03-25

    Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 10⁵ CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

  13. Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety.

    Science.gov (United States)

    Mangal, Manisha; Bansal, Sangita; Sharma, Satish K; Gupta, Ram K

    2016-07-03

    Food safety is a global health concern. For the prevention and recognition of problems related to health and safety, detection of foodborne pathogen is of utmost importance at all levels of food production chain. For several decades, a lot of research has been targeted at the development of rapid methodology as reducing the time needed to complete pathogen detection tests has been the primary goal of food microbiologists. With the result, food microbiology laboratories now have a wide array of detection methods and automated technologies such as enzyme immunoassay, polymerase chain reaction, and microarrays, which can cut test times considerably. Nucleic acid amplification strategies and advances in amplicon detection methodologies have been the key factors in the progress of molecular microbiology. A comprehensive literature survey has been carried out to give an overview in the field of foodborne pathogen detection. In this paper, we describe the conventional methods, as well as recent developments in food pathogen detection, identification, and quantification, with a major emphasis on molecular detection methods.

  14. The rapid identification of lactic acid bacteria present in Chilean winemaking processes using culture-independent analysis

    OpenAIRE

    Ilabaca, Carolina; Jara, Carla; Romero, Jaime

    2014-01-01

    A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S ribosomal RNA (rRNA) genes was developed to identify lactic acid bacteria (LAB) that are commonly present in winemaking processes (Oenococcus, Pediococcus, Lactobacillus, and Leuconostoc). This culture-independent approach revealed the presence of Oenococcus in the spontaneous malolactic fermentation in industrial Chilean wines. Electronic supplementary material The online version of this article (...

  15. Rapid Quantification of Bacteria in Infected Root Canals Using Fluorescence Reagents and a Membrane Filter: A Pilot Study on Its Clinical Application to the Evaluation of the Outcomes of Endodontic Treatment

    Directory of Open Access Journals (Sweden)

    Takuichi Sato

    2012-01-01

    Full Text Available Objective. The bacterial examination has been performed during the course of the root canal treatment. In the present pilot study, the new developed method, using fluorescence reagents and a membrane filter, was applied to the detection and quantification of bacteria in infected root canals, in order to evaluate the outcomes of the treatment. Methods. Six infected root canals with periapical lesions from 5 subjects were included. Informed consent was obtained from all subjects (age ranges, 23–79 years. Samples from infected root canals were collected at the beginning of the treatment (termed #25 First, the end of the first day of treatment (termed #55 First, and the next appointment day (termed #55 Second. Then, the bacterial count (CFU was measured using fluorescence reagents (4′,6′-diamidino-2-phenylindole and propidium iodide and the polycarbonate membrane filter by Bioplorer. Results. The mean ± SD of CFU in the sample of “#25 First” was (1.0±1.4×105. As the root canal treatment progressed, the CFU decreased as 7.9×103 (#55 First and 4.3×102 (#55 Second. Conclusion. In the present pilot study, rapid detection and quantification of bacteria in infected root canals were found to be successfully performed using fluorescence reagents and a membrane filter (Bioplorer analysis.

  16. Marine environmental pollution stress detection through direct viable counts of bacteria

    Digital Repository Service at National Institute of Oceanography (India)

    Ramaiah, N.; Kenkre, V.D.; Verlecar, X.N.

    Direct viable counts (DVC) of bacteria were quantified from polluted and relatively less/non-polluted coastal locations during different seasons to assess whether they can be routinely monitored for an understanding of environmental stress(es...

  17. Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

    Science.gov (United States)

    Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

    2017-02-01

    An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

  18. Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Li, Juan; Zhao, Guang-Hui; Lin, RuiQing; Blair, David; Sugiyama, Hiromu; Zhu, Xing-Quan

    2015-11-01

    Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

  19. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

    Science.gov (United States)

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane.

  20. Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Dinggang eZhou

    2016-03-01

    Full Text Available Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was ten-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100% by LAMP and 97/100 cases (97% by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable and cost-effective for detection of the bar specific transgenic sugarcane.

  1. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    Science.gov (United States)

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  2. Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence.

    Science.gov (United States)

    Qiu, Wanwei; Xu, Hui; Takalkar, Sunitha; Gurung, Anant S; Liu, Bin; Zheng, Yafeng; Guo, Zebin; Baloda, Meenu; Baryeh, Kwaku; Liu, Guodong

    2015-02-15

    In this article, we describe a carbon nanotube (CNT)-based lateral flow biosensor (LFB) for rapid and sensitive detection of DNA sequence. Amine-modified DNA detection probe was covalently immobilized on the shortened multi-walled carbon nanotubes (MWCNTs) via diimide-activated amidation between the carboxyl groups on the CNT surface and amine groups on the detection DNA probes. Sandwich-type DNA hybridization reactions were performed on the LFB and the captured MWCNTs on test zone and control zone of LFB produced the characteristic black bands, enabling visual detection of DNA sequences. Combining the advantages of lateral flow chromatographic separation with unique physical properties of MWCNT (large surface area), the optimized LFB was capable of detecting of 0.1 nM target DNA without instrumentation. Quantitative detection could be realized by recording the intensity of the test line with the Image J software, and the detection limit of 40 pM was obtained. This detection limit is 12.5 times lower than that of gold nanoparticle (GNP)-based LFB (0.5 nM, Mao et al. Anal. Chem. 2009, 81, 1660-1668). Another important feature is that the preparation of MWCNT-DNA conjugates was robust and the use of MWCNT labels avoided the aggregation of conjugates and tedious preparation time, which were often met in the traditional GNP-based nucleic acid LFB. The applications of MWCNT-based LFB can be extended to visually detect protein biomarkers using MWCNT-antibody conjugates. The MWCNT-based LFB thus open a new door to prepare a new generation of LFB, and shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Rapid Purification of Salmonella DNA in Minced Meat and Detection by Real-time PCR

    DEFF Research Database (Denmark)

    Jenikova, G.; Jensen, Annette Nygaard; Demnerova, K.

    2001-01-01

    of DNeasy was found to be 6-8 CFU in just 19 end-point fluorescence (C-t) values, while this was 22 C-t for a combination of DNeasy and BactXtractor. Extraction by DNeasy resulted in C-t cells per 25 g, when the samples were inoculated with Salmonella......Four rapid and simple DNA purification and sample treatment protocols were evaluated for detection of Salmonella enterica in spiked minced meat, using a fluorogenic 5' nuclease (TaqMan) PCR assay in an ABI-Prism 7700 Sequence Detector. The detection limit with the single separation treatment...... before the overnight preenrichment. The method is currently being adapted to a BioRobot 3000 platform. However, the use of paramagnetic beads (DNA Direct) resulted in poor and variable detection limit....

  4. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Changhua; Mao, Mao [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Yuan, Hang [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Shen, Huaibin [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Wu, Feng; Ma, Lan, E-mail: malan@sz.tsinghua.edu.cn [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Li, Lin Song, E-mail: lsli@henu.edu.cn [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China)

    2013-09-15

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 Degree-Sign C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

  5. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    Science.gov (United States)

    Zhou, Changhua; Mao, Mao; Yuan, Hang; Shen, Huaibin; Wu, Feng; Ma, Lan; Li, Lin Song

    2013-09-01

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 °C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

  6. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Y.; Dunn, J.; Gao, S.; Bruno, J. F.; Luft, B. J.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

  7. Rapid subsurface detection of nanoscale defects in live microprocessors by functional infrared emission spectral microscopy.

    Science.gov (United States)

    Saloma, Caesar; Tarun, Alvarado; Bailon, Michelle; Soriano, Maricor

    2005-12-01

    We demonstrate the rapid and nondestructive detection of subsurface nanometer-size defects in 90 nm technology live microprocessors with a new technique called functional infrared emission spectral microscopy. Broken, leaky, and good transistors with similar photoemission images are identified from each other by their different emission spectra that are calculated as linear combinations of weighted basis spectra. The basis spectra are derived from a spectral library by principal component analysis. Leaky transistors do not exhibit apparent morphological damage and are undetectable by optical or scanning probe microscopy alone. The emission signals from two or more transistors combined incoherently, and defect detection is primarily limited by the signal-to-noise ratio of the detected spectrum and not by the separation distance of neighboring transistors.

  8. A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins.

    Science.gov (United States)

    Banerjee, Pratik; Lenz, Dominik; Robinson, Joseph Paul; Rickus, Jenna L; Bhunia, Arun K

    2008-02-01

    Cell-based biosensors (CBBs) are becoming important tools for biosecurity applications and rapid diagnostics in food microbiology for their unique capability of detecting physiologically hazardous materials. A multi-well plate-based biosensor containing B-cell hybridoma, Ped-2E9, encapsulated in type I collagen matrix, was developed for rapid detection of viable cells of pathogenic Listeria, the toxin listeriolysin O, and the enterotoxin from Bacillus species. This sensor measures the alkaline phosphatase release from infected Ped-2E9 cells colorimetrically. Pathogenic L. monocytogenes cells and toxin preparations from L. monocytogenes or B. cereus showed cytotoxicity ranging from 24 to 98% at 3-6 h postinfection. In contrast, nonpathogenic L. innocua (F4247) and B. subtilis induced minimal cytotoxicity, ranging only 0.4-7.6%. Laser scanning cytometry and cryo-nano scanning electron microscopy confirmed the live or dead status of the infected Ped-2E9 cells in gel matrix. This paper presents the first example of a cell-based sensing system using collagen-encapsulated mammalian cells for rapid detection of pathogenic bacteria or toxin, and demonstrates a potential for onsite use as a portable detection system.

  9. Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

    Science.gov (United States)

    Muniroh, M S; Sariah, M; Zainal Abidin, M A; Lima, N; Paterson, R R M

    2014-05-01

    Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Rapid detection of economic adulterants in fresh milk by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Abernethy, Grant; Higgs, Kerianne

    2013-05-03

    A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods. Copyright © 2013 Elsevier B.V. All rights reserved.