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Sample records for detecting pathogenic african

  1. The use of ITS1 rDNA PCR in detecting pathogenic African trypanosomes.

    PubMed

    Njiru, Z K; Constantine, C C; Guya, S; Crowther, J; Kiragu, J M; Thompson, R C A; Dávila, A M R

    2005-02-01

    There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei, T. evansi, T. b. rhodesiense and T. b. gambiense) gave a constant product of approximately 480 bp; T. congolense, savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp, T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon, T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi. The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.

  2. Application of a multiplex PCR assay for the detection of gastrointestinal pathogens in a rural African setting.

    PubMed

    Eibach, Daniel; Krumkamp, Ralf; Hahn, Andreas; Sarpong, Nimako; Adu-Sarkodie, Yaw; Leva, Amelie; Käsmaier, Julia; Panning, Marcus; May, Jürgen; Tannich, Egbert

    2016-04-14

    Despite high morbidity and mortality, the laboratory diagnosis of gastrointestinal infections is largely neglected in tropical African settings. This study aims to apply the Luminex multiplex PCR assay for the diagnosis of gastrointestinal pathogens in rural Ghana to evaluate its usefulness as a routine method. A case-control study was conducted at the Agogo Presbyterian Hospital in Ghana. Stool samples were collected from children below 6 years of age with (cases) and without (controls) diarrhoea. Samples were screened for 15 different diarrhoeal pathogens by the Luminex xTAG GPP assay and associations between diarrhoea and gastrointestinal infections and fractions attributable to diarrhea (AF) were determined. The Luminex PCR assay identified organisms in 96.6% (n = 428) of 443 cases and in 92.5% (n = 221) of 239 selected controls. A mean of 2.5 (standard deviation [SD]: ± 1.3) and 2.3 (SD: ± 1.3) organisms per sample were detected in cases and controls respectively. An association with diarrhoea was found for rotavirus (adjusted odds ratio [aOR] = 7.2; 95% confidence interval [CI]: 2.9-18.1), norovirus (aOR = 2.7; 95% CI: 1.4-5.3) and Shigella spp. (aOR = 1.7; 95% CI: 1.2-2.4) with respective AFs of 12.5% (95% CI: 9.6-15.3), 7.9% (95% CI: 3.8-11.7) and 16.9% (95% CI: 6.9-25.9). The high proportion of pathogen-positive stool samples with a high number of co-infections in cases and controls suggests a substantial amount of transient or colonizing microorganisms for which treatment is not necessarily implicated. The use of sequential diagnostic algorithms with pathogen specific or quantitative PCRs might be most appropriate for diagnosing gastrointestinal infections.

  3. Molecular detection of zoonotic tick-borne pathogens from ticks collected from ruminants in four South African provinces.

    PubMed

    Mtshali, Khethiwe; Khumalo, Zth; Nakao, Ryo; Grab, Dennis J; Sugimoto, Chihiro; Thekisoe, Omm

    2016-01-01

    Ticks carry and transmit a remarkable array of pathogens including bacteria, protozoa and viruses, which may be of veterinary and/or of medical significance. With little to no information regarding the presence of tick-borne zoonotic pathogens or their known vectors in southern Africa, the aim of our study was to screen for Anaplasma phagocytophilum, Borrelia burgdorferi, Coxiella burnetii, Rickettsia species and Ehrlichia ruminantium in ticks collected and identified from ruminants in the Eastern Cape, Free State, KwaZulu-Natal and Mpumalanga Provinces of South Africa. The most abundant tick species identified in this study were Rhipicephalus evertsi evertsi (40%), Rhipicephalus species (35%), Amblyomma hebraeum (10%) and Rhipicephalus decoloratus (14%). A total of 1634 ticks were collected. DNA was extracted, and samples were subjected to PCR amplification and sequencing. The overall infection rates of ticks with the target pathogens in the four Provinces were as follows: A. phagocytophilum, 7%; C. burnetii, 7%; E. ruminantium, 28%; and Rickettsia spp., 27%. The presence of B. burgdorferi could not be confirmed. The findings of this study show that zoonotic pathogens are present in ticks in the studied South African provinces. This information will aid in the epidemiology of tick-borne zoonotic diseases in the country as well as in raising awareness about such diseases in the veterinary, medical and tourism sectors, as they may be the most affected.

  4. Molecular detection of zoonotic tick-borne pathogens from ticks collected from ruminants in four South African provinces

    PubMed Central

    MTSHALI, Khethiwe; KHUMALO, Zamantungwa T. H.; NAKAO, Ryo; GRAB, Dennis J.; SUGIMOTO, Chihiro; THEKISOE, Oriel M. M.

    2015-01-01

    Ticks carry and transmit a remarkable array of pathogens including bacteria, protozoa and viruses, which may be of veterinary and/or of medical significance. With little to no information regarding the presence of tick-borne zoonotic pathogens or their known vectors in southern Africa, the aim of our study was to screen for Anaplasma phagocytophilum, Borrelia burgdorferi, Coxiella burnetii, Rickettsia species and Ehrlichia ruminantium in ticks collected and identified from ruminants in the Eastern Cape, Free State, KwaZulu-Natal and Mpumalanga Provinces of South Africa. The most abundant tick species identified in this study were Rhipicephalus evertsi evertsi (40%), Rhipicephalus species (35%), Amblyomma hebraeum (10%) and Rhipicephalus decoloratus (14%). A total of 1634 ticks were collected. DNA was extracted, and samples were subjected to PCR amplification and sequencing. The overall infection rates of ticks with the target pathogens in the four Provinces were as follows: A. phagocytophilum, 7%; C. burnetii, 7%; E. ruminantium, 28%; and Rickettsia spp., 27%. The presence of B. burgdorferi could not be confirmed. The findings of this study show that zoonotic pathogens are present in ticks in the studied South African provinces. This information will aid in the epidemiology of tick-borne zoonotic diseases in the country as well as in raising awareness about such diseases in the veterinary, medical and tourism sectors, as they may be the most affected. PMID:26227797

  5. Portable pathogen detection system

    DOEpatents

    Colston, Billy W.; Everett, Matthew; Milanovich, Fred P.; Brown, Steve B.; Vendateswaran, Kodumudi; Simon, Jonathan N.

    2005-06-14

    A portable pathogen detection system that accomplishes on-site multiplex detection of targets in biological samples. The system includes: microbead specific reagents, incubation/mixing chambers, a disposable microbead capture substrate, and an optical measurement and decoding arrangement. The basis of this system is a highly flexible Liquid Array that utilizes optically encoded microbeads as the templates for biological assays. Target biological samples are optically labeled and captured on the microbeads, which are in turn captured on an ordered array or disordered array disposable capture substrate and then optically read.

  6. Rapid Detection of Pathogens

    SciTech Connect

    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. Multiplex detection of agricultural pathogens

    DOEpatents

    Siezak, Thomas R.; Gardner, Shea; Torres, Clinton; Vitalis, Elizabeth; Lenhoff, Raymond J.

    2013-01-15

    Described are kits and methods useful for detection of agricultural pathogens in a sample. Genomic sequence information from agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay and/or an array assay to successfully identify the presence or absence of pathogens in a sample.

  8. Multiplex detection of agricultural pathogens

    DOEpatents

    McBride, Mary Teresa; Slezak, Thomas Richard; Messenger, Sharon Lee

    2010-09-14

    Described are kits and methods useful for detection of seven agricultural pathogens (BPSV; BHV; BVD; FMDV; BTV; SVD; and VESV) in a sample. Genomic sequence information from 7 agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  9. Multiplex detection of respiratory pathogens

    DOEpatents

    McBride, Mary [Brentwood, CA; Slezak, Thomas [Livermore, CA; Birch, James M [Albany, CA

    2012-07-31

    Described are kits and methods useful for detection of respiratory pathogens (influenza A (including subtyping capability for H1, H3, H5 and H7 subtypes) influenza B, parainfluenza (type 2), respiratory syncytial virus, and adenovirus) in a sample. Genomic sequence information from the respiratory pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  10. Biosensors for plant pathogen detection.

    PubMed

    Khater, Mohga; de la Escosura-Muñiz, Alfredo; Merkoçi, Arben

    2017-07-15

    Infectious plant diseases are caused by pathogenic microorganisms such as fungi, bacteria, viruses, viroids, phytoplasma and nematodes. Worldwide, plant pathogen infections are among main factors limiting crop productivity and increasing economic losses. Plant pathogen detection is important as first step to manage a plant disease in greenhouses, field conditions and at the country boarders. Current immunological techniques used to detect pathogens in plant include enzyme-linked immunosorbent assays (ELISA) and direct tissue blot immunoassays (DTBIA). DNA-based techniques such as polymerase chain reaction (PCR), real time PCR (RT-PCR) and dot blot hybridization have also been proposed for pathogen identification and detection. However these methodologies are time-consuming and require complex instruments, being not suitable for in-situ analysis. Consequently, there is strong interest for developing new biosensing systems for early detection of plant diseases with high sensitivity and specificity at the point-of-care. In this context, we revise here the recent advancement in the development of advantageous biosensing systems for plant pathogen detection based on both antibody and DNA receptors. The use of different nanomaterials such as nanochannels and metallic nanoparticles for the development of innovative and sensitive biosensing systems for the detection of pathogens (i.e. bacteria and viruses) at the point-of-care is also shown. Plastic and paper-based platforms have been used for this purpose, offering cheap and easy-to-use really integrated sensing systems for rapid on-site detection. Beside devices developed at research and development level a brief revision of commercially available kits is also included in this review. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Pathogen detection using engineered bacteriophages.

    PubMed

    Smartt, Abby E; Xu, Tingting; Jegier, Patricia; Carswell, Jessica J; Blount, Samuel A; Sayler, Gary S; Ripp, Steven

    2012-04-01

    Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.

  12. Spectroscopic Detection of Pathogens

    SciTech Connect

    ALAM,M. KATHLEEN; TIMLIN,JERILYN A.; MARTIN,LAURA E.; HJELLE,DRIAN; LYONS,RICK; GARRISON,KRISTIN

    2000-11-01

    The goal of this LDRD Research project was to provide a preliminary examination of the use of infrared spectroscopy as a tool to detect the changes in cell cultures upon activation by an infectious agent. Due to a late arrival of funding, only 5 months were available to transfer and setup equipment at UTTM,develop cell culture lines, test methods of in-situ activation and collect kinetic data from activated cells. Using attenuated total reflectance (ATR) as a sampling method, live cell cultures were examined prior to and after activation. Spectroscopic data were collected from cells immediately after activation in situ and, in many cases for five successive hours. Additional data were collected from cells activated within a test tube (pre-activated), in both transmission mode as well as in ATR mode. Changes in the infrared data were apparent in the transmission data collected from the pre-activated cells as well in some of the pre-activated ATR data. Changes in the in-situ activated spectral data were only occasionally present due to (1) the limited time cells were studied and (2) incomplete activation. Comparison of preliminary data to infrared bands reported in the literature suggests the primary changes seen are due an increase in ribonucleic acid (RNA) production. This work will be continued as part of a 3 year DARPA grant.

  13. APDS: Autonomous Pathogen Detection System

    SciTech Connect

    Langlois, R G; Brown, S; Burris, L; Colston, B; Jones, L; Makarewicz, T; Mariella, R; Masquelier, D; McBride, M; Milanovich, F; Masarabadi, S; Venkateswaran, K; Marshall, G; Olson, D; Wolcott, D

    2002-02-14

    An early warning system to counter bioterrorism, the Autonomous Pathogen Detection System (APDS) continuously monitors the environment for the presence of biological pathogens (e.g., anthrax) and once detected, it sounds an alarm much like a smoke detector warns of a fire. Long before September 11, 2001, this system was being developed to protect domestic venues and events including performing arts centers, mass transit systems, major sporting and entertainment events, and other high profile situations in which the public is at risk of becoming a target of bioterrorist attacks. Customizing off-the-shelf components and developing new components, a multidisciplinary team developed APDS, a stand-alone system for rapid, continuous monitoring of multiple airborne biological threat agents in the environment. The completely automated APDS samples the air, prepares fluid samples in-line, and performs two orthogonal tests: immunoassay and nucleic acid detection. When compared to competing technologies, APDS is unprecedented in terms of flexibility and system performance.

  14. Use of cross-reactive serological assays for detecting novel pathogens in wildlife: assessing an appropriate cutoff for henipavirus assays in African bats.

    PubMed

    Peel, Alison J; McKinley, Trevelyan J; Baker, Kate S; Barr, Jennifer A; Crameri, Gary; Hayman, David T S; Feng, Yan-Ru; Broder, Christopher C; Wang, Lin-Fa; Cunningham, Andrew A; Wood, James L N

    2013-11-01

    Reservoir hosts of novel pathogens are often identified or suspected as such on the basis of serological assay results, prior to the isolation of the pathogen itself. Serological assays might therefore be used outside of their original, validated scope in order to infer seroprevalences in reservoir host populations, until such time that specific diagnostic assays can be developed. This is particularly the case in wildlife disease research. The absence of positive and negative control samples and gold standard diagnostic assays presents challenges in determining an appropriate threshold, or 'cutoff', for the assay that enables differentiation between seronegative and seropositive individuals. Here, multiple methods were explored to determine an appropriate cutoff for a multiplexed microsphere assay that is used to detect henipavirus antibody binding in fruit bat plasma. These methods included calculating multiples of 'negative' control assay values, receiver operating characteristic curve analyses, and Bayesian mixture models to assess the distribution of assay outputs for classifying seropositive and seronegative individuals within different age classes. As for any diagnostic assay, the most appropriate cutoff determination method and value selected must be made according to the aims of the study. This study is presented as an example for others where reference samples, and assays that have been characterised previously, are absent.

  15. Bacteriophage-Based Pathogen Detection

    NASA Astrophysics Data System (ADS)

    Ripp, Steven

    Considered the most abundant organism on Earth, at a population approaching 1031, bacteriophage, or phage for short, mediate interactions with myriad bacterial hosts that has for decades been exploited in phage typing schemes for signature identification of clinical, food-borne, and water-borne pathogens. With over 5,000 phage being morphologically characterized and grouped as to susceptible host, there exists an enormous cache of bacterial-specific sensors that has more recently been incorporated into novel bio-recognition assays with heightened sensitivity, specificity, and speed. These assays take many forms, ranging from straightforward visualization of labeled phage as they attach to their specific bacterial hosts to reporter phage that genetically deposit trackable signals within their bacterial hosts to the detection of progeny phage or other uniquely identifiable elements released from infected host cells. A comprehensive review of these and other phage-based detection assays, as directed towards the detection and monitoring of bacterial pathogens, will be provided in this chapter.

  16. The Autonomous Pathogen Detection System

    SciTech Connect

    Dzenitis, J M; Makarewicz, A J

    2009-01-13

    We developed, tested, and now operate a civilian biological defense capability that continuously monitors the air for biological threat agents. The Autonomous Pathogen Detection System (APDS) collects, prepares, reads, analyzes, and reports results of multiplexed immunoassays and multiplexed PCR assays using Luminex{copyright} xMAP technology and flow cytometer. The mission we conduct is particularly demanding: continuous monitoring, multiple threat agents, high sensitivity, challenging environments, and ultimately extremely low false positive rates. Here, we introduce the mission requirements and metrics, show the system engineering and analysis framework, and describe the progress to date including early development and current status.

  17. Autonomous pathogen detection system 2001

    SciTech Connect

    Langlois, R G; Wang, A; Colston, B; Masquelier, D; Jones, L; Venkateswaran, K S; Nasarabadi, S; Brown, S; Ramponi, A; Milanovich, F P

    2001-01-09

    The objective of this project is to design, fabricate and field-demonstrate a fully Autonomous Pathogen Detector (identifier) System (APDS). This will be accomplished by integrating a proven flow cytometer and real-time polymerase chain reaction (PCR) detector with sample collection, sample preparation and fluidics to provide a compact, autonomously operating instrument capable of simultaneously detecting multiple pathogens and/or toxins. The APDS will be designed to operate in fixed locations, where it continuously monitors air samples and automatically reports the presence of specific biological agents. The APDS will utilize both multiplex immuno and nucleic acid assays to provide ''quasi-orthogonal'', multiple agent detection approaches to minimize false positives and increase the reliability of identification. Technical advancements across several fronts must first be made in order to realize the full extent of the APDS. Commercialization will be accomplished through three progressive generations of instruments. The APDS is targeted for domestic applications in which (1) the public is at high risk of exposure to covert releases of bioagent such as in major subway systems and other transportation terminals, large office complexes, and convention centers; and (2) as part of a monitoring network of sensors integrated with command and control systems for wide area monitoring of urban areas and major gatherings (e.g., inaugurations, Olympics, etc.). In this latter application there is potential that a fully developed APDS could add value to Defense Department monitoring architectures.

  18. Rapid Waterborne Pathogen Detection with Mobile Electronics

    PubMed Central

    Wu, Tsung-Feng; Chen, Yu-Chen; Wang, Wei-Chung; Kucknoor, Ashwini S.; Lin, Che-Jen; Lo, Yu-Hwa; Yao, Chun-Wei; Lian, Ian

    2017-01-01

    Pathogen detection in water samples, without complex and time consuming procedures such as fluorescent-labeling or culture-based incubation, is essential to public safety. We propose an immunoagglutination-based protocol together with the microfluidic device to quantify pathogen levels directly from water samples. Utilizing ubiquitous complementary metal–oxide–semiconductor (CMOS) imagers from mobile electronics, a low-cost and one-step reaction detection protocol is developed to enable field detection for waterborne pathogens. 10 mL of pathogen-containing water samples was processed using the developed protocol including filtration enrichment, immune-reaction detection and imaging processing. The limit of detection of 10 E. coli O157:H7 cells/10 mL has been demonstrated within 10 min of turnaround time. The protocol can readily be integrated into a mobile electronics such as smartphones for rapid and reproducible field detection of waterborne pathogens. PMID:28598391

  19. Real Time Detection of Foodborne Pathogens

    NASA Astrophysics Data System (ADS)

    Velusamy, V.; Arshak, K.; Korostynka, O.; Vaseashta, Ashok; Adley, C.

    Contamination of foods by harmful bacteria by natural events or malicious intent poses a serious threat to public health and safety. This review introduces current technologies in detecting pathogens in food and foodborne illnesses. Causes of foodborne diseases and trends impacting foodborne diseases such as globalization and changes in micro-organisms, human populations, lifestyles, and climates are addressed. In addition, a review of the limitations in detecting pathogens with conventional technologies is presented. Finally, a review of nanostructured and nanomaterials based sensing technologies by pathogen, detection limits, and advantages is described.

  20. Digital PCR for detection of citrus pathogens

    USDA-ARS?s Scientific Manuscript database

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  1. Waterborne Pathogens: Detection Methods and Challenges

    PubMed Central

    Ramírez-Castillo, Flor Yazmín; Loera-Muro, Abraham; Jacques, Mario; Garneau, Philippe; Avelar-González, Francisco Javier; Harel, Josée; Guerrero-Barrera, Alma Lilián

    2015-01-01

    Waterborne pathogens and related diseases are a major public health concern worldwide, not only by the morbidity and mortality that they cause, but by the high cost that represents their prevention and treatment. These diseases are directly related to environmental deterioration and pollution. Despite the continued efforts to maintain water safety, waterborne outbreaks are still reported globally. Proper assessment of pathogens on water and water quality monitoring are key factors for decision-making regarding water distribution systems’ infrastructure, the choice of best water treatment and prevention waterborne outbreaks. Powerful, sensitive and reproducible diagnostic tools are developed to monitor pathogen contamination in water and be able to detect not only cultivable pathogens but also to detect the occurrence of viable but non-culturable microorganisms as well as the presence of pathogens on biofilms. Quantitative microbial risk assessment (QMRA) is a helpful tool to evaluate the scenarios for pathogen contamination that involve surveillance, detection methods, analysis and decision-making. This review aims to present a research outlook on waterborne outbreaks that have occurred in recent years. This review also focuses in the main molecular techniques for detection of waterborne pathogens and the use of QMRA approach to protect public health. PMID:26011827

  2. Waterborne pathogens: detection methods and challenges.

    PubMed

    Ramírez-Castillo, Flor Yazmín; Loera-Muro, Abraham; Jacques, Mario; Garneau, Philippe; Avelar-González, Francisco Javier; Harel, Josée; Guerrero-Barrera, Alma Lilián

    2015-05-21

    Waterborne pathogens and related diseases are a major public health concern worldwide, not only by the morbidity and mortality that they cause, but by the high cost that represents their prevention and treatment. These diseases are directly related to environmental deterioration and pollution. Despite the continued efforts to maintain water safety, waterborne outbreaks are still reported globally. Proper assessment of pathogens on water and water quality monitoring are key factors for decision-making regarding water distribution systems' infrastructure, the choice of best water treatment and prevention waterborne outbreaks. Powerful, sensitive and reproducible diagnostic tools are developed to monitor pathogen contamination in water and be able to detect not only cultivable pathogens but also to detect the occurrence of viable but non-culturable microorganisms as well as the presence of pathogens on biofilms. Quantitative microbial risk assessment (QMRA) is a helpful tool to evaluate the scenarios for pathogen contamination that involve surveillance, detection methods, analysis and decision-making. This review aims to present a research outlook on waterborne outbreaks that have occurred in recent years. This review also focuses in the main molecular techniques for detection of waterborne pathogens and the use of QMRA approach to protect public health.

  3. Detection of Pathogens Using AFM and SPR

    NASA Astrophysics Data System (ADS)

    Vaseashta, Ashok

    2005-03-01

    A priori detection of pathogens in food and water has become a subject of paramount importance. Several recent incidents have resulted in the government passing stringent regulations for tolerable amounts of contamination of food products. Identification and/or monitoring of bacterial contamination in food are critical. The conventional methods of pathogen detection require time-consuming steps to arrive disembark at meaningful measurement in a timely manner as the detection time exceeds the time in which perishable food recycles through the food chain distribution. The aim of this presentation is to outline surface plasmon resonance (SPR) and atomic force microscopy (AFM) as two methods for fast detect6ion of pathogens. Theoretical basis of SPR and experimental results of SPR and AFM on E. coli O157:H7 and prion are presented.

  4. Tuberculosis Detection by Giant African Pouched Rats

    ERIC Educational Resources Information Center

    Poling, Alan; Weetjens, Bart; Cox, Christophe; Beyene, Negussie; Durgin, Amy; Mahoney, Amanda

    2011-01-01

    In recent years, operant discrimination training procedures have been used to teach giant African pouched rats to detect tuberculosis (TB) in human sputum samples. This article summarizes how the rats are trained and used operationally, as well as their performance in studies published to date. Available data suggest that pouched rats, which can…

  5. Tuberculosis Detection by Giant African Pouched Rats

    ERIC Educational Resources Information Center

    Poling, Alan; Weetjens, Bart; Cox, Christophe; Beyene, Negussie; Durgin, Amy; Mahoney, Amanda

    2011-01-01

    In recent years, operant discrimination training procedures have been used to teach giant African pouched rats to detect tuberculosis (TB) in human sputum samples. This article summarizes how the rats are trained and used operationally, as well as their performance in studies published to date. Available data suggest that pouched rats, which can…

  6. Automated Methods for Multiplexed Pathogen Detection

    SciTech Connect

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However

  7. Automated methods for multiplexed pathogen detection.

    PubMed

    Straub, Timothy M; Dockendorff, Brian P; Quiñonez-Díaz, Maria D; Valdez, Catherine O; Shutthanandan, Janani I; Tarasevich, Barbara J; Grate, Jay W; Bruckner-Lea, Cynthia J

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However

  8. Development of an Autonomous Pathogen Detection System

    SciTech Connect

    Langlosi, S.; Brown, S.; Colston, B.; Jones, L.; Masquelier, D.; Meyer, P.; McBride, M.; Nasarabad, S.; Ramponi, A.J.; Venkatseswarm, K.; Milanovich, F.

    2000-10-12

    An Autonomous Pathogen Detection System (APDS) is being designed and evaluated for use in domestic counter-terrorism. The goal is a fully automated system that utilizes both flow cytometry and polymerase chain reaction (PCR) to continuously monitor the air for BW pathogens in major buildings or high profile events. A version 1 APDS system consisting of an aerosol collector, a sample preparation subsystem, and a flow cytometer for detecting the antibody-labeled target organisms has been completed and evaluated. Improved modules are under development for a version 2 APDS including a Lawrence Livermore National Laboratory-designed aerosol preconcentrator, a multiplex flow cytometer, and a flow-through PCR detector.

  9. Statistical methodology for pathogen detection.

    PubMed

    Ogliari, Paulo José; de Andrade, Dalton Francisco; Pacheco, Juliano Anderson; Franchin, Paulo Rogério; Batista, Cleide Rosana Vieira

    2007-08-01

    The main goal of the present study was to discuss the application of the McNemar test to the comparison of proportions in dependent samples. Data were analyzed from studies conducted to verify the suitability of replacing a conventional method with a new one for identifying the presence of Salmonella. It is shown that, in most situations, the McNemar test does not provide all the elements required by the microbiologist to make a final decision and that appropriate functions of the proportions need to be considered. Sample sizes suitable to guarantee a test with a high power in the detection of significant differences regarding the problem studied are obtained by simulation. Examples of functions that are of great value to the microbiologist are presented.

  10. Metagenomics for pathogen detection in public health

    PubMed Central

    2013-01-01

    Traditional pathogen detection methods in public health infectious disease surveillance rely upon the identification of agents that are already known to be associated with a particular clinical syndrome. The emerging field of metagenomics has the potential to revolutionize pathogen detection in public health laboratories by allowing the simultaneous detection of all microorganisms in a clinical sample, without a priori knowledge of their identities, through the use of next-generation DNA sequencing. A single metagenomics analysis has the potential to detect rare and novel pathogens, and to uncover the role of dysbiotic microbiomes in infectious and chronic human disease. Making use of advances in sequencing platforms and bioinformatics tools, recent studies have shown that metagenomics can even determine the whole-genome sequences of pathogens, allowing inferences about antibiotic resistance, virulence, evolution and transmission to be made. We are entering an era in which more novel infectious diseases will be identified through metagenomics-based methods than through traditional laboratory methods. The impetus is now on public health laboratories to integrate metagenomics techniques into their diagnostic arsenals. PMID:24050114

  11. DETECTION OF PATHOGENS BY FLOURESCENCE SPECTROSCOPY

    USDA-ARS?s Scientific Manuscript database

    Abstract This study was performed as a preliminary study for rapid detection of food bone pathogens by fluorescence spectroscopy. E coli, Salmonella and Campylobactor which are the most commonly found in food were used in this study. Bacteria were grown in agar plate and diluted in saline and prepa...

  12. Light Scattering based detection of food pathogens

    USDA-ARS?s Scientific Manuscript database

    The current methods for detecting foodborne pathogens are mostly destructive (i.e., samples need to be pretreated), and require time, personnel, and laboratories for analyses. Optical methods including light scattering based techniques have gained a lot of attention recently due to its their rapid a...

  13. Quantitative multiplex detection of pathogen biomarkers

    DOEpatents

    Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I.; Martinez, Jennifer; Grace, Wynne K.

    2016-02-09

    The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

  14. Quantitative multiplex detection of pathogen biomarkers

    DOEpatents

    Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I; Martinez, Jennifer; Grace, Wynne K

    2014-10-14

    The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

  15. Detection of foodborne pathogens using microarray technology

    USDA-ARS?s Scientific Manuscript database

    Assays based on the polymerase chain reaction (PCR) are now accepted methods for rapidly confirming the presence or absence of specific pathogens in foods and other types of samples. Conventional PCR requires the use of agarose gel electrophoresis to detect the PCR product; whereas, real-time PCR c...

  16. Phylogenetic origins of African and Neotropical Beauveria bassiana s.l. pathogens of the coffee berry borer, Hypothenemus hampei.

    PubMed

    Rehner, Stephen A; Posada, Francisco; Buckley, Ellen P; Infante, Francisco; Castillo, Alfredo; Vega, Fernando E

    2006-09-01

    A phylogenetic epidemiological study of Beauveria bassiana s.l. was conducted for African and Neotropical pathogens of the coffee berry borer (CBB), Hypothenemus hampei, based on inferences from two nuclear intergenic regions, EFutr and Bloc. CBB pathogens were distributed among four terminal clades, however, the majority of African and Neotropical isolates cluster in a well-supported monophyletic group, informally designated AFNEO_1. Although the relationship between African and Neotropical AFNEO_1 is unresolved, the majority of alleles detected were exclusive to either the African or the Neotropical populations. These fixed genetic differences suggest that their disjunction predates the world trade in coffee. Neotropical AFNEO_1 have a broad host range and CBB pathogens are intermixed phylogenetically with isolates from diverse indigenous insects. Several Neotropical AFNEO_1 isolates were isolated from coffee plants as epiphytes or endophytes, thus plants themselves may potentially serve as reservoirs of pathogens against their insect pests. Topological incongruence between the EFutr and Bloc phylogenies of Neotropical AFNEO_1 may signify that individuals within this population are recombining.

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

    SciTech Connect

    Jaing, C.

    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.

  18. Tracing enteric pathogen contamination in sub-Saharan African groundwater.

    PubMed

    Sorensen, J P R; Lapworth, D J; Read, D S; Nkhuwa, D C W; Bell, R A; Chibesa, M; Chirwa, M; Kabika, J; Liemisa, M; Pedley, S

    2015-12-15

    Quantitative PCR (qPCR) can rapidly screen for an array of faecally-derived bacteria, which can be employed as tracers to understand groundwater vulnerability to faecal contamination. A microbial DNA qPCR array was used to examine 45 bacterial targets, potentially relating to enteric pathogens, in 22 groundwater supplies beneath the city of Kabwe, Zambia in both the dry and subsequent wet season. Thermotolerant (faecal) coliforms, sanitary risks, and tryptophan-like fluorescence, an emerging real-time reagentless faecal indicator, were also concurrently investigated. There was evidence for the presence of enteric bacterial contamination, through the detection of species and group specific 16S rRNA gene fragments, in 72% of supplies where sufficient DNA was available for qPCR analysis. DNA from the opportunistic pathogen Citrobacter freundii was most prevalent (69% analysed samples), with Vibrio cholerae also perennially persistent in groundwater (41% analysed samples). DNA from other species such as Bifidobacterium longum and Arcobacter butzleri was more seasonally transient. Bacterial DNA markers were most common in shallow hand-dug wells in laterite/saprolite implicating rapid subsurface pathways and vulnerability to pollution at the surface. Boreholes into the underlying dolomites were also contaminated beneath the city highlighting that a laterite/saprolite overburden, as occurs across much of sub-Saharan aquifer, does not adequately protect underlying bedrock groundwater resources. Nevertheless, peri-urban boreholes all tested negative establishing there is limited subsurface lateral transport of enteric bacteria outside the city limits. Thermotolerant coliforms were present in 97% of sites contaminated with enteric bacterial DNA markers. Furthermore, tryptophan-like fluorescence was also demonstrated as an effective indicator and was in excess of 1.4μg/L in all contaminated sites. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Antibody conjugated graphene nanocomposites for pathogen detection

    NASA Astrophysics Data System (ADS)

    Sign, Chandan; Sumana, Gajjala

    2016-04-01

    Graphene oxide (GO), due to its excellent electrochemical properties and large surface area, known to be highly suitable material for biosensing application. Here, we report in situ synthesis of silver nanopaticles (AgNPs) onto the GO sheets for the electrochemical detection of Salmonella typhimurium (S.typhimurium). The GO-AgNPs composites have been deposited onto the indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. Carbodiimide coupling (EDC-NHS) has been used for the immobilization of antibodies of Salmonella typhimurium (anti-S.typhimurium) for detection of S.typhimurium. The electron microscopy and UV-visible studies reveal successful synthesis GO-AgNPs composites while FT-IR studies suggest the proper immobilization of anti-S.typhi. The cyclic voltammetry (CV) has been utilized for detection using anti-S.typhi/GOAgNPs/ITO based immunoelectrode as a function of S.typhimurium concentration. The fabricated immunosensor shows improved sensitivity of 33.04 μACFU-1mLcm-2 in a wide detection range of 101 to 106 CFUmL-1. This immunosensor may be utilized for the detection of other food borne pathogens like aflatoxin and E.coli also.

  20. Biosensors for the Detection of Food Pathogens

    PubMed Central

    Poltronieri, Palmiro; Mezzolla, Valeria; Primiceri, Elisabetta; Maruccio, Giuseppe

    2014-01-01

    Food pathogens frequently cause foodborne diseases. There is a need to rapidly identify the source of the bacteria in order to contain their spread and epidemics. A pre-enrichment culture or a direct culture on agar plate are standard microbiological methods. In this review, we present an update on alternative molecular methods to nucleic acid-based detection for species identification. Biosensor-based methods rely on the recognition of antigen targets or receptors by antibodies, aptamers or high-affinity ligands. The captured antigens may be then directly or indirectly detected through an antibody or high-affinity and high-specificity recognition molecule. Various different detection methods are discussed, from label-free sensors and immunosensors to fluorescence-based ones. Each method shows advantages and disadvantages in terms of equipment, sensitivity, simplicity and cost-effectiveness. Finally, lab-on-a-chip (LOC) devices are introduced briefly, with the potential to be fast, sensitive and useful for on-site bacteria detection in food processing laboratories to check potential contamination by sample monitoring combined with a rapid pre-enrichment step. PMID:28234334

  1. APDS: The Autonomous Pathogen Detection System

    SciTech Connect

    Hindson, B; Makarewicz, A; Setlur, U; Henderer, B; McBride, M; Dzenitis, J

    2004-10-04

    We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system was developed to provide early warning to civilians in the event of a bioterrorism incident and can be used at high profile events for short-term, intensive monitoring or in major public buildings or transportation nodes for long-term monitoring. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic-acid based polymerase chain reaction (PCR) amplification and detection. Highly multiplexed antibody-based and duplex nucleic acid-based assays are combined to reduce false positives to a very low level, lower reagent costs, and significantly expand the detection capabilities of this biosensor. This article provides an overview of the current design and operation of the APDS. Certain sub-components of the ADPS are described in detail, including the aerosol collector, the automated sample preparation module that performs multiplexed immunoassays with confirmatory PCR, and the data monitoring and communications system. Data obtained from an APDS that operated continuously for seven days in a major U.S. transportation hub is reported.

  2. Field application of pathogen detection technologies

    SciTech Connect

    Straub, Tim M.; Call, Douglas R.; Bruckner-Lea, Cindy J.; Colburn, Heather A.; Baird, Cheryl L.; Bartholomew, Rachel A.; Ozanich, Richard M.; Jarman, Kristin H.

    2016-06-29

    Over the last 10 years there has been a significant increase in commercial products designed for field-based detection of microbial pathogens. This is due, in part, to the anthrax attacks in the United States in 2001, and the need for first responders to quickly identify the composition of suspected white powders and other potential biothreats. Demand for rapid detection is also driven by the need to ensure safe food, water, and environmental systems. From a technology perspective, rapid identification methods have largely capitalized on PCR and other molecular recognition techniques that can be deployed as robust field instrumentation. Examples of the relevant needs include the ability to: 1) declare a water distribution system free of microbial pathogens after a pipe/main break repair; 2) assess risks of contamination such as when produce production and processing plants are located near concentrated animal feeing operations; 3) evaluate the safety of ready-to-eat products; 4) determine the extent of potential serious disease outbreaks in remote and/or disaster stricken areas where access to clinical laboratories is not an immediate option; and 5) quickly assess credible biological terrorism events. Many of the principles underlying rapid detection methods are derived from methods for environmental microbiology, but there is a dearth of literature describing and evaluating field-based detection systems. Thus, the aims of this chapter are to: 1) summarize the different kinds of commercially available sampling kits and field-based biological detectors; 2) highlight some of the continued challenges of sample preparation to stimulate new research towards minimizing the impact of inhibitors on PCR-based detection systems; 3) describe our general rationale and statistically-based approach for instrument evaluation; 4) provide statistical and spatial guidelines for developing valid sampling plans; and 5) summarize some current needs and emerging technologies. This

  3. Rapid Detection of Human Pathogenic Orthobunyaviruses

    PubMed Central

    Weidmann, Manfred; Rudaz, Veronique; Nunes, Marcio R. T.; Vasconcelos, Pedro F. C.; Hufert, Frank T.

    2003-01-01

    Modern detection and identification tools can help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging diseases. We developed highly sensitive one-step TaqMan reverse transcription-PCR assays with sensitivities ranging from 104 to 101 molecules for 11 human pathogens of the orthobunyaviruses. We compared the performances of these assays on three currently available cyclers (ABI-PRISM 7700, LightCycler, and SmartCycler). The assay for Oropouche virus (OROV) was tested using sera collected from days 1 to 5 after onset of OROV disease and was found to be greatly superior to an established nested PCR system. A mean copy number of 1.31 × 107 OROV RNA/ml of serum was detected. Diagnostic RNA detection can be used as early as day 1 after onset of OROV disease. The use of a mobile SmartCycler and a hands-on time of less than 3 h could help to intensify outbreak surveillance and control, especially in field studies. PMID:12843078

  4. Autonomous system for pathogen detection and identification

    SciTech Connect

    Belgrader, P.; Benett, W.; Bergman, W.; Langlois, R.; Mariella, R.; Milanovich, F.; Miles, R.; Venkateswaran, K.; Long, G.; Nelson, W.

    1998-09-24

    This purpose of this project is to build a prototype instrument that will, running unattended, detect, identify, and quantify BW agents. In order to accomplish this, we have chosen to start with the world' s leading, proven, assays for pathogens: surface-molecular recognition assays, such as antibody-based assays, implemented on a high-performance, identification (ID)-capable flow cytometer, and the polymerase chain reaction (PCR) for nucleic-acid based assays. With these assays, we must integrate the capability to: l collect samples from aerosols, water, or surfaces; l perform sample preparation prior to the assays; l incubate the prepared samples, if necessary, for a period of time; l transport the prepared, incubated samples to the assays; l perform the assays; l interpret and report the results of the assays. Issues such as reliability, sensitivity and accuracy, quantity of consumables, maintenance schedule, etc. must be addressed satisfactorily to the end user. The highest possible sensitivity and specificity of the assay must be combined with no false alarms. Today, we have assays that can, in under 30 minutes, detect and identify simulants for BW agents at concentrations of a few hundred colony-forming units per ml of solution. If the bio-aerosol sampler of this system collects 1000 Ymin and concentrates the respirable particles into 1 ml of solution with 70% processing efficiency over a period of 5 minutes, then this translates to a detection/ID capability of under 0.1 agent-containing particle/liter of air.

  5. Filter-based pathogen enrichment technology for detection of multiple viable foodborne pathogens in 1 day.

    PubMed

    Murakami, Taku

    2012-09-01

    Conventional foodborne pathogen assays currently used in the food industry often require long culture enrichments to increase pathogen levels so they can be detected. Even using sensitive real-time PCR assays, culture enrichment at least overnight is necessary especially for detection of pathogens with slow growth rates such as Listeria monocytogenes. To eliminate this cumbersome enrichment step and detect minute amounts of pathogens within 1 day, filter-based pathogen enrichment technology was developed utilizing a unique combination of glass fiber depth filter and porous filter aid materials to efficiently separate pathogens from food homogenates and avoid filter clogging by food particles. After pathogen immobilization in depth filters, only viable pathogens were selectively collected in a small volume of growth medium via microbial multiplication and migration; nonviable pathogens remained inside the filters. By assaying viable pathogens using real-time PCRs, multiple species of foodborne pathogens were detected, including L. monocytogenes, Salmonella enterica, and Escherichia coli O157:H7, at around 1 CFU/ml or 1 CFU/g in various food samples. This filter-based pathogen enrichment technology is a unique bacterial enrichment alternative to the conventional culture enrichment step and can significantly shorten the time necessary to obtain assay results.

  6. The Autonomous Pathogen Detection System (APDS)

    SciTech Connect

    Morris, J; Dzenitis, J

    2004-09-22

    Shaped like a mailbox on wheels, it's been called a bioterrorism ''smoke detector.'' It can be found in transportation hubs such as airports and subways, and it may be coming to a location near you. Formally known as the Autonomous Pathogen Detection System, or APDS, this latest tool in the war on bioterrorism was developed at Lawrence Livermore National Laboratory to continuously sniff the air for airborne pathogens and toxins such as anthrax or plague. The APDS is the modern day equivalent of the canaries miners took underground with them to test for deadly carbon dioxide gas. But this canary can test for numerous bacteria, viruses, and toxins simultaneously, report results every hour, and confirm positive samples and guard against false positive results by using two different tests. The fully automated system collects and prepares air samples around the clock, does the analysis, and interprets the results. It requires no servicing or human intervention for an entire week. Unlike its feathered counterpart, when an APDS unit encounters something deadly in the air, that's when it begins singing, quietly. The APDS unit transmits a silent alert and sends detailed data to public health authorities, who can order evacuation and begin treatment of anyone exposed to toxic or biological agents. It is the latest in a series of biodefense detectors developed at DOE/NNSA national laboratories. The manual predecessor to APDS, called BASIS (for Biological Aerosol Sentry and Information System), was developed jointly by Los Alamos and Lawrence Livermore national laboratories. That system was modified to become BioWatch, the Department of Homeland Security's biological urban monitoring program. A related laboratory instrument, the Handheld Advanced Nucleic Acid Analyzer (HANAA), was first tested successfully at LLNL in September 1997. Successful partnering with private industry has been a key factor in the rapid advancement and deployment of biodefense instruments such as these

  7. Microbiology: Detection of Bacterial Pathogens and Their Occurrence.

    ERIC Educational Resources Information Center

    Reasoner, Donald J.

    1978-01-01

    Presents a literature review of bacterial pathogens that are related to water pollution, covering publications from 1976-77. This review includes: (1) bacterial pathogens in animals; and (2) detection and identification of waterborne bacterial pathogens. A list of 129 references is also presented. (HM)

  8. Aptamer-Based Technologies in Foodborne Pathogen Detection.

    PubMed

    Teng, Jun; Yuan, Fang; Ye, Yingwang; Zheng, Lei; Yao, Li; Xue, Feng; Chen, Wei; Li, Baoguang

    2016-01-01

    Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX); and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make the accurate assessments on the risks of infections (humans and animals) or contaminations (foods and other commodities) caused by various pathogens. This article reviews the development in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development in aptamer-based biosensors including optical biosensors for multiple pathogen detection by multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors and lateral chromatography test strips, and their applications in pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening remain to be overcome.

  9. Aptamer-Based Technologies in Foodborne Pathogen Detection

    PubMed Central

    Teng, Jun; Yuan, Fang; Ye, Yingwang; Zheng, Lei; Yao, Li; Xue, Feng; Chen, Wei; Li, Baoguang

    2016-01-01

    Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX); and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make the accurate assessments on the risks of infections (humans and animals) or contaminations (foods and other commodities) caused by various pathogens. This article reviews the development in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development in aptamer-based biosensors including optical biosensors for multiple pathogen detection by multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors and lateral chromatography test strips, and their applications in pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening remain to be overcome. PMID:27672383

  10. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    PubMed

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Oplatowska, Michalina; Gajanandana, Oraprapai; Grant, Irene R; Karoonuthaisiri, Nitsara; Elliott, Christopher T

    2013-01-01

    Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  11. [Application of pyrosequencing in detection of common pathogens in sepsis].

    PubMed

    Hu, Ziyou; Han, Hui; Zeng, Yong; Wu, Bingyi

    2013-07-01

    To apply pyrosequencing technique in the detection of the common pathogens in sepsis. The primers for amplification and sequencing in pyrosequencing were designed according to alignment of the bacterial 16S rRNA sequence. Bacterial genomic DNA was extracted for pyrosequencing, and the pathogen species were determined according to the sequencing data obtained. Pyrosequencing effectively yielded the sequencing data of the 28 bp sequences of the pathogens and clearly distinguished the pathogen species of Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria meningitides, and Salmonella, but failed to distinguish Staphylococcus epidermidis from Staphylococcus aureus. Pyrosequencing technique can effectively distinguish the common pathogens in sepsis at the species level.

  12. Impedance measurements for detecting pathogens attached to antibodies

    DOEpatents

    Miles, Robin R.; Venkateswaran, Kodumudi S.; Fuller, Christopher K.

    2004-12-28

    The use of impedance measurements to detect the presence of pathogens attached to antibody-coated beads. In a fluidic device antibodies are immobilized on a surface of a patterned interdigitated electrode. Pathogens in a sample fluid streaming past the electrode attach to the immobilized antibodies, which produces a change in impedance between two adjacent electrodes, which impedance change is measured and used to detect the presence of a pathogen. To amplify the signal, beads coated with antibodies are introduced and the beads would stick to the pathogen causing a greater change in impedance between the two adjacent electrodes.

  13. Method for detecting pathogens attached to specific antibodies

    DOEpatents

    Miles, Robin R.; Venkateswaran, Kodumudi S.; Fuller, Christopher K.

    2005-01-25

    The use of impedance measurements to detect the presence of pathogens attached to antibody-coated beads. In a fluidic device antibodies are immobilized on a surface of a patterned interdigitated electrode. Pathogens in a sample fluid streaming past the electrode attach to the immobilized antibodies, which produces a change in impedance between two adjacent electrodes, which impedance change is measured and used to detect the presence of a pathogen. To amplify the signal, beads coated with antibodies are introduced and the beads would stick to the pathogen causing a greater change in impedance between the two adjacent electrodes.

  14. Contact with Domestic Dogs Increases Pathogen Exposure in Endangered African Wild Dogs (Lycaon pictus)

    PubMed Central

    Woodroffe, Rosie; Prager, Katherine C.; Conrad, Patricia A.; Dubovi, Edward J.; Mazet, Jonna A. K.

    2012-01-01

    Background Infectious diseases have contributed to the decline and local extinction of several wildlife species, including African wild dogs (Lycaon pictus). Mitigating such disease threats is challenging, partly because uncertainty about disease dynamics makes it difficult to identify the best management approaches. Serious impacts on susceptible populations most frequently occur when generalist pathogens are maintained within populations of abundant (often domestic) “reservoir” hosts, and spill over into less abundant host species. If this is the case, disease control directed at the reservoir host might be most appropriate. However, pathogen transmission within threatened host populations may also be important, and may not be controllable by managing another host species. Methodology/Principal Findings We investigated interspecific and intraspecific transmission routes, by comparing African wild dogs' exposure to six canine pathogens with behavioural measures of their opportunities for contact with domestic dogs and with other wild dogs. Domestic dog contact was associated with exposure to canine parvovirus, Ehrlichia canis, Neospora caninum and perhaps rabies virus, but not with exposure to canine distemper virus or canine coronavirus. Contact with other wild dogs appeared not to increase the risk of exposure to any of the pathogens. Conclusions/Significance These findings, combined with other data, suggest that management directed at domestic dogs might help to protect wild dog populations from rabies virus, but not from canine distemper virus. However, further analyses are needed to determine the management approaches – including no intervention – which are most appropriate for each pathogen. PMID:22238695

  15. Detection of bacterial pathogens including potential new species in human head lice from Mali

    PubMed Central

    Amanzougaghene, Nadia; Fenollar, Florence; Sangaré, Abdoul Karim; Sissoko, Mahamadou S.; Doumbo, Ogobara K.; Raoult, Didier

    2017-01-01

    In poor African countries, where no medical and biological facilities are available, the identification of potential emerging pathogens of concern at an early stage is challenging. Head lice, Pediculus humanus capitis, have a short life, feed only on human blood and do not transmit pathogens to their progeny. They are, therefore, a perfect tool for the xenodiagnosis of current or recent human infection. This study assessed the occurrence of bacterial pathogens from head lice collected in two rural villages from Mali, where a high frequency of head lice infestation had previously been reported, using molecular methods. Results show that all 600 head lice, collected from 117 individuals, belonged to clade E, specific to West Africa. Bartonella quintana, the causative agent of trench fever, was identified in three of the 600 (0.5%) head lice studied. Our study also shows, for the first time, the presence of the DNA of two pathogenic bacteria, namely Coxiella burnetii (5.1%) and Rickettsia aeschlimannii (0.6%), detected in human head lice, as well as the DNA of potential new species from the Anaplasma and Ehrlichia genera of unknown pathogenicity. The finding of several Malian head lice infected with B. quintana, C. burnetii, R. aeschlimannii, Anaplasma and Ehrlichia is alarming and highlights the need for active survey programs to define the public health consequences of the detection of these emerging bacterial pathogens in human head lice. PMID:28931077

  16. Bio-Functional Au/Si Nanorods for Pathogen Detection

    USDA-ARS?s Scientific Manuscript database

    Technical Abstract Nanotechnology applications for food safety and biosecurity, especially development of nanoscale sensors for foodborne pathogen measurement are emerging. A novel bio-functional nanosensor for Salmonella detection was developed using hetero-nanorods. The silica nanorods were fabr...

  17. Advances in rapid detection methods for foodborne pathogens.

    PubMed

    Zhao, Xihong; Lin, Chii-Wann; Wang, Jun; Oh, Deog Hwan

    2014-03-28

    Food safety is increasingly becoming an important public health issue, as foodborne diseases present a widespread and growing public health problem in both developed and developing countries. The rapid and precise monitoring and detection of foodborne pathogens are some of the most effective ways to control and prevent human foodborne infections. Traditional microbiological detection and identification methods for foodborne pathogens are well known to be time consuming and laborious as they are increasingly being perceived as insufficient to meet the demands of rapid food testing. Recently, various kinds of rapid detection, identification, and monitoring methods have been developed for foodborne pathogens, including nucleic-acid-based methods, immunological methods, and biosensor-based methods, etc. This article reviews the principles, characteristics, and applications of recent rapid detection methods for foodborne pathogens.

  18. Gene expression analysis of whole blood RNA from pigs infected with low and high pathogenic African swine fever viruses

    DOE PAGES

    Jaing, Crystal; Rowland, Raymond R. R.; Allen, Jonathan E.; ...

    2017-08-31

    African swine fever virus (ASFV) is a macrophage-tropic virus responsible for ASF, a transboundary disease that threatens swine production world-wide. Since there are no vaccines available to control ASF after an outbreak, obtaining an understanding of the virus-host interaction is important for developing new intervention strategies. In this study, a whole transcriptomic RNA-Seq method was used to characterize differentially expressed genes in pigs infected with a low pathogenic ASFV isolate, OUR T88/3 (OURT), or the highly pathogenic Georgia 2007/1 (GRG). After infection, pigs infected with OURT showed no or few clinical signs; whereas, GRG produced clinical signs consistent with acutemore » ASF. RNA-Seq detected the expression of ASFV genes from the whole blood of the GRG, but not the OURT pigs, consistent with the pathotypes of these strains and the replication of GRG in circulating monocytes. Even though GRG and OURT possess different pathogenic properties, there was significant overlap in the most upregulated host genes. A small number of differentially expressed microRNAs were also detected in GRG and OURT pigs. These data confirm previous studies describing the response of macrophages and lymphocytes to ASFV infection, as well as reveal unique gene pathways upregulated in response to infection with GRG.« less

  19. Towards a unified system for detecting waterborne pathogens

    SciTech Connect

    Straub, Tim M.; Chandler, Darrell P.

    2003-03-01

    Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large volume water samples to detect low target concentrations of certain pathogen groups, removing co-concentrated inhibitors from the sample, and standardizing a culture-independent endpoint detection method. Integrating the disparate technologies into a single, universal, simple method and detection system would represent a significant advance in public health and microbiological water quality analysis. Recent advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. This review discusses some of the challenges in developing a universal pathogen detection method, current technology that may be employed to overcome these challenges, and the remaining needs for developing an integrated pathogen detection and monitoring system for source or finished water.

  20. Using DNA Microarrays to Detect Multiple Pathogen Threats in Water.

    SciTech Connect

    Straub, Tim M.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Call, Douglas R.; Chandler, Darrell P.

    2004-06-01

    Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large volume water samples to detect low target concentrations of certain pathogen groups, removing co-concentrated inhibitors from the sample, and standardizing a culture-independent endpoint detection method. Integrating the disparate technologies into a single, universal, simple method and detection system would represent a significant advance in public health and microbiological water quality analysis. Recent advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. This review discusses some of the challenges in developing a universal pathogen detection method, current technology that may be employed to overcome these challenges, and the remaining needs for developing an integrated pathogen detection and monitoring system for source or finished water.

  1. A Review of Membrane-Based Biosensors for Pathogen Detection

    PubMed Central

    van den Hurk, Remko; Evoy, Stephane

    2015-01-01

    Biosensors are of increasing interest for the detection of bacterial pathogens in many applications such as human, animal and plant health, as well as food and water safety. Membranes and membrane-like structures have been integral part of several pathogen detection platforms. Such structures may serve as simple mechanical support, function as a part of the transduction mechanism, may be used to filter out or concentrate pathogens, and may be engineered to specifically house active proteins. This review focuses on membrane materials, their associated biosensing applications, chemical linking procedures, and transduction mechanisms. The sensitivity of membrane biosensors is discussed, and the state of the field is evaluated and summarized. PMID:26083229

  2. Parasitic, bacterial, and viral enteric pathogens associated with diarrhea in the Central African Republic.

    PubMed Central

    Georges, M C; Wachsmuth, I K; Meunier, D M; Nebout, N; Didier, F; Siopathis, M R; Georges, A J

    1984-01-01

    A total of 1,197 diarrheic children less than 15 years old were investigated for parasitic, bacterial, and viral enteropathogens from March 1981 through February 1982 in the Central African Republic. One or more pathogens were identified from 49.4% of the patients. Rotavirus was the most frequently identified pathogen among children less than 18 months old. Enteropathogenic Escherichia coli was the second most frequently isolated pathogen (12.1%) in children less than 2 years of age. Campylobacter jejuni was also isolated frequently from diarrheic children less than 5 years of age (10.9%). Entamoeba histolytica was identified in very young children and was found to be the most frequent enteropathogen associated with diarrhea in children over the age of 2 years. Enterotoxigenic Escherichia coli was rarely isolated (ca. 2%). There was a peak in the incidence of rotavirus during the dry season and in the incidence of Campylobacter jejuni during the rainy season. PMID:6330161

  3. Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

    PubMed Central

    Pava-Ripoll, Monica; Pearson, Rachel E.G.; Miller, Amy K.; Ziobro, George C.

    2015-01-01

    There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how

  4. Detection of foodborne bacterial pathogens from individual filth flies.

    PubMed

    Pava-Ripoll, Monica; Pearson, Rachel E G; Miller, Amy K; Ziobro, George C

    2015-02-13

    There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how

  5. Resequencing Pathogen Microarray (RPM) for prospective detection and identification of emergent pathogen strains and variants

    NASA Astrophysics Data System (ADS)

    Tibbetts, Clark; Lichanska, Agnieszka M.; Borsuk, Lisa A.; Weslowski, Brian; Morris, Leah M.; Lorence, Matthew C.; Schafer, Klaus O.; Campos, Joseph; Sene, Mohamadou; Myers, Christopher A.; Faix, Dennis; Blair, Patrick J.; Brown, Jason; Metzgar, David

    2010-04-01

    High-density resequencing microarrays support simultaneous detection and identification of multiple viral and bacterial pathogens. Because detection and identification using RPM is based upon multiple specimen-specific target pathogen gene sequences generated in the individual test, the test results enable both a differential diagnostic analysis and epidemiological tracking of detected pathogen strains and variants from one specimen to the next. The RPM assay enables detection and identification of pathogen sequences that share as little as 80% sequence similarity to prototype target gene sequences represented as detector tiles on the array. This capability enables the RPM to detect and identify previously unknown strains and variants of a detected pathogen, as in sentinel cases associated with an infectious disease outbreak. We illustrate this capability using assay results from testing influenza A virus vaccines configured with strains that were first defined years after the design of the RPM microarray. Results are also presented from RPM-Flu testing of three specimens independently confirmed to the positive for the 2009 Novel H1N1 outbreak strain of influenza virus.

  6. xMAP Technology: Applications in Detection of Pathogens

    PubMed Central

    Reslova, Nikol; Michna, Veronika; Kasny, Martin; Mikel, Pavel; Kralik, Petr

    2017-01-01

    xMAP technology is applicable for high-throughput, multiplex and simultaneous detection of different analytes within a single complex sample. xMAP multiplex assays are currently available in various nucleic acid and immunoassay formats, enabling simultaneous detection and typing of pathogenic viruses, bacteria, parasites and fungi and also antigen or antibody interception. As an open architecture platform, the xMAP technology is beneficial to end users and therefore it is used in various pharmaceutical, clinical and research laboratories. The main aim of this review is to summarize the latest findings and applications in the field of pathogen detection using microsphere-based multiplex assays. PMID:28179899

  7. Nano/Micro and Spectroscopic Approaches to Food Pathogen Detection

    NASA Astrophysics Data System (ADS)

    Cho, Il-Hoon; Radadia, Adarsh D.; Farrokhzad, Khashayar; Ximenes, Eduardo; Bae, Euiwon; Singh, Atul K.; Oliver, Haley; Ladisch, Michael; Bhunia, Arun; Applegate, Bruce; Mauer, Lisa; Bashir, Rashid; Irudayaraj, Joseph

    2014-06-01

    Despite continuing research efforts, timely and simple pathogen detection with a high degree of sensitivity and specificity remains an elusive goal. Given the recent explosion of sensor technologies, significant strides have been made in addressing the various nuances of this important global challenge that affects not only the food industry but also human health. In this review, we provide a summary of the various ongoing efforts in pathogen detection and sample preparation in areas related to Fourier transform infrared and Raman spectroscopy, light scattering, phage display, micro/nanodevices, and nanoparticle biosensors. We also discuss the advantages and potential limitations of the detection methods and suggest next steps for further consideration.

  8. DNA extraction protocol for rapid PCR detection of pathogenic bacteria

    USDA-ARS?s Scientific Manuscript database

    Virtually all current assays for foodborne pathogens, including PCR assays, are conducted after lengthy cultural enrichment of the sample to increase the concentration of the target organism. This delays detection by many hours, prevents quantitation, and limits the ability to detect multiple organ...

  9. DNA chip based sensor for amperometric detection of infectious pathogens.

    PubMed

    Singh, Swati; Kaushal, Ankur; Khare, Shashi; Kumar, Ashok

    2017-10-01

    Several infectious pathogens are found in human whose detection is essential for rapid cure of diseases. The most commonly found pathogen in human is Streptococcus pyogenes which leads to a wide range of infections from mild pharyngitis to rheumatic heart disease. An ultrasensitive DNA chip based sensor was developed for quick identification of pathogen S. pyogenes from patient throat swab samples. The amperometric response was measured after hybridization of specific probe with single stranded genomic DNA (ssG-DNA) from the patient samples. The DNA chip was characterized by FTIR, SEM and validated with suspected patient real samples. The sensitivity of the DNA chip based sensor was found 951.34(μA/cm(2))/ng DNA and lower limit of detection (LOD) was 130fg/6μL samples. The DNA chip based sensor is highly specific and takes only 30min for identification of specific pathogen. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Reassortant low-pathogenic avian influenza H5N2 viruses in African wild birds.

    PubMed

    Snoeck, Chantal J; Adeyanju, Adeniyi T; De Landtsheer, Sébastien; Ottosson, Ulf; Manu, Shiiwua; Hagemeijer, Ward; Mundkur, Taej; Muller, Claude P

    2011-05-01

    To investigate the presence and persistence of avian influenza virus in African birds, we monitored avian influenza in wild and domestic birds in two different regions in Nigeria. We found low-pathogenic avian influenza (LPAI) H5N2 viruses in three spur-winged geese (Plectropterus gambensis) in the Hadejia-Nguru wetlands. Phylogenetic analyses revealed that all of the genes, except the non-structural (NS) genes, of the LPAI H5N2 viruses were more closely related to genes recently found in wild and domestic birds in Europe. The NS genes formed a sister group to South African and Zambian NS genes. This suggested that the Nigerian LPAI H5N2 viruses found in wild birds were reassortants exhibiting an NS gene that circulated for at least 7 years in African birds and is part of the African influenza gene pool, and genes that were more recently introduced into Africa from Eurasia, most probably by intercontinental migratory birds. Interestingly the haemagglutinin and neuraminidase genes formed a sister branch to highly pathogenic avian influenza (HPAI) H5N2 strains found in the same wild bird species in the same wetland only 1 year earlier. However, they were not the closest known relatives of each other, suggesting that their presence in the wetland resulted from two separate introductions. The presence of LPAI H5N2 in wild birds in the Hadejia-Nguru wetlands, where wild birds and poultry occasionally mix, provides ample opportunity for infection across species boundaries, with the potential risk of generating HPAI viruses after extensive circulation in poultry.

  11. Methods for detecting pathogens in the beef food chain: detecting particular pathogens

    USDA-ARS?s Scientific Manuscript database

    The main food-borne pathogens of concern in the beef food chain are Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp.; however, the presence of other pathogens, including Listeria monocytogenes, Campylobacter spp., Clostridium spp., Bacillus cereus, and Mycobacterium avium subsp. par...

  12. Waveguide-based biosensors for pathogen detection.

    PubMed

    Mukundan, Harshini; Anderson, Aaron S; Grace, W Kevin; Grace, Karen M; Hartman, Nile; Martinez, Jennifer S; Swanson, Basil I

    2009-01-01

    Optical phenomena such as fluorescence, phosphorescence, polarization, interference and non-linearity have been extensively used for biosensing applications. Optical waveguides (both planar and fiber-optic) are comprised of a material with high permittivity/high refractive index surrounded on all sides by materials with lower refractive indices, such as a substrate and the media to be sensed. This arrangement allows coupled light to propagate through the high refractive index waveguide by total internal reflection and generates an electromagnetic wave-the evanescent field-whose amplitude decreases exponentially as the distance from the surface increases. Excitation of fluorophores within the evanescent wave allows for sensitive detection while minimizing background fluorescence from complex, "dirty" biological samples. In this review, we will describe the basic principles, advantages and disadvantages of planar optical waveguide-based biodetection technologies. This discussion will include already commercialized technologies (e.g., Corning's EPIC(®) Ô, SRU Biosystems' BIND(™), Zeptosense(®), etc.) and new technologies that are under research and development. We will also review differing assay approaches for the detection of various biomolecules, as well as the thin-film coatings that are often required for waveguide functionalization and effective detection. Finally, we will discuss reverse-symmetry waveguides, resonant waveguide grating sensors and metal-clad leaky waveguides as alternative signal transducers in optical biosensing.

  13. Waveguide-Based Biosensors for Pathogen Detection

    PubMed Central

    Mukundan, Harshini; Anderson, Aaron S.; Grace, W. Kevin; Grace, Karen M.; Hartman, Nile; Martinez, Jennifer S.; Swanson, Basil I.

    2009-01-01

    Optical phenomena such as fluorescence, phosphorescence, polarization, interference and non-linearity have been extensively used for biosensing applications. Optical waveguides (both planar and fiber-optic) are comprised of a material with high permittivity/high refractive index surrounded on all sides by materials with lower refractive indices, such as a substrate and the media to be sensed. This arrangement allows coupled light to propagate through the high refractive index waveguide by total internal reflection and generates an electromagnetic wave—the evanescent field—whose amplitude decreases exponentially as the distance from the surface increases. Excitation of fluorophores within the evanescent wave allows for sensitive detection while minimizing background fluorescence from complex, “dirty” biological samples. In this review, we will describe the basic principles, advantages and disadvantages of planar optical waveguide-based biodetection technologies. This discussion will include already commercialized technologies (e.g., Corning’s EPIC® Ô, SRU Biosystems’ BIND™, Zeptosense®, etc.) and new technologies that are under research and development. We will also review differing assay approaches for the detection of various biomolecules, as well as the thin-film coatings that are often required for waveguide functionalization and effective detection. Finally, we will discuss reverse-symmetry waveguides, resonant waveguide grating sensors and metal-clad leaky waveguides as alternative signal transducers in optical biosensing. PMID:22346727

  14. Metagenomic approaches to disclose disease-associated pathogens: detection of viral pathogens in honeybees.

    PubMed

    Granberg, Fredrik; Karlsson, Oskar E; Belák, Sándor

    2015-01-01

    Metagenomic approaches have become invaluable for culture-independent and sequence-independent detection and characterization of disease-associated pathogens. Here, the sequential steps from sampling to verification of results are described for a metagenomic-based approach to detect potential pathogens in honeybees. The pre-sequencing steps are given in detail, but due to the rapid development of sequencing technologies, all platform-specific procedures, as well as subsequent bioinformatics analysis, are more generally described. It should also be noted that this approach could, with minor modifications, be adapted for other organisms and sample matrices.

  15. Point detection of bacterial and viral pathogens using oral samples

    NASA Astrophysics Data System (ADS)

    Malamud, Daniel

    2008-04-01

    Oral samples, including saliva, offer an attractive alternative to serum or urine for diagnostic testing. This is particularly true for point-of-use detection systems. The various types of oral samples that have been reported in the literature are presented here along with the wide variety of analytes that have been measured in saliva and other oral samples. The paper focuses on utilizing point-detection of infectious disease agents, and presents work from our group on a rapid test for multiple bacterial and viral pathogens by monitoring a series of targets. It is thus possible in a single oral sample to identify multiple pathogens based on specific antigens, nucleic acids, and host antibodies to those pathogens. The value of such a technology for detecting agents of bioterrorism at remote sites is discussed.

  16. Innovative applications of bacteriophages in rapid detection and identification of foodborne pathogens

    USDA-ARS?s Scientific Manuscript database

    Relative to traditional microbiological approaches, biosensors are a rapid method for foodborne bacterial pathogen detection. Biosensors function by detecting the interaction of the target pathogen, or pathogen derived molecule, with a biological recognition component which must have sufficient aff...

  17. Advances and Challenges in Viability Detection of Foodborne Pathogens

    PubMed Central

    Zeng, Dexin; Chen, Zi; Jiang, Yuan; Xue, Feng; Li, Baoguang

    2016-01-01

    Foodborne outbreaks are a serious public health and food safety concern worldwide. There is a great demand for rapid, sensitive, specific, and accurate methods to detect microbial pathogens in foods. Conventional methods based on cultivation of pathogens have been the gold standard protocols; however, they take up to a week to complete. Molecular assays such as polymerase chain reaction (PCR), sequencing, microarray technologies have been widely used in detection of foodborne pathogens. Among molecular assays, PCR technology [conventional and real-time PCR (qPCR)] is most commonly used in the foodborne pathogen detection because of its high sensitivity and specificity. However, a major drawback of PCR is its inability to differentiate the DNA from dead and viable cells, and this is a critical factor for the food industry, regulatory agencies and the consumer. To remedy this shortcoming, researchers have used biological dyes such as ethidium monoazide and propidium monoazide (PMA) to pretreat samples before DNA extraction to intercalate the DNA of dead cells in food samples, and then proceed with regular DNA preparation and qPCR. By combining PMA treatment with qPCR (PMA-qPCR), scientists have applied this technology to detect viable cells of various bacterial pathogens in foods. The incorporation of PMA into PCR-based assays for viability detection of pathogens in foods has increased significantly in the last decade. On the other hand, some downsides with this approach have been noted, particularly to achieve complete suppression of signal of DNA from the dead cells present in some particular food matrix. Nowadays, there is a tendency of more and more researchers adapting this approach for viability detection; and a few commercial kits based on PMA are available in the market. As time goes on, more scientists apply this approach to a broader range of pathogen detections, this viability approach (PMA or other chemicals such as platinum compound) may eventually

  18. Advances and Challenges in Viability Detection of Foodborne Pathogens.

    PubMed

    Zeng, Dexin; Chen, Zi; Jiang, Yuan; Xue, Feng; Li, Baoguang

    2016-01-01

    Foodborne outbreaks are a serious public health and food safety concern worldwide. There is a great demand for rapid, sensitive, specific, and accurate methods to detect microbial pathogens in foods. Conventional methods based on cultivation of pathogens have been the gold standard protocols; however, they take up to a week to complete. Molecular assays such as polymerase chain reaction (PCR), sequencing, microarray technologies have been widely used in detection of foodborne pathogens. Among molecular assays, PCR technology [conventional and real-time PCR (qPCR)] is most commonly used in the foodborne pathogen detection because of its high sensitivity and specificity. However, a major drawback of PCR is its inability to differentiate the DNA from dead and viable cells, and this is a critical factor for the food industry, regulatory agencies and the consumer. To remedy this shortcoming, researchers have used biological dyes such as ethidium monoazide and propidium monoazide (PMA) to pretreat samples before DNA extraction to intercalate the DNA of dead cells in food samples, and then proceed with regular DNA preparation and qPCR. By combining PMA treatment with qPCR (PMA-qPCR), scientists have applied this technology to detect viable cells of various bacterial pathogens in foods. The incorporation of PMA into PCR-based assays for viability detection of pathogens in foods has increased significantly in the last decade. On the other hand, some downsides with this approach have been noted, particularly to achieve complete suppression of signal of DNA from the dead cells present in some particular food matrix. Nowadays, there is a tendency of more and more researchers adapting this approach for viability detection; and a few commercial kits based on PMA are available in the market. As time goes on, more scientists apply this approach to a broader range of pathogen detections, this viability approach (PMA or other chemicals such as platinum compound) may eventually

  19. A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

    PubMed

    Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

    2014-01-01

    Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

  20. Automated RNA Extraction and Purification for Multiplexed Pathogen Detection

    SciTech Connect

    Bruzek, Amy K.; Bruckner-Lea, Cindy J.

    2005-01-01

    Pathogen detection has become an extremely important part of our nation?s defense in this post 9/11 world where the threat of bioterrorist attacks are a grim reality. When a biological attack takes place, response time is critical. The faster the biothreat is assessed, the faster countermeasures can be put in place to protect the health of the general public. Today some of the most widely used methods for detecting pathogens are either time consuming or not reliable [1]. Therefore, a method that can detect multiple pathogens that is inherently reliable, rapid, automated and field portable is needed. To that end, we are developing automated fluidics systems for the recovery, cleanup, and direct labeling of community RNA from suspect environmental samples. The advantage of using RNA for detection is that there are multiple copies of mRNA in a cell, whereas there are normally only one or two copies of DNA [2]. Because there are multiple copies of mRNA in a cell for highly expressed genes, no amplification of the genetic material may be necessary, and thus rapid and direct detection of only a few cells may be possible [3]. This report outlines the development of both manual and automated methods for the extraction and purification of mRNA. The methods were evaluated using cell lysates from Escherichia coli 25922 (nonpathogenic), Salmonella typhimurium (pathogenic), and Shigella spp (pathogenic). Automated RNA purification was achieved using a custom sequential injection fluidics system consisting of a syringe pump, a multi-port valve and a magnetic capture cell. mRNA was captured using silica coated superparamagnetic beads that were trapped in the tubing by a rare earth magnet. RNA was detected by gel electrophoresis and/or by hybridization of the RNA to microarrays. The versatility of the fluidics systems and the ability to automate these systems allows for quick and easy processing of samples and eliminates the need for an experienced operator.

  1. Rapid detection, characterization, and enrumeration of food-borne pathogens

    USDA-ARS?s Scientific Manuscript database

    In recent years, there has been much research activity on the development of methodologies that are rapid, accurate, and ultrasensitive for detecting pathogenic microorganisms in food. Rapid methods include immunological systems such as the lateral flow assays and enzyme-linked immunosorbent assays...

  2. Bio-functional Au/Si Nanrods for Pathogen Detection

    USDA-ARS?s Scientific Manuscript database

    Nanotechnology applications for food safety and biosecurity, especially development of nanoscale sensors for foodborne pathogen measurement are emerging. A novel bio-functional nanosensor for Salmonella detection was developed using hetero-nanorods. The silica nanorods were fabricated by glancing a...

  3. Hyperspectral Imaging for Detecting Pathogens Grown on Agar Plates

    USDA-ARS?s Scientific Manuscript database

    This paper is concerned with the development of a hyperspectral imaging technique for detecting and identifying one of the most common foodborne pathogens, Campylobacter. Direct plating using agars is an effective tool for laboratory tests and analyses of microorganisms. The morphology (size, growth...

  4. Hyperspectral imaging using RGB color for foodborne pathogen detection

    USDA-ARS?s Scientific Manuscript database

    This paper reports the latest development of a color vision technique for detecting colonies of foodborne pathogens grown on agar plates with a hyperspectral image classification model that was developed using full hyperspectral data. The hyperspectral classification model depended on reflectance sp...

  5. Detection Methodologies for Pathogen and Toxins: A Review.

    PubMed

    Alahi, Md Eshrat E; Mukhopadhyay, Subhas Chandra

    2017-08-16

    Pathogen and toxin-contaminated foods and beverages are a major source of illnesses, even death, and have a significant economic impact worldwide. Human health is always under a potential threat, including from biological warfare, due to these dangerous pathogens. The agricultural and food production chain consists of many steps such as harvesting, handling, processing, packaging, storage, distribution, preparation, and consumption. Each step is susceptible to threats of environmental contamination or failure to safeguard the processes. The production process can be controlled in the food and agricultural sector, where smart sensors can play a major role, ensuring greater food quality and safety by low cost, fast, reliable, and profitable methods of detection. Techniques for the detection of pathogens and toxins may vary in cost, size, and specificity, speed of response, sensitivity, and precision. Smart sensors can detect, analyse and quantify at molecular levels contents of different biological origin and ensure quality of foods against spiking with pesticides, fertilizers, dioxin, modified organisms, anti-nutrients, allergens, drugs and so on. This paper reviews different methodologies to detect pathogens and toxins in foods and beverages.

  6. Detection Methodologies for Pathogen and Toxins: A Review

    PubMed Central

    Mukhopadhyay, Subhas Chandra

    2017-01-01

    Pathogen and toxin-contaminated foods and beverages are a major source of illnesses, even death, and have a significant economic impact worldwide. Human health is always under a potential threat, including from biological warfare, due to these dangerous pathogens. The agricultural and food production chain consists of many steps such as harvesting, handling, processing, packaging, storage, distribution, preparation, and consumption. Each step is susceptible to threats of environmental contamination or failure to safeguard the processes. The production process can be controlled in the food and agricultural sector, where smart sensors can play a major role, ensuring greater food quality and safety by low cost, fast, reliable, and profitable methods of detection. Techniques for the detection of pathogens and toxins may vary in cost, size, and specificity, speed of response, sensitivity, and precision. Smart sensors can detect, analyse and quantify at molecular levels contents of different biological origin and ensure quality of foods against spiking with pesticides, fertilizers, dioxin, modified organisms, anti-nutrients, allergens, drugs and so on. This paper reviews different methodologies to detect pathogens and toxins in foods and beverages. PMID:28813028

  7. Hyperspectral Reflectance Imaging for Detecting a Foodborne Pathogen: Campylobacter

    USDA-ARS?s Scientific Manuscript database

    This paper is concerned with the development of a hyperspectral reflectance imaging technique for detecting and identifying one of the most common foodborne pathogens, Campylobacter. Direct plating using agars is an effective tool for laboratory tests and analyses of microorganisms. The morphology (...

  8. Multiple Pathogen Detection Using Biosensors: Advancements and Challenges

    USDA-ARS?s Scientific Manuscript database

    Advancements in biosensor research have considerably impacted clinical diagnostics for human health. Efforts in capitalizing on the sensitivity of biosensors for food pathogen detection are evident in the food safety/security research community. For practical application with foods that normally h...

  9. Integrated Detection of Pathogens and Host Biomarkers for Wounds

    DTIC Science & Technology

    2012-04-01

    of readily-cultured species in chronic wounds [4]. It is becoming clear that the community of microorganisms inhabiting the human body, collectively...from which bacterial species were cultured by NMRC, we detected nosocomial pathogens in nine samples. In four of these samples we detected an organism...of these bacterial species (See Appendix A). Our team has recently updated the LLMDA with newly sequenced viruses, bacteria, fungi, archaea and

  10. Portable Microfluidic Integrated Plasmonic Platform for Pathogen Detection

    PubMed Central

    Tokel, Onur; Yildiz, Umit Hakan; Inci, Fatih; Durmus, Naside Gozde; Ekiz, Okan Oner; Turker, Burak; Cetin, Can; Rao, Shruthi; Sridhar, Kaushik; Natarajan, Nalini; Shafiee, Hadi; Dana, Aykutlu; Demirci, Utkan

    2015-01-01

    Timely detection of infectious agents is critical in early diagnosis and treatment of infectious diseases. Conventional pathogen detection methods, such as enzyme linked immunosorbent assay (ELISA), culturing or polymerase chain reaction (PCR) require long assay times, and complex and expensive instruments, which are not adaptable to point-of-care (POC) needs at resource-constrained as well as primary care settings. Therefore, there is an unmet need to develop simple, rapid, and accurate methods for detection of pathogens at the POC. Here, we present a portable, multiplex, inexpensive microfluidic-integrated surface plasmon resonance (SPR) platform that detects and quantifies bacteria, i.e., Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) rapidly. The platform presented reliable capture and detection of E. coli at concentrations ranging from ~105 to 3.2 × 107 CFUs/mL in phosphate buffered saline (PBS) and peritoneal dialysis (PD) fluid. The multiplexing and specificity capability of the platform was also tested with S. aureus samples. The presented platform technology could potentially be applicable to capture and detect other pathogens at the POC and primary care settings. PMID:25801042

  11. Portable microfluidic integrated plasmonic platform for pathogen detection.

    PubMed

    Tokel, Onur; Yildiz, Umit Hakan; Inci, Fatih; Durmus, Naside Gozde; Ekiz, Okan Oner; Turker, Burak; Cetin, Can; Rao, Shruthi; Sridhar, Kaushik; Natarajan, Nalini; Shafiee, Hadi; Dana, Aykutlu; Demirci, Utkan

    2015-03-24

    Timely detection of infectious agents is critical in early diagnosis and treatment of infectious diseases. Conventional pathogen detection methods, such as enzyme linked immunosorbent assay (ELISA), culturing or polymerase chain reaction (PCR) require long assay times, and complex and expensive instruments, which are not adaptable to point-of-care (POC) needs at resource-constrained as well as primary care settings. Therefore, there is an unmet need to develop simple, rapid, and accurate methods for detection of pathogens at the POC. Here, we present a portable, multiplex, inexpensive microfluidic-integrated surface plasmon resonance (SPR) platform that detects and quantifies bacteria, i.e., Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) rapidly. The platform presented reliable capture and detection of E. coli at concentrations ranging from ~10(5) to 3.2 × 10(7) CFUs/mL in phosphate buffered saline (PBS) and peritoneal dialysis (PD) fluid. The multiplexing and specificity capability of the platform was also tested with S. aureus samples. The presented platform technology could potentially be applicable to capture and detect other pathogens at the POC and primary care settings.

  12. Evaluation of coral pathogen growth rates after exposure to atmospheric African dust samples

    USGS Publications Warehouse

    Lisle, John T.; Garrison, Virginia H.; Gray, Michael A.

    2014-01-01

    Laboratory experiments were conducted to assess if exposure to atmospheric African dust stimulates or inhibits the growth of four putative bacterial coral pathogens. Atmospheric dust was collected from a dust-source region (Mali, West Africa) and from Saharan Air Layer masses over downwind sites in the Caribbean [Trinidad and Tobago and St. Croix, U.S. Virgin Islands (USVI)]. Extracts of dust samples were used to dose laboratory-grown cultures of four putative coral pathogens: Aurantimonas coralicida (white plague type II), Serratia marcescens (white pox), Vibrio coralliilyticus, and V. shiloi (bacteria-induced bleaching). Growth of A. coralicida and V. shiloi was slightly stimulated by dust extracts from Mali and USVI, respectively, but unaffected by extracts from the other dust sources. Lag time to the start of log-growth phase was significantly shortened for A. coralicida when dosed with dust extracts from Mali and USVI. Growth of S. marcescens and V. coralliilyticus was neither stimulated nor inhibited by any of the dust extracts. This study demonstrates that constituents from atmospheric dust can alter growth of recognized coral disease pathogens under laboratory conditions.

  13. Bio-functional Au/Si nanorods for pathogen detection

    NASA Astrophysics Data System (ADS)

    Park, Bosoon; Fu, Junxue; Zhao, Yiping; Siragusa, Gregory R.; Cho, Yong-Jin; Lawrence, Kurt C.; Windham, William R.

    2007-09-01

    Nanotechnology applications for food safety and biosecurity, especially development of nanoscale sensors for foodborne pathogen measurement are emerging. A novel bio-functional nanosensor for Salmonella detection was developed using hetero-nanorods. The silica nanorods were fabricated by glancing angle deposition method and the gold was sputtered onto the silica nanorods. Alexa488-succinimide dye was immobilized onto the annealed Si nanorods via the attachment between dye ester and primary amine group supplied by the 3-Aminopropyltriethoxysilane. The anti-Salmonella was conjugated to gold via Dithiobis[succinimidylpropionate] self-assembly monolayer. Due to the high aspect ratio nature of the Si nanorods, hundreds or thousands of dye molecules attached to the Si nanorods produced enhanced fluorescence signal. These biologically functionalized nanorods can be used to detect Salmonella with fluorescent microscopic imaging. This new nanoscale biosensor will be able to detect other foodborne pathogenic bacteria for food safety and security applications.

  14. Application of bacteriophages for detection of foodborne pathogens.

    PubMed

    Schmelcher, Mathias; Loessner, Martin J

    2014-01-01

    Bacterial contamination of food products presents a challenge for the food industry and poses a high risk for the consumer. Despite increasing awareness and improved hygiene measures, foodborne pathogens remain a threat for public health, and novel methods for detection of these organisms are needed. Bacteriophages represent ideal tools for diagnostic assays because of their high target cell specificity, inherent signal-amplifying properties, easy and inexpensive production, and robustness. Every stage of the phage lytic multiplication cycle, from the initial recognition of the host cell to the final lysis event, may be harnessed in several ways for the purpose of bacterial detection. Besides intact phage particles, phage-derived affinity molecules such as cell wall binding domains and receptor binding proteins can serve for this purpose. This review provides an overview of existing phage-based technologies for detection of foodborne pathogens, and highlights the most recent developments in this field, with particular emphasis on phage-based biosensors.

  15. Multiplexed detection of waterborne pathogens in circular microfluidics.

    PubMed

    Agrawal, Shailaja; Morarka, Amit; Bodas, Dhananjay; Paknikar, K M

    2012-07-01

    Microfluidic lab-on-a-chip presents an ideal solution for bacterial sensing and identification due to its advantages like large surface-to-volume ratio, requirement of low sample volume and multiplexing possibility. The present work deals with the development of an immunosensor chip using circular microchannels fabricated directly with microdimensional copper wire and permanent magnet for capture of Fe(3)O(4) magnetic nanoparticle (MNP) conjugate. The MNP facilitate capture of the antigen in a confined space and hence, enhanced fluorescence signal for detection. The multiplexed microfluidic chip permits visual detection and quantification of waterborne pathogens viz. Escherichia coli and Salmonella typhimurium simultaneously. CdTe quantum dots (QDs) with different emission wavelengths were conjugated with anti-E. coli and anti-S. typhimurium antibodies for concurrent fluorescence detection. The present technique provides an inexpensive yet powerful tool to image and quantify pathogens at low numbers with passage of large sample volumes.

  16. Application of bacteriophages for detection of foodborne pathogens

    PubMed Central

    Schmelcher, Mathias; Loessner, Martin J

    2014-01-01

    Bacterial contamination of food products presents a challenge for the food industry and poses a high risk for the consumer. Despite increasing awareness and improved hygiene measures, foodborne pathogens remain a threat for public health, and novel methods for detection of these organisms are needed. Bacteriophages represent ideal tools for diagnostic assays because of their high target cell specificity, inherent signal-amplifying properties, easy and inexpensive production, and robustness. Every stage of the phage lytic multiplication cycle, from the initial recognition of the host cell to the final lysis event, may be harnessed in several ways for the purpose of bacterial detection. Besides intact phage particles, phage-derived affinity molecules such as cell wall binding domains and receptor binding proteins can serve for this purpose. This review provides an overview of existing phage-based technologies for detection of foodborne pathogens, and highlights the most recent developments in this field, with particular emphasis on phage-based biosensors. PMID:24533229

  17. Aptamer-assisted novel technologies for detecting bacterial pathogens.

    PubMed

    Alizadeh, Naser; Memar, Mohammad Yousef; Moaddab, Seyyed Reza; Kafil, Hossein Samadi

    2017-09-01

    Nowadays, all people are at risk of infectious diseases that are mainly caused by bacteria causing infection. There is a permanent demand for an appropriate detection method that is affordable, practical, careful, rapid, sensitive, efficient and economical. Aptamers are single stranded DNA or RNA oligonucleotides, which can be recognized specifically and bind to their target molecules and also, be exploited in diagnostic applications. Recently, aptamer-based systems have offered great potentials in applications for the recognition of several important bacterial pathogens from clinical and food specimens. There are several reports appraising the diagnostic applicability of aptamer-based systems for the detection of pathogens. As for its excellent sensitivity, as well as its rapid and efficient detectability, this technique may be practical to indicate bacterial targets with less sample size and may consume less time than traditional methods These systems offer a promising approach for the sensitive and quick detection of food-borne and clinical agents. This review provides an overview of aptamer-based methods as a novel approach for detecting bacterial pathogens. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Optimization and clinical validation of a pathogen detection microarray

    PubMed Central

    Wong, Christopher W; Heng, Charlie Lee Wah; Wan Yee, Leong; Soh, Shirlena WL; Kartasasmita, Cissy B; Simoes, Eric AF; Hibberd, Martin L; Sung, Wing-Kin; Miller, Lance D

    2007-01-01

    DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics. PMID:17531104

  19. Prevalence of foodborne pathogens in food from selected African countries - A meta-analysis.

    PubMed

    Paudyal, Narayan; Anihouvi, Victor; Hounhouigan, Joseph; Matsheka, Maitshwarelo Ignatius; Sekwati-Monang, Bonno; Amoa-Awua, Wisdom; Atter, Amy; Ackah, Nina Bernice; Mbugua, Samuel; Asagbra, Agnes; Abdelgadir, Warda; Nakavuma, Jesca; Jakobsen, Mogens; Fang, Weihuan

    2017-05-16

    Food safety information in the African region is insufficient and fragmented due to lack of surveillance, documentation and reporting, thereby resulting in inefficient utilization of resources, duplication of activities, and lack of synergy among the countries of the region. This paper reviews the prevalence of foodborne pathogens in seven African countries (Benin, Botswana, Ghana, Kenya, Nigeria, Sudan and Uganda) from papers in regional or international journals published between January 2000 and December 2015. One hundred and sixteen publications that dealt with food microbiology were reviewed for general analysis, while 66 papers on contamination of pathogenic bacteria were used for meta-analysis of prevalence. The food items were split into two categories: raw foods and ready-to-eat (RTE) foods (including street food and beverages) for meta-analysis. Majority of the reviewed studies (67.2%, 78/116) dealt with food of animal origin: 38.8% for meat and eggs, 17.2% for dairy products and 11.2% for aquatic products. Only 8.6% examined foods of plant origin (fruits and vegetables). The remaining 24.1% was the composite RTE food and beverages. Enterobacteriaceae, Escherichia coli, Salmonella, Staphylococcus aureus and Listeria monocytogenes were the most frequently reported organisms in those studies. Although the data were highly heterogeneous, a striking feature is high prevalence of the major pathogens in RTE foods, almost as high as in raw foods. E. coli averaged at 37.6% in raw foods and 31.6% in RTE foods. The corresponding prevalence for Salmonella was 19.9% vs 21.7%; S. aureus, 27.8% vs 25.1% and L. monocytogenes, 19.5% vs 6.7%. The average prevalence of foodborne pathogens in these countries was 34.2% (29.0-39.3%). Differences in food types as well as non-uniform protocols for sampling and identification might have contributed to high heterogeneity (I(2) >97%) although some high prevalence data could be factual with extensive varieties of raw and RTE foods

  20. Investigation of magnetic microdiscs for bacterial pathogen detection

    NASA Astrophysics Data System (ADS)

    Castillo-Torres, Keisha Y.; Garraud, Nicolas; Arnold, David P.; McLamore, Eric S.

    2016-05-01

    Despite strict regulations to control the presence of human pathogens in our food supply, recent foodborne outbreaks have heightened public concern about food safety and created urgency to improve methods for pathogen detection. Herein we explore a potentially portable, low-cost system that uses magnetic microdiscs for the detection of bacterial pathogens in liquid samples. The system operates by optically measuring the rotational dynamics of suspended magnetic microdiscs functionalized with pathogen-binding aptamers. The soft ferromagnetic (Ni80Fe20) microdiscs exhibit a closed magnetic spin arrangement (i.e. spin vortex) with zero magnetic stray field, leading to no disc agglomeration when in free suspension. With very high surface area for functionalization and volumes 10,000x larger than commonly used superparamagnetic nanoparticles, these 1.5-μm-diameter microdiscs are well suited for tagging, trapping, actuating, or interrogating bacterial targets. This work reports a wafer-level microfabrication process for fabrication of 600 million magnetic microdiscs per substrate and measurement of their rotational dynamics response. Additionally, the biofunctionalization of the microdiscs with DNA aptamers, subsequent binding to E. coli bacteria, and their magnetic manipulation is reported.

  1. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  2. MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis

    PubMed Central

    Stijlemans, Benoît; Brys, Lea; Korf, Hannelie; Bieniasz-Krzywiec, Pawel; Sparkes, Amanda; Vansintjan, Liese; Leng, Lin; Vanbekbergen, Nele; Mazzone, Massimiliano; Caljon, Guy; Van Den Abbeele, Jan; Odongo, Steven; De Trez, Carl; Magez, Stefan; Van Ginderachter, Jo A.; Beschin, Alain; Bucala, Richard; De Baetselier, Patrick

    2016-01-01

    Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis. PMID:27632207

  3. Microfluidics-based integrated airborne pathogen detection systems

    NASA Astrophysics Data System (ADS)

    Northrup, M. Allen; Alleman-Sposito, Jennifer; Austin, Todd; Devitt, Amy; Fong, Donna; Lin, Phil; Nakao, Brian; Pourahmadi, Farzad; Vinas, Mary; Yuan, Bob

    2006-09-01

    Microfluidic Systems is focused on building microfluidic platforms that interface front-end mesofluidics to handle real world sample volumes for optimal sensitivity coupled to microfluidic circuitry to process small liquid volumes for complex reagent metering, mixing, and biochemical analysis, particularly for pathogens. MFSI is the prime contractor on two programs for the US Department of Homeland Security: BAND (Bioagent Autonomous Networked Detector) and IBADS (Instantaneous Bio-Aerosol Detection System). The goal of BAND is to develop an autonomous system for monitoring the air for known biological agents. This consists of air collection, sample lysis, sample purification, detection of DNA, RNA, and toxins, and a networked interface to report the results. For IBADS, MFSI is developing the confirmatory device which must verify the presence of a pathogen with 5 minutes of an air collector/trigger sounding an alarm. Instrument designs and biological assay results from both BAND and IBADS will be presented.

  4. Detection of pathogenic gram negative bacteria using infrared thermography

    NASA Astrophysics Data System (ADS)

    Lahiri, B. B.; Divya, M. P.; Bagavathiappan, S.; Thomas, Sabu; Philip, John

    2012-11-01

    Detection of viable bacteria is of prime importance in all fields of microbiology and biotechnology. Conventional methods of enumerating bacteria are often time consuming and labor-intensive. All living organisms generate heat due to metabolic activities and hence, measurement of heat energy is a viable tool for detection and quantification of bacteria. In this article, we employ a non-contact and real time method - infrared thermography (IRT) for measurement of temperature variations in four clinically significant gram negative pathogenic bacteria, viz. Vibrio cholerae, Vibrio mimicus, Proteus mirabilis and Pseudomonas aeruginosa. We observe that, the energy content, defined as the ratio of heat generated by bacterial metabolic activities to the heat lost from the liquid medium to the surrounding, vary linearly with the bacterial concentration in all the four pathogenic bacteria. The amount of energy content observed in different species is attributed to their metabolisms and morphologies that affect the convection velocity and hence heat transport in the medium.

  5. Sample preparation and assay refinements for pathogen detection platforms

    NASA Astrophysics Data System (ADS)

    Lim, Daniel V.; Kearns, Elizabeth A.; Leskinen, Stephaney D.; Magaña, Sonia; Stroot, Joyce M.; Hunter, Dawn M.; Schlemmer, Sarah M.

    2009-02-01

    Food-borne and waterborne microbial pathogens are a potential problem in biowarfare and public health. Such pathogens can affect the health, combat readiness, and effectiveness of the warfighter in a battlefield environment and present potential threats to the civilian population through intentional or natural contamination of food and water. Conventional procedures to detect and identify microbial pathogens in food, water, and other materials can take days to perform and may provide inconclusive information. Research at the University of South Florida's Advanced Biosensors Laboratory (ABL) focuses on development of sample processing procedures and biosensor-based assays for rapid detection of biothreat agents. Rapid processing methods, including use of an automated concentrator of microorganisms in water, have been developed for complex matrix samples including ground beef, apple juice, produce, potable water and recreational water, enabling such samples to be directly tested by biosensor assays for target analytes. Bacillus atrophaeus spores and other bacteria can be concentrated from potable and recreational water at low levels with a dead-end hollow-fiber ultrafiltration concentration system. Target bacteria recovered by these processing procedures can be identified by evanescent wave, fiber optic biosensors or other detection platforms. Fiber optic biosensor assays have been improved to include subsequent PCR analysis and viability determination of captured target bacteria using broth enrichment and/or ATP luminescence.

  6. Detection of pathogenic DNA at the single-molecule level

    NASA Astrophysics Data System (ADS)

    Yahiatène, Idir; Klamp, Tobias; Schüttpelz, Mark; Sauer, Markus

    2011-03-01

    We demonstrate ultrasensitive detection of pathogenic DNA in a homogeneous assay at the single-molecule level applying two-color coincidence analysis. The target molecule we quantify is a 100 nucleotide long synthetic single-stranded oligonucleotide adapted from Streptococcus pneumoniae, a bacterium causing lower respiratory tract infections. Using spontaneous hybridization of two differently fluorescing Molecular Beacons we demonstrate a detection sensitivity of 100 fM (10-13M) in 30 seconds applying a simple microfluidic device with a 100 μm channel and confocal two-color fluorescence microscopy.

  7. Fluorogenic Substrate Detection of Viable Intracellular and Extracellular Pathogenic Protozoa

    NASA Astrophysics Data System (ADS)

    Jackson, Peter R.; Pappas, Michael G.; Hansen, Brian D.

    1985-01-01

    Viable Leishmania promastigotes and amastigotes were detected by epifluorescence microscopy with fluorescein diacetate being used to mark living parasites and the nucleic acid-binding compound ethidium bromide to stain dead cells. This procedure is superior to other assays because it is faster and detects viable intracellular as well as extracellular Leishmania. Furthermore, destruction of intracellular pathogens by macrophages is more accurately determined with fluorescein diacetate than with other stains. The procedure may have applications in programs to develop drugs and vaccines against protozoa responsible for human and animal disease.

  8. Coxiella burnetii detected in three species of endangered North African gazelles that recently aborted.

    PubMed

    García, Elena; Espeso, Gerardo; Fernández, Rocío; Gómez-Martín, Ángel; Rodríguez-Linde, José María; De la Fe, Christian

    2017-01-15

    Coxiella (C.) burnetii is the etiological agent of the zoonotic disease known a Q fever. This agent can infect multiple hosts although its pathogenic potential in wild ruminants has been poorly studied. The polymerase chain reaction and the serological test detected the presence of C. burnetii in a population of North African gazelles (n = 355), comprising dorcas gazelle (Gazella dorcas neglecta), dama gazelle (Nanger dama mhorr) and Cuvier's gazelle (Gazella cuvieri) which, some of them, they recently aborted. Serological tests for Brucella spp., C. burnetii, Chlamydophila abortus, border disease pestivirus, and Toxoplasma spp. were performed together with specific cultures to detect Salmonella spp., Listeria spp., and Campylobacter spp. and a polymerase chain reaction for C. burnetii on serum and vaginal swabs samples collected from a representative number of animals (n = 65). These tests only detected the presence of C. burnetii in 18 specimens (27.3%). C. burnetii was the only pathogen detected, with eight animals testing positive on the polymerase chain reaction, 15 on the serological test, and five on both the tests. This article reveals the presence of C. burnetii during a medium and late-stage abortions occurred in a population of North African gazelles. The presence of C. burnetii as causal agent of abortions in Cuvier's gazelles has never been reported. The consequences of the findings are discussed here, showing the need to adopt urgent control measures to prevent the spread of C. burnetii in captive populations that are essential for the conservation of these endangered species. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

    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

  10. Detection of Blastocystis hominis: a controversial human pathogen.

    PubMed

    Basak, Silpi; Rajurkar, Monali N; Mallick, Sanjay Kumar

    2014-01-01

    Blastocystis hominis has been reclassified as a protozoan parasite. Its role as a human pathogen is somewhat controversial. There has been a dramatic increase in the frequency of B. hominis infection in association with diarrhea especially in immunocompromised hosts like AIDS patients, travelers, homosexuals, day care children, animal handlers especially zoo keepers, etc. Recent reports suggest that B. hominis is an emerging pathogen; hence, we have undertaken this study to detect B. hominis from stool samples of patients attending our hospital. About 200 stool samples were tested by light microscopic examination, for observing wet mounts with saline and Lugol's iodine. Permanent staining of fecal smear by Gram's staining and modified acid fast staining was done. The stool sample which was microscopically positive for B. hominis was cultured on Lowenstein-Jensen's (LJ) medium. In one patient, the vacuolated form of B. hominis was observed in wet mount with saline preparation of stool sample. This was very clearly seen in wet mount with Lugol's iodine. In Gram's stained preparation, also the vacuolated form was observed. Detection of B. hominis was not possible by modified acid fast staining. B. hominis was also grown on LJ medium which is an egg-containing medium. Clinical microbiology laboratories should start screening of stool samples for B. hominis as it is an emerging pathogen.

  11. Multiplex PCR to detect four different tomato-infecting pathogens.

    PubMed

    Quintero-Vásquez, Gabriela Alejandra; Bazán-Tejeda, María Luisa; Martínez-Peñafiel, Eva; Kameyama-Kawabe, Luis; Bermúdez-Cruz, Rosa María

    2013-07-01

    This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

  12. Integrated Detection of Pathogens and Host Biomarkers for Wounds

    SciTech Connect

    Jaing, C

    2012-03-19

    The increasing incidence and complications arising from combat wounds has necessitated a reassessment of methods for effective treatment. Infection, excessive inflammation, and incidence of drug-resistant organisms all contribute toward negative outcomes for afflicted individuals. The organisms and host processes involved in wound progression, however, are incompletely understood. We therefore set out, using our unique technical resources, to construct a profile of combat wounds which did or did not successfully resolve. We employed the Lawrence Livermore Microbial Detection Array and identified a number of nosocomial pathogens present in wound samples. Some of these identities corresponded with bacterial isolates previously cultured, while others were not obtained via standard microbiology. Further, we optimized proteomics protocols for the identification of host biomarkers indicative of various stages in wound progression. In combination with our pathogen data, our biomarker discovery efforts will provide a profile corresponding to wound complications, and will assist significantly in treatment of these complex cases.

  13. 9 CFR 113.36 - Detection of pathogens by the chicken inoculation test.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Detection of pathogens by the chicken... REQUIREMENTS Standard Procedures § 113.36 Detection of pathogens by the chicken inoculation test. The test for detection of extraneous pathogens provided in this section shall be conducted when such a test is prescribed...

  14. Nanoparticles based DNA conjugates for detection of pathogenic microorganisms

    NASA Astrophysics Data System (ADS)

    Jamdagni, Pragati; Khatri, Poonam; Rana, J. S.

    2016-01-01

    Infectious diseases have been on rise in the recent past. Early diagnosis plays a role as important as proper treatment and prophylaxis. The current practices of detection are time consuming which may result in unnecessary delays in treatment. Advances in nanodiagnostic approaches have been in focus lately. The rising interest and better understanding of nanoparticles have led to opening up of new frontiers in the concerned area. Optical properties of nanoparticles are being exploited to design detection systems that can provide fast, one-step and reliable results. Based on conserved DNA sequences unique to the target organism, the results offer accuracy comparable to conventional tests. Further, visual or spectrophotometric analysis omits the need of costly apparatus for result interpretation. The present review aims at putting together the information on nanoparticles based DNA conjugate systems for detection of pathogenic microorganisms.

  15. Comparison of different methods of detection of enteric pathogenic protozoa.

    PubMed

    Ahmed, N H; Chowdhary, A

    2013-01-01

    The study was conducted to compare different methods of detection of pathogenic protozoan parasites in stool specimens of People Living with HIV/AIDS (PLHA). Stool specimens of 242 HIV sero-positive patients were examined using the wet mount technique, modified Ziehl-Neelsen's (ZN) staining, auto-fluorescence and auramine fluorescence staining. Patient specimens, 94 and 40 out of 242, were also subjected to Giardia antigen detection using an enzyme immunoassay and Cryptosporidium antigen detection by immuno-chromatography, respectively. For calculation of sensitivity, specificity, positive and negative predictive values, light microscopy of wet mounts and modified ZN stained smears for Giardia and Coccidia, respectively, were considered as gold standards. Sensitivity of auto-fluorescence, auramine-O staining and antigen detection techniques was found to be 100% as compared to the routine standards. The specificity of auto-fluorescence was 90.6% and 100% for Cyclospora and Isospora, respectively; that of auramine-O staining was 98.9% for Cryptosporidium, 99.30% for Cyclospora and 100% for Isospora; and that of antigen detection was 90.6% and 97.7% for Cryptosporidium and Giardia, respectively. In laboratories requiring screening of large number of stool specimens for detection of protozoan parasites, fluorescence microscopy and antigen detection can be useful techniques. Confirmation of positive results, however, needs to be done with the standard techniques.

  16. Autonomous Pathogen Detection System - FY02 Annual Progress Report

    SciTech Connect

    Colston, B; Brown, S; Burris, K; Elkin, C; Hindson, B; Langlois, R; Masquelier, D; McBride, M; Metz, T; Nasarabadi, S; Makarewicz, T; Milznovich, F; Venkateswaran, K S; Visuri, S

    2002-11-11

    The objective of this project is to design, fabricate and field demonstrate a biological agent detection and identification capability, the Autonomous Pathogen Detector System (APDS). Integrating a flow cytometer and real-time polymerase chain reaction (PCR) detector with sample collection, sample preparation and fluidics will provide a compact, autonomously operating instrument capable of simultaneously detecting multiple pathogens and/or toxins. The APDS will operate in fixed locations, continuously monitoring air samples and automatically reporting the presence of specific biological agents. The APDS will utilize both multiplex immunoassays and nucleic acid assays to provide ''quasi-orthogonal'' multiple agent detection approaches to minimize false positives and increase the reliability of identification. Technical advances across several fronts must occur, however, to realize the full extent of the APDS. The end goal of a commercially available system for civilian biological weapon defense will be accomplished through three progressive generations of APDS instruments. The APDS is targeted for civilian applications in which the public is at high risk of exposure to covert releases of bioagent, such as major subway systems and other transportation terminals, large office complexes and convention centers. APDS is also designed to be part of a monitoring network of sensors integrated with command and control systems for wide-area monitoring of urban areas and major public gatherings. In this latter application there is potential that a fully developed APDS could add value to DoD monitoring architectures.

  17. Detection of 3-hydroxykynurenine in a plant pathogenic fungus.

    PubMed Central

    Wilson, T J Greer; Thomsen, Karl Kristian; Petersen, Bent O; Duus, Jens Ø; Oliver, Richard P

    2003-01-01

    A redox-active compound has been purified from the barley powdery mildew fungus Blumeria ( Erysiphe ) graminis f. sp. hordei. A combination of spectrophotometry, MS and NMR has identified it as 3-hydroxykynurenine (3OHKyn). This compound, never previously detected in any fungus or pathogen, is best known for its role in vertebrate cataracts. It is found abundantly in developing and germinating spores and also in runner hyphae. Two roles for 3OHKyn are discussed: first, the presence of active oxygen species would enable 3OHKyn to cross-link the spore chemically with the plant. Secondly, it may be acting as an UV protectant and an antioxidant. PMID:12556224

  18. Sexually transmitted pathogens in pregnant women in a rural South African community.

    PubMed Central

    O'Farrell, N; Hoosen, A A; Kharsany, A B; van den Ende, J

    1989-01-01

    One hundred and ninety three consecutive pregnant women attending peripheral antenatal clinics attached to Ngwelezana Hospital, Empangeni, Kwa-Zulu, were examined for evidence of sexually transmitted pathogens. The following incidences were found: Trichomonas vaginalis 49.2% (95), Candida spp 38.3% (74), Chlamydia trachomatis 11.4% (22), Gardnerella vaginalis 6.2% (12), Neisseria gonorrhoeae 5.7% (11), positive syphilis serology results 11.9% (23), hepatitis B surface antigen 4.1% (eight). No woman had antibody to human immunodeficiency virus (HIV). Dyskaryotic smears were found in 20 (10.4%). Human papillomavirus (HPV) was detected cytologically in 11 (5.7%). The range of sexually transmitted pathogens found in this rural community was similar to that found in urban groups studied in South Africa. PMID:2807289

  19. Human pathogens in plant biofilms: Formation, physiology, and detection.

    PubMed

    Ximenes, Eduardo; Hoagland, Lori; Ku, Seockmo; Li, Xuan; Ladisch, Michael

    2017-01-09

    Fresh produce, viewed as an essential part of a healthy life style is usually consumed in the form of raw or minimally processed fruits and vegetables, and is a potentially important source of food-borne human pathogenic bacteria and viruses. These are passed on to the consumer since the bacteria can form biofilms or otherwise populate plant tissues, thereby using plants as vectors to infect animal hosts. The life cycle of the bacteria in plants differs from those in animals or humans and results in altered physiochemical and biological properties (e.g., physiology, immunity, native microflora, physical barriers, mobility, and temperature). Mechanisms by which healthy plants may become contaminated by microorganisms, develop biofilms, and then pass on their pathogenic burden to people are explored in the context of hollow fiber microfiltration by which plant-derived microorganisms may be recovered and rapidly concentrated to facilitate study of their properties. Enzymes, when added to macerated plant tissues, hydrolyze or alter macromolecules that would otherwise foul hollow-fiber microfiltration membranes. Hence, microfiltration may be used to quickly increase the concentration of microorganisms to detectable levels. This review discusses microbial colonization of vegetables, formation and properties of biofilms, and how hollow fiber microfiltration may be used to concentrate microbial targets to detectable levels. The use of added enzymes helps to disintegrate biofilms and minimize hollow fiber membrane fouling, thereby providing a new tool for more time effectively elucidating mechanisms by which biofilms develop and plant tissue becomes contaminated with human pathogens. Biotechnol. Bioeng. 2016;9999: 1-16. © 2017 Wiley Periodicals, Inc.

  20. New quantitative detection of pathogens in heterogeneous environmental samples

    NASA Astrophysics Data System (ADS)

    Lee, Eun-Hee; Wang, Xiaofang; Mitchell, Kristi; Chae, Seon-Ha; Son, Ahjeong

    2015-04-01

    Quantum dots and magnetic beads based genomic assay (NanoGene assay) has been developed for sensitive and inhibition resistant gene quantification to achieve in-situ bacteria monitoring in environmental samples. In this study, eaeA gene of pathogenic E. coli O157:H7 was quantified. The result demonstrated the excellent sensitivity (i.e., limit of detection: 87 gene copies for dsDNA and 890 zeptomolar for ssDNA) in the presence of nonspecific microbial populations (Kim et al., 2010; 2011a). The feasibility of the developed gene quantification for non-laboratory environment usage (in-situ use) was investigated. Therefore, DNA hybridization was achieved at ambient temperature and minimum agitation, and the analysis was completed within hours. Most importantly, the NanoGene assay demonstrated the resistance to the presence of naturally occurring inhibitors (humic acids, cations) and residual reagents (surfactants, alcohols) from DNA extraction (Kim et al., 2011b). The assay was also applied to humic acids laden soils (7 types of soils with various amount of organic matters) and successfully quantified 105 to 108 CFU of E. coli O157:H7 per gram soil (R2 = 0.99). The results indicate that the presented NanoGene assay is suitable for further development as an in-situ bacteria monitoring method for working with heterogeneous environmental samples (Wang et al., 2013). Another aspect of the method is to transform the NanoGene assay into a portable device that can be used as a pathogenic bacteria detector in environment. The project consisted of the first inline fluidic components development and characterization as well as the first integration effort on a briefcase platform for the in-situ pathogen detection system (IPDS) (Mitchell et al., 2014). Our long term vision is to further miniaturize the briefcase platform implementation of the IPDS and to commercialize the handheld version of the IPDS.

  1. Humic substances interfere with detection of pathogenic prion protein

    USGS Publications Warehouse

    Smith, Christen B.; Booth, Clarissa J.; Wadzinski, Tyler J.; Legname, Giuseppe; Chappell, Rick; Johnson, Christopher J.; Pedersen, Joel A.

    2014-01-01

    Studies examining the persistence of prions (the etiological agent of transmissible spongiform encephalopathies) in soil require accurate quantification of pathogenic prion protein (PrPTSE) extracted from or in the presence of soil particles. Here, we demonstrate that natural organic matter (NOM) in soil impacts PrPTSE detection by immunoblotting. Methods commonly used to extract PrPTSE from soils release substantial amounts of NOM, and NOM inhibited PrPTSE immunoblot signal. The degree of immunoblot interference increased with increasing NOM concentration and decreasing NOM polarity. Humic substances affected immunoblot detection of prion protein from both deer and hamsters. We also establish that after interaction with humic acid, PrPTSE remains infectious to hamsters inoculated intracerebrally, and humic acid appeared to slow disease progression. These results provide evidence for interactions between PrPTSE and humic substances that influence both accurate measurement of PrPTSE in soil and disease transmission.

  2. A review on detection methods used for foodborne pathogens

    PubMed Central

    Priyanka, B.; Patil, Rajashekhar K.; Dwarakanath, Sulatha

    2016-01-01

    Foodborne pathogens have been a cause of a large number of diseases worldwide and more so in developing countries. This has a major economic impact. It is important to contain them, and to do so, early detection is very crucial. Detection and diagnostics relied on culture-based methods to begin with and have developed in the recent past parallel to the developments towards immunological methods such as enzyme-linked immunosorbent assays (ELISA) and molecular biology-based methods such as polymerase chain reaction (PCR). The aim has always been to find a rapid, sensitive, specific and cost-effective method. Ranging from culturing of microbes to the futuristic biosensor technology, the methods have had this common goal. This review summarizes the recent trends and brings together methods that have been developed over the years. PMID:28139531

  3. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    PubMed

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. Published by Elsevier Inc.

  4. Hyperspectral imaging for detecting pathogens grown on agar plates

    NASA Astrophysics Data System (ADS)

    Yoon, Seung Chul; Lawrence, Kurt C.; Siragusa, Gregory R.; Line, John E.; Park, Bosoon; Windham, William R.

    2007-09-01

    This paper is concerned with the development of a hyperspectral imaging technique for detecting and identifying one of the most common foodborne pathogens, Campylobacter. Direct plating using agars is an effective tool for laboratory tests and analyses of microorganisms. The morphology (size, growth pattern, color, etc.) of colonies grown on agar plates has been widely used to tentatively differentiate organisms. However, it is sometimes difficult to differentiate target organisms like Campylobacters from other contaminants grown together on the same agar plates. A hyperspectral imaging system operating at the visible and near infrared (VNIR) spectral region from 400 nm to 900 nm was set up to measure spectral signatures of 17 different Campylobacter and non-Campylobacter subspecies. Protocols for culturing, imaging samples and for calibrating measured data were developed. The VNIR spectral library of all 17 organisms commonly encountered in poultry was established from calibrated hyperspectral images. A classification algorithm was developed to locate and identify Campylobacters, non-Campylobacter contaminants, and background agars with 99.29% accuracy. This research has a potential to be expanded to detect other pathogens grown on agar media.

  5. A liquid-crystal-based DNA biosensor for pathogen detection

    PubMed Central

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-01-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection. PMID:26940532

  6. A liquid-crystal-based DNA biosensor for pathogen detection

    NASA Astrophysics Data System (ADS)

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-03-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection.

  7. Detection of Biological Pathogens Using Multiple Wireless Magnetoelastic Biosensors

    NASA Astrophysics Data System (ADS)

    Shen, Wen

    A number of recent, high-profile incidences of food-borne illness spreading through the food supply and the use of anthrax by terrorists after the September 11, 2001 attacks have demonstrated the need for new technologies that can rapidly detect the presence of biological pathogens. A bevy of biosensors show excellent detection sensitivity and specificity. However, false positive and false negative signals remain one of the primary reasons that many of these newly developed biosensors have not found application in the marketplace. The research described in this dissertation focuses on developing a free-standing magnetoelastic based bio-sensing system using a pulse method. This method allows fast detection, eliminates the bias magnetic field that is necessary in current methods, makes the system more simply and suitable for in-field detection. This system has two pairs of transformer coils, where a measurement sensor and a control sensor can be put in each pair of coils. The control sensor is used to compensate for environmental variables. The effect of pulse power on the performance of the magnetoelastic sensors in the pulse system is studied. The system is found to have excellent stability, good detection repeatability when used with multiple sensors. This research has investigated and demonstrated a multiple sensors approach. Because it will involve the simultaneous measurement of many sensors, it will significantly reduce problems encountered with false positive indications. The positioning and interference of sensors are investigated. By adding a multi-channel structure to the pulse detection system, the effect of sensor interference is minimized. The result of the repeatability test shows that the standard deviation when measuring three 1 mm magnetoelastic sensors is around 500 Hz, which is smaller than the minimum requirement for actual spores/bacteria detection. Magnetoelastic sensors immobilized with JRB7 phages and E2 phages have been used to specifically

  8. Detection of pathogenic organisms in food, water, and body fluids

    NASA Astrophysics Data System (ADS)

    Wallace, William H.; Henley, Michael V.; Sayler, Gary S.

    2002-06-01

    The construction of specific bioluminescent bacteriophage for detection of pathogenic organism can be developed to overcome interferences in complex matrices such as food, water and body fluids. Detection and identification of bacteria often require several days and frequently weeks by standard methods of isolation, growth and biochemical test. Immunoassay detection often requires the expression of the bacterial toxin, which can lead to non-detection of cells that may express the toxin under conditions different from testing protocols. Immunoassays require production of a specific antibody to the agent for detection and interference by contaminants frequently affects results. PCR based detection may be inhibited by substances in complex matrices. Modified methods of the PCR technique, such as magnetic capture-hybridization PCR (MCH-PCR), appear to improve the technique by removing the DNA products away from the inhibitors. However, the techniques required for PCR-based detection are slow and the procedures require skilled personnel working with labile reagents. Our approach is based on transferring bioluminescence (lux) genes into a selected bacteriophage. Bacteriophages are bacterial viruses that are widespread in nature and often are genus and species specific. This specificity eliminates or reduces false positives in a bacteriophage assay. The phage recognizes a specific receptor molecule on the surface of a susceptible bacterium, attaches and then injects the viral nucleic acid into the cell. The injected viral genome is expressed and then replicated, generating numerous exact copies of the viral genetic material including the lux genes, often resulting in an increase in bioluminescence by several hundred fold.

  9. System for rapid detection of antibiotic resistance of airborne pathogens

    NASA Astrophysics Data System (ADS)

    Fortin, M.; Noiseux, I.; Mouslinkina, L.; Vernon, M. L.; Laflamme, C.; Filion, G.; Duchaine, C.; Ho, J.

    2009-05-01

    This project uses function-based detection via a fundamental understanding of the genetic markers of AR to distinguish harmful organisms from innocuous ones. This approach circumvents complex analyses to unravel the taxonomic details of 1399 pathogen species, enormously simplifying detection requirements. Laval Hospital's fast permeabilization strategy enables AR revelation in <1hr. Packaging the AR protocols in liquid-processing cartridges and coupling these to our in-house miniature fiber optic flow cell (FOFC) provides first responders with timely information on-site. INO's FOFC platform consists of a specialty optical fiber through which a hole is transversally bored by laser micromachining. The analyte solution is injected into the hole of the fiber and the particles are detected and counted. The advantage with respect to classic free space FC is that alignment occurs in the fabrication process only and complex excitation and collection optics are replaced by optical fibers. Moreover, we use a sheathless configuration which has the advantage of increase the portability of the system, to reduce excess biohazard material and the need for weekly maintenance. In this paper we present the principle of our FOFC along with a, demonstration of the basic capability of the platform for detection of bacillus cereus spores using permeabilized staining.

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

    USDA-ARS?s Scientific Manuscript database

    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) ...

  11. AOTF hyperspectral microscopic imaging for foodborne pathogenic bacteria detection

    NASA Astrophysics Data System (ADS)

    Park, Bosoon; Lee, Sangdae; Yoon, Seung-Chul; Sundaram, Jaya; Windham, William R.; Hinton, Arthur, Jr.; Lawrence, Kurt C.

    2011-06-01

    Hyperspectral microscope imaging (HMI) method which provides both spatial and spectral information can be effective for foodborne pathogen detection. The AOTF-based hyperspectral microscope imaging method can be used to characterize spectral properties of biofilm formed by Salmonella enteritidis as well as Escherichia coli. The intensity of spectral imagery and the pattern of spectral distribution varied with system parameters (integration time and gain) of HMI system. The preliminary results demonstrated determination of optimum parameter values of HMI system and the integration time must be no more than 250 ms for quality image acquisition from biofilm formed by S. enteritidis. Among the contiguous spectral imagery between 450 and 800 nm, the intensity of spectral images at 498, 522, 550 and 594 nm were distinctive for biofilm; whereas, the intensity of spectral images at 546 nm was distinctive for E. coli. For more accurate comparison of intensity from spectral images, a calibration protocol, using neutral density filters and multiple exposures, need to be developed to standardize image acquisition. For the identification or classification of unknown food pathogen samples, ground truth regions-of-interest pixels need to be selected for "spectrally pure fingerprints" for the Salmonella and E. coli species.

  12. A fractal analysis of pathogen detection by biosensors

    NASA Astrophysics Data System (ADS)

    Doke, Atul M.; Sadana, Ajit

    2006-05-01

    A fractal analysis is presented for the detection of pathogens such as Franscisela tularensis, and Yersinia pestis (the bacterium that causes plague) using a CANARY (cellular analysis and notification of antigens risks and yields) biosensor (Rider et al., 2003). In general, the binding and dissociation rate coefficients may be adequately described by either a single- or a dual-fractal analysis. An attempt is made to relate the binding rate coefficient to the degree of heterogeneity (fractal dimension value) present on the biosensor surface. Binding and dissociation rate coefficient values obtained are presented. The kinetics aspects along with the affinity values presented are of interest, and should along with the rate coefficients presented for the binding and the dissociation phase be of significant interest in help designing better biosensors for an application area that is bound to gain increasing importance in the future.

  13. Evaluation of 3M molecular detection system and ANSR pathogen detection system for rapid detection of salmonella from egg products

    USDA-ARS?s Scientific Manuscript database

    Loop-mediated isothermal amplification (LAMP) is a novel simple detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to evaluate the effectiveness of 3M Molecular Detection System (MDS) and ANSR Pathogen Det...

  14. Prospective Detection of Respiratory Pathogens in Symptomatic Children with Cancer

    PubMed Central

    Srinivasan, Ashok; Gu, Zhengming; Smith, Teresa; Morgenstern, Markus; Sunkara, Anusha; Kang, Guolian; Srivastava, Deo K.; Gaur, Aditya H.; Leung, Wing; Hayden, Randall T.

    2016-01-01

    Background The data on human rhinovirus (HRV), coronavirus (hCoV), bocavirus (hBoV), metapneumovirus (hMPV), Chlamydophila pneumoniae, Mycoplasma pneumoniae and Bordetella pertussis infections in children with cancer is limited. Methods We sought to determine prospectively the prevalence of respiratory pathogens in these children, using multiplexed-PCR. Results We enrolled 253 children with upper, or lower respiratory tract infection (URTI/LRTI) during a one year period. A respiratory virus was detected in 193 (76%) patients; 156 (81%) patients had URTI. Human rhinovirus was the most common virus detected in 97 (62%) and 24 (65%) patients with URTI and LRTI, respectively. Leukemia or lymphoma (LL) was the most common underlying diagnosis in 95 (49%) patients followed by solid tumor 47 (24%), post-hematopoietic stem cell transplant (HCT) 28 (15%), and brain tumor in 23 (12%) patients. By multiple logistic regression analysis hBoV was the most commonly detected respiratory virus in patients with LRTI (P = 0.008; odds ratio, 4.52; 95% confidence interval, 1.48-13.79). Co-infection with more than 1 virus was present in 47 (24%) patients, and did not increase the risk for LRTI. Two (0.7%) patients succumbed to LRTI from parainfluenza virus (PIV)-3 and respiratory syncytial virus/HRV infection, respectively. C.pneumoniae and M.pneumoniae were detected in 4 and 3 patients, respectively. Conclusions HRV was the most common virus detected in children with cancer and post-HCT hospitalized with an acute respiratory illness, and was not associated with increased morbidity. Prospective studies with viral load determination and asymptomatic controls are needed to study the association of these emerging respiratory viruses with LRTI in children with cancer and post-HCT. PMID:23190778

  15. Evaluation of the xTAG Gastrointestinal Pathogen Panel Assay for the Detection of Enteric Pathogens in Kuwait.

    PubMed

    Albert, Manuel John; Rotimi, Vincent Olubunmi; Iqbal, Jamshed; Chehadeh, Wassim

    To evaluate the utility of the Luminex xTAG gastrointestinal pathogen panel (GPP) assay in the detection of enteric pathogens from diarrheal stool samples in Kuwait. The Luminex xTAG GPP assay was used according to the manufacturer's instructions to evaluate single diarrheal stool samples from 109 hospitalized patients at Mubarak Al-Kabeer Hospital, Kuwait, from March 2014 to June 2015. The assay procedure involved nucleic acid extraction from stool samples, amplification of the target by reverse transcriptase polymerase chain reaction, hybridization of the amplified target by probe, detection of the target by the Luminex instrument and computerized data analysis. Conventional microbiological assays were used as the gold standard for comparison. From the 109 diarrheal stool samples, 20 (18.4%) pathogens were detected by the xTAG GPP assay compared to 10 (9.2%) pathogens using conventional assays. Both methods detected 3 Salmonella spp., 3 Clostridium difficile, 2 rotavirus and 2 norovirus. In addition, the xTAG GPP assay detected 1 Shigella sp., 6 Campylobacter spp., 1 Cryptosporidium sp. and 2 Giardia lamblia which were missed by conventional assays. In this study, xTAG GPP detected twice as many pathogens as the conventional assays. We recommend the introduction of this assay in routine diagnostic laboratories for a rapid and better diagnosis and treatment of diarrheal disease. © 2016 S. Karger AG, Basel.

  16. Detection of pathogenic micro-organisms--a contribution to discussion.

    PubMed

    Glaeser, H

    2004-05-01

    When applying methods for the detection of pathogenic micro-organisms in foodstuffs, information on the distribution of the target organism in the foodstuff submitted to a test should be available. The sampling plan used should allow to detect the presence of low levels of the target organism with a high probability. The individual steps of a detection procedure (pre-enrichment, selective enrichment, isolation and confirmation) need careful examination. It is important that inoculation of low levels of the target organism leads to successful enrichment even in the presence of relatively large numbers of competing organisms. In cases where competing micro-organisms form suspect colonies, there is a risk that false-negative results are obtained, because colonies of the target organism may not be isolated. Collaborative trials have to be carried out to assess the performance of presence/absence tests. Meaningful results are obtained only, if the test samples contain low levels of the target organism and if the effect of competing micro-organisms is checked. While it can hardly be disputed that the determination of the sensitivity and specificity of the test method provides valuable information, this cannot be said for "accordance" and "concordance", two recently proposed parameters which correspond to repeatability and reproducibility in quantitative tests. A better alternative may be to specify the probability to obtain two positive results, when analysing samples containing the target organism under repeatability or reproducibility conditions. In an analogous way, the probability to obtain two negative results with samples containing competing micro-organisms, but not the target organism, could be specified.

  17. Exploiting mosquito sugar feeding to detect mosquito-borne pathogens

    PubMed Central

    Hall-Mendelin, Sonja; Ritchie, Scott A.; Johansen, Cheryl A.; Zborowski, Paul; Cortis, Giles; Dandridge, Scott; Hall, Roy A.; van den Hurk, Andrew F.

    2010-01-01

    Arthropod-borne viruses (arboviruses) represent a global public health problem, with dengue viruses causing millions of infections annually, while emerging arboviruses, such as West Nile, Japanese encephalitis, and chikungunya viruses have dramatically expanded their geographical ranges. Surveillance of arboviruses provides vital data regarding their prevalence and distribution that may be utilized for biosecurity measures and the implementation of disease control strategies. However, current surveillance methods that involve detection of virus in mosquito populations or sero-conversion in vertebrate hosts are laborious, expensive, and logistically problematic. We report a unique arbovirus surveillance system to detect arboviruses that exploits the process whereby mosquitoes expectorate virus in their saliva during sugar feeding. In this system, infected mosquitoes captured by CO2-baited updraft box traps are allowed to feed on honey-soaked nucleic acid preservation cards within the trap. The cards are then analyzed for expectorated virus using real-time reverse transcription-PCR. In field trials, this system detected the presence of Ross River and Barmah Forest viruses in multiple traps deployed at two locations in Australia. Viral RNA was preserved for at least seven days on the cards, allowing for long-term placement of traps and continuous collection of data documenting virus presence in mosquito populations. Furthermore no mosquito handling or processing was required and cards were conveniently shipped to the laboratory overnight. The simplicity and efficacy of this approach has the potential to transform current approaches to vector-borne disease surveillance by streamlining the monitoring of pathogens in vector populations. PMID:20534559

  18. Rapid SNP Detection and Genotyping of Bacterial Pathogens by Pyrosequencing.

    PubMed

    Amoako, Kingsley K; Thomas, Matthew C; Janzen, Timothy W; Goji, Noriko

    2017-01-01

    Bacterial identification and typing are fixtures of microbiology laboratories and are vital aspects of our response mechanisms in the event of foodborne outbreaks and bioterrorist events. Whole genome sequencing (WGS) is leading the way in terms of expanding our ability to identify and characterize bacteria through the identification of subtle differences between genomes (e.g. single nucleotide polymorphisms (SNPs) and insertions/deletions). Modern high-throughput technologies such as pyrosequencing can facilitate the typing of bacteria by generating short-read sequence data of informative regions identified by WGS analyses, at a fraction of the cost of WGS. Thus, pyrosequencing systems remain a valuable asset in the laboratory today. Presented in this chapter are two methods developed in the Amoako laboratory that detail the identification and genotyping of bacterial pathogens. The first targets canonical single nucleotide polymorphisms (canSNPs) of evolutionary importance in Bacillus anthracis, the causative agent of Anthrax. The second assay detects Shiga-toxin (stx) genes, which are associated with virulence in Escherichia coli and Shigella spp., and differentiates the subtypes of stx-1 and stx-2 based on SNP loci. These rapid methods provide end users with important information regarding virulence traits as well as the evolutionary and biogeographic origin of isolates.

  19. Molecular detection of Marteilia sydneyi, pathogen of Sydney rock oysters.

    PubMed

    Kleeman, S N; Adlard, R D

    2000-03-14

    The life cycle of Marteilia sydneyi, the aetiological agent of QX disease in the Sydney rock oyster Saccostrea commercialis, is not known. We have developed and optimised 2 diagnostic assays, the polymerase chain reaction (PCR) and in situ hybridisation, for use in investigating the role of possible alternative hosts in the life cycle of this pathogen. PCR primers, designed within the ITS1 rDNA of M. sydneyi, amplified a 195 bp fragment. Sensitivity of the PCR assay was assessed using DNA extracted from known numbers of sporonts purified from infected oyster digestive gland. DNA equivalent to 0.01 sporonts was detectable following agarose gel electrophoresis. The potential inhibitory effect of the presence of host DNA on the PCR assay was tested by the addition of oyster genomic DNA during amplification. Concentrations of host DNA in excess of 50 ng per 20 microliters reaction reduced the sensitivity of the test. Environmental validation of the PCR assay was demonstrated by the amplification of M. sydneyi DNA from 50 ng of genomic DNA extracted from QX-infected oysters. A DNA probe was constructed using the M. sydneyi unique primers and was able to detect 10 pg of M. sydneyi PCR amplified DNA in dot-blot hybridisations. The probe hybridised with presporulating and sporulating M. sydneyi stages in paraffin sections of oyster digestive gland. No non-specific binding was observed. Hybridisation consistency and signal intensity decreased as sporonts matured. While the high sensitivity and specificity of the PCR test will allow rapid screening of large numbers of potential alternative hosts for the presence of parasite DNA, it does not actually identify infective stages. In situ hybridisation conducted on paraffin sections will determine the location of the parasite within the host for morphological characterisation.

  20. Survey of ixodid ticks and two tick-borne pathogens in African buffaloes, Syncerus caffer, from the Caprivi Strip, Namibia.

    PubMed

    Pascucci, Ilaria; Capobianco Dondona, Andrea; Cammà, Cesare; Marcacci, Maurilia; Di Domenico, Marco; Lelli, Rossella; Scacchia, Massimo; Jago, Mark; Khaiseb, Siegfried; Hager, Anna Laure; Tjipura-Zaire, Georgina; Caporale, Vincenzo

    2011-12-01

    A capture operation to ascertain health status in free-ranging buffaloes from six different areas in the Caprivi Strip in the northeast corner of Namibia was conducted in October 2009. Basic information on the ticks and tick-borne pathogens normally found in wildlife from this area are scarce. The objective of this study was to assess the host status of African buffaloes, Syncerus caffer, for ixodid ticks and two selected tick-borne pathogens in the Caprivi Strip, a key area bordering Angola, Zambia, Botswana, and Zimbabwe. Four different tick species have been identified among the 233 collected specimens, and, of 95 tested buffaloes, 54 (57%) were positive for Theileria parva, whereas only 3 (3%) showed evidence of being infected with Ehrlichia ruminantium.

  1. DETECTION AND ENUMERATION OF PATHOGENS AND INDICATOR MICROORGANISMS

    EPA Science Inventory

    Pathogenic microorganisms are routinely discharged to collection systems throughout the world along with a myriad of commensal organisms, organic and inorganic wastes. It is not surprising then that the density of any given pathogen is relatively small in relationship to the popu...

  2. DETECTION AND ENUMERATION OF PATHOGENS AND INDICATOR MICROORGANISMS

    EPA Science Inventory

    Pathogenic microorganisms are routinely discharged to collection systems throughout the world along with a myriad of commensal organisms, organic and inorganic wastes. It is not surprising then that the density of any given pathogen is relatively small in relationship to the popu...

  3. Simultaneous detection of viral and bacterial enteric pathogens using the Seeplex® Diarrhea ACE detection system.

    PubMed

    Coupland, L J; McElarney, I; Meader, E; Cowley, K; Alcock, L; Naunton, J; Gray, J

    2013-10-01

    A panel of 223 faecal samples was analysed to determine the clinical utility of the Seeplex® Diarrhea ACE Detection multiplex PCR system (Seeplex system; Seegene, Korea), a qualitative multiplexing PCR technology that enables simultaneous multi-pathogen detection of four viruses and/or ten bacteria associated with acute gastroenteritis. Conventional diagnostic methods and a norovirus-specific multiplex real-time RT–PCR detected 98 pathogens in 96 samples. The Seeplex system detected 81 pathogens in 75 samples. All samples positive for adenovirus, norovirus, Campylobacter spp., Escherichia coli O157, Shigella spp. or Vibrio spp. were detected by the Seeplex system. Rotavirus, Clostridium difficile toxin B, and Salmonella spp. were not detected in 12.5%, 50% and 15.8% of samples, respectively. Additional multiple infections were detected in 19 samples by the Seeplex system. The Seeplex system provides significant additional diagnostic capability for the syndromic diagnosis of acute gastroenteritis with increased sensitivity for the majority of pathogens.

  4. Detection and treatment of chemical weapons and/or biological pathogens

    DOEpatents

    Mariella Jr., Raymond P.

    2004-09-07

    A system for detection and treatment of chemical weapons and/or biological pathogens uses a detector system, an electrostatic precipitator or scrubber, a circulation system, and a control. The precipitator or scrubber is activated in response to a signal from the detector upon the detection of chemical weapons and/or biological pathogens.

  5. 9 CFR 113.36 - Detection of pathogens by the chicken inoculation test.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Detection of pathogens by the chicken... REQUIREMENTS Standard Procedures § 113.36 Detection of pathogens by the chicken inoculation test. The test for...,000 doses. (b) At least 25 healthy susceptible young chickens, properly identified and obtained...

  6. 9 CFR 113.36 - Detection of pathogens by the chicken inoculation test.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Detection of pathogens by the chicken... REQUIREMENTS Standard Procedures § 113.36 Detection of pathogens by the chicken inoculation test. The test for...,000 doses. (b) At least 25 healthy susceptible young chickens, properly identified and obtained...

  7. 9 CFR 113.36 - Detection of pathogens by the chicken inoculation test.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Detection of pathogens by the chicken... REQUIREMENTS Standard Procedures § 113.36 Detection of pathogens by the chicken inoculation test. The test for...,000 doses. (b) At least 25 healthy susceptible young chickens, properly identified and obtained...

  8. LITERATURE REVIEW OF MOLECULAR METHODS FOR SIMULTANEOUS DETECTION OF PATHOGENS IN WATER

    EPA Science Inventory

    This literature search is a review of molecular technologies (qPCR, microarray, microfluidics and lab-on-a-chip) for simultaneous detection of multiple waterborne pathogens in order to understand the state of the technology. The search content focuses on: pathogen detection witho...

  9. LITERATURE REVIEW OF MOLECULAR METHODS FOR SIMULTANEOUS DETECTION OF PATHOGENS IN WATER

    EPA Science Inventory

    This literature search is a review of molecular technologies (qPCR, microarray, microfluidics and lab-on-a-chip) for simultaneous detection of multiple waterborne pathogens in order to understand the state of the technology. The search content focuses on: pathogen detection witho...

  10. Characterization of novel sufraces by FTIR spectroscopy and atomic force microscopy for food pathogen detection

    USDA-ARS?s Scientific Manuscript database

    Single molecular detection of pathogens and toxins of interest to food safety is within grasp using technology such as Atomic Force Microscopy. Using antibodies or specific aptamers connected to the AFM tip make it possible to detect a pathogen molecule on a surface. However, it also becomes necess...

  11. Detecting human bacterial pathogens in wastewater treatment plants by a high-throughput shotgun sequencing technique.

    PubMed

    Cai, Lin; Zhang, Tong

    2013-05-21

    Human pathogens are one of the major threats to global public health. Wastewater treatment plants (WWTPs) serve as city guts to receive and digest various human pathogens. Several techniques have been developed to detect human pathogens in WWTPs and to assess potential environmental risks. In this study, we employed 24 metagenomic DNA data sets derived from a high-throughput shotgun sequencing technique to more accurately and efficiently detect human bacterial pathogens in influent, activated sludge, and effluent of two Hong Kong WWTPs. Each data set was quality-filtered and normalized to 12,500,000 DNA sequences with a length of 150-190 bp. Then, a BLASTN search against Greengenes general 16S rRNA gene database and human pathogenic bacteria 16S rRNA gene database, a BLASTX search against human pathogenic bacteria virulence factor database, as well as MetaPhlAn analysis were conducted to survey the distribution, diversity, and abundance of human bacterial pathogens. The results revealed that (i) nine bacterial pathogens were detected; (ii) the overall pathogenic bacteria abundance was estimated as 0.06-3.20% in the total bacteria population using 16S rRNA gene fingerprinting; (iii) pathogenic bacteria detected in activated sludge and effluent shared similar profiles but were different from influent based on both 16S rRNA gene and virulence factor fingerprintings; (iv) Mycobacterium tuberculosis -like species may present potential pathogenic risks because it was detected with high abundance in both activated sludge and effluent. These findings provided a comprehensive profile of commonly concerned human pathogens in two Hong Kong WWTPs and demonstrated that the high-throughput shotgun sequencing technique is a feasible and effectual approach for environmental detection of human bacterial pathogens.

  12. Erythrophore cell response to food-associated pathogenic bacteria: implications for detection.

    PubMed

    Hutchison, Janine R; Dukovcic, Stephanie R; Dierksen, Karen P; Carlyle, Calvin A; Caldwell, Bruce A; Trempy, Janine E

    2008-09-01

    Cell-based biosensors have been proposed for use as function-based detectors of toxic agents. We report the use of Betta splendens chromatophore cells, specifically erythrophore cells, for detection of food-associated pathogenic bacteria. Evaluation of erythrophore cell response, using Bacillus spp., has revealed that this response can distinguish pathogenic Bacillus cereus from a non-pathogenic B. cereus ΔplcR deletion mutant and a non-pathogenic Bacillus subtilis. Erythrophore cells were exposed to Salmonella enteritidis, Clostridium perfringens and Clostridium botulinum. Each bacterial pathogen elicited a response from erythrophore cells that was distinguished from the corresponding bacterial growth medium, and this observed response was unique for each bacterial pathogen. These findings suggest that erythrophore cell response has potential for use as a biosensor in the detection and toxicity assessment for food-associated pathogenic bacteria.

  13. Erythrophore cell response to food‐associated pathogenic bacteria: implications for detection

    PubMed Central

    Hutchison, Janine R.; Dukovcic, Stephanie R.; Dierksen, Karen P.; Carlyle, Calvin A.; Caldwell, Bruce A.; Trempy, Janine E.

    2008-01-01

    Summary Cell‐based biosensors have been proposed for use as function‐based detectors of toxic agents. We report the use of Betta splendens chromatophore cells, specifically erythrophore cells, for detection of food‐associated pathogenic bacteria. Evaluation of erythrophore cell response, using Bacillus spp., has revealed that this response can distinguish pathogenic Bacillus cereus from a non‐pathogenic B. cereus ΔplcR deletion mutant and a non‐pathogenic Bacillus subtilis. Erythrophore cells were exposed to Salmonella enteritidis, Clostridium perfringens and Clostridium botulinum. Each bacterial pathogen elicited a response from erythrophore cells that was distinguished from the corresponding bacterial growth medium, and this observed response was unique for each bacterial pathogen. These findings suggest that erythrophore cell response has potential for use as a biosensor in the detection and toxicity assessment for food‐associated pathogenic bacteria. PMID:21261862

  14. Anaerobic culture to detect periodontal and caries pathogens

    PubMed Central

    Tanner, Anne C. R.

    2014-01-01

    Background Anaerobic culture has been critical in our understanding of the oral microbiotas. Highlight Studies in advanced periodontitis in the 1970’s revealed microbial complexes that associated with different clinical presentations. Taxonomy studies identified species newly-observed in periodontitis as Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter (Wolinella) rectus and other Campylobacter species, and Tannerella (Bacteroides) forsythia. Anaerobic culture of initial periodontitis showed overlap in the microbiota with gingivitis, and added Selenomonas noxia and Filifactor alocis as putative periodontal pathogens. Porphyromonas gingivalis and T. forsythia were found to be associated with initial periodontitis in adults. The dominant microbiota of dental caries differs from that of periodontitis. The major cariogenic species are acidogenic and acid tolerant species particularly Streptococcus mutans, and Lactobacillus and Bifidobacterium species. Anaerobic culture of severe early childhood caries revealed a widely diverse microbiota, comparable to that observed using cloning and sequencing. The PCR-based cloning approach, however, underestimated Actinobacteria compared with culture. Only a subset of the caries-associated microbiota was acid tolerant, with different segments of the microbiota cultured on blood agar compared to a low pH acid agar. While the major caries-associated species was S. mutans, a new species, Scardovia wiggsiae, was significantly associated with early childhood caries. Higher counts of S. wiggsiae were also observed in initial white spot carious lesions in adolescents. Conclusion In periodontitis and dental caries, anaerobic culture studies of advanced disease provided a comprehensive analysis of the microbiota of these infections. Anaerobic culture highlighted the limitation of PCR with standard primers that underestimate detection of Actinobacteria. PMID:25678835

  15. Canine Detection of the Volatilome: A Review of Implications for Pathogen and Disease Detection

    PubMed Central

    Angle, Craig; Waggoner, Lowell Paul; Ferrando, Arny; Haney, Pamela; Passler, Thomas

    2016-01-01

    The volatilome is the entire set of volatile organic compounds (VOC) produced by an organism. The accumulation of VOC inside and outside of the body reflects the unique metabolic state of an organism. Scientists are developing technologies to non-invasively detect VOC for the purposes of medical diagnosis, therapeutic monitoring, disease outbreak containment, and disease prevention. Detection dogs are proven to be a valuable real-time mobile detection technology for the detection of VOC related to explosives, narcotics, humans, and many other targets of interests. Little is known about what dogs are detecting when searching for biological targets. It is important to understand where biological VOC originates and how dogs might be able to detect biological targets. This review paper discusses the recent scientific literature involving VOC analysis and postulates potential biological targets for canine detection. Dogs have shown their ability to detect pathogen and disease-specific VOC. Future research will determine if dogs can be employed operationally in hospitals, on borders, in underserved areas, on farms, and in other operational environments to give real-time feedback on the presence of a biological target. PMID:27446935

  16. Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species

    PubMed Central

    Mackie, Ryan S.; Harrison, Thomas; Shariat, Basir; Kind, Trey; Kehl, Timo; Löchelt, Martin; Boucher, Christina

    2017-01-01

    ABSTRACT Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings. PMID:28330894

  17. Novel relapsing fever Borrelia detected in African penguins (Spheniscus demersus) admitted to two rehabilitation centers in South Africa.

    PubMed

    Yabsley, Michael J; Parsons, Nola J; Horne, Elizabeth C; Shock, Barbara C; Purdee, Michaelle

    2012-03-01

    The African penguin, Spheniscus demersus, the only penguin species that breeds in Africa, is endangered, and several diseases including avian malaria, babesiosis, and aspergillosis are common in some populations. From 2002 to 2010, spirochetes morphologically consistent with Borrelia were observed on thin blood smears from 115 of 8,343 (1.4%) African penguins admitted to rehabilitation centers in the Western Cape and Eastern Cape provinces of South Africa. Prevalence rates were significantly higher among chicks and juveniles compared with adults and for birds sampled during the summer months of October to February compared with winter months. The majority of infected birds were ultimately released, despite lack of antibiotic treatment; however, at least one bird is believed to have died of borreliosis based on characteristic gross and microscopic lesions. Analysis of partial flaB gene sequences indicated this was a relapsing fever Borrelia most similar to a Borrelia sp. detected in soft ticks from a seabird colony in Japan. This represents the fourth report of a relapsing fever Borrelia sp. in an avian species and highlights the need for additional studies of potentially pathogenic organisms infecting the African penguin in South Africa.

  18. Detection of pathogenic leptospires in urine from naturally infected cattle by nested PCR.

    PubMed

    Bomfim, Maria Rosa Quaresma; Barbosa-Stancioli, Edel Figueiredo; Koury, Matilde Cota

    2008-11-01

    A nested polymerase chain reaction (PCR) using primers from the LipL32 sequence of Leptospira spp. was used to detect shedding of pathogenic leptospires in urine from naturally infected cattle. Amplicons (497bp) were obtained from 21 pathogenic reference serovars belonging to four species (L. interrogans, L. borgpetersenii, L. santarosai, L. kirschneri). DNA was amplified from 26/30 urine samples taken from cattle with suspected leptospirosis and from leptospires cultivated from 10 of these samples. The limit of detection of DNA in the clinical samples was 200pg and the nested PCR detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified using DNA from other common bacterial species from the bovine urogenital tract or urine, or from the non-pathogenic L. biflexa Andamana serovar. The nested PCR exhibited high specificity and sensitivity for detection of pathogenic serovars in urine from cattle.

  19. Novel strategies to enhance lateral flow immunoassay sensitivity for detecting foodborne pathogens.

    PubMed

    Shan, Shan; Lai, Weihua; Xiong, Yonghua; Wei, Hua; Xu, Hengyi

    2015-01-28

    Food contaminated by foodborne pathogens causes diseases, affects individuals, and even kills those affected individuals. As such, rapid and sensitive detection methods should be developed to screen pathogens in food. One current detection method is lateral flow immunoassay, an efficient technique because of several advantages, including rapidity, simplicity, stability, portability, and sensitivity. This review presents the format and principle of lateral flow immunoassay strip and the development of conventional lateral flow immunoassay for detecting foodborne pathogens. Furthermore, novel strategies that can be applied to enhance the sensitivity of lateral flow immunoassay to detect foodborne pathogens are presented; these strategies include innovating new label application, designing new formats of lateral flow immunoassay, combining with other methods, and developing signal amplification systems. With these advancements, detection sensitivity and detection time can be greatly improved.

  20. Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array

    PubMed Central

    Devault, Alison M.; McLoughlin, Kevin; Jaing, Crystal; Gardner, Shea; Porter, Teresita M.; Enk, Jacob M.; Thissen, James; Allen, Jonathan; Borucki, Monica; DeWitte, Sharon N.; Dhody, Anna N.; Poinar, Hendrik N.

    2014-01-01

    Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis (“Black Death” plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time. PMID:24603850

  1. Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array.

    PubMed

    Devault, Alison M; McLoughlin, Kevin; Jaing, Crystal; Gardner, Shea; Porter, Teresita M; Enk, Jacob M; Thissen, James; Allen, Jonathan; Borucki, Monica; DeWitte, Sharon N; Dhody, Anna N; Poinar, Hendrik N

    2014-03-06

    Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis ("Black Death" plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time.

  2. Molecular inversion probe: a new tool for highly specific detection of plant pathogens.

    PubMed

    Lau, Han Yih; Palanisamy, Ramkumar; Trau, Matt; Botella, Jose R

    2014-01-01

    Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.

  3. Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens

    NASA Astrophysics Data System (ADS)

    Seo, Youngmin; Kim, Ji-Eun; Jeong, Yoon; Lee, Kwan Hong; Hwang, Jangsun; Hong, Jongwook; Park, Hansoo; Choi, Jonghoon

    2016-01-01

    Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens.

  4. Detection of TORCH pathogens in children with congenital cataracts

    PubMed Central

    Lu, Bin; Yang, Yabo

    2016-01-01

    The aim of the present study was to investigate the correlation between infection rates with TORCH pathogens including toxoplasma, rubella virus, cytomegalovirus, and herpes simplex virus (HSV) I and II and congenital cataracts. In total, the data from 69 children with congenital cataract treated at the Children's Hospital of the Zhejiang University School of Medicine between May 2006 and September 2013 were examined, including the complete serum test results for immunoglobulin (Ig)G and IgM that target TORCH pathogenic antibodies. These results were compared with the antibody levels of 5,914 children in a control group. Using SPSS 19.0 software, variance equation Levene tests, mean equation t tests, and completely randomized design of four tables χ2 tests were applied. The HSV II IgG positivity rates significantly differed between the cataract and control groups. These results suggested that HSV may be one of the pathogenic viruses that leads to congenital cataracts. PMID:27446337

  5. Detecting Directional Selection in the Presence of Recent Admixture in African-Americans

    PubMed Central

    Lohmueller, Kirk E.; Bustamante, Carlos D.; Clark, Andrew G.

    2011-01-01

    We investigate the performance of tests of neutrality in admixed populations using plausible demographic models for African-American history as well as resequencing data from African and African-American populations. The analysis of both simulated and human resequencing data suggests that recent admixture does not result in an excess of false-positive results for neutrality tests based on the frequency spectrum after accounting for the population growth in the parental African population. Furthermore, when simulating positive selection, Tajima's D, Fu and Li's D, and haplotype homozygosity have lower power to detect population-specific selection using individuals sampled from the admixed population than from the nonadmixed population. Fay and Wu's H test, however, has more power to detect selection using individuals from the admixed population than from the nonadmixed population, especially when the selective sweep ended long ago. Our results have implications for interpreting recent genome-wide scans for positive selection in human populations. PMID:21196524

  6. Detecting directional selection in the presence of recent admixture in African-Americans.

    PubMed

    Lohmueller, Kirk E; Bustamante, Carlos D; Clark, Andrew G

    2011-03-01

    We investigate the performance of tests of neutrality in admixed populations using plausible demographic models for African-American history as well as resequencing data from African and African-American populations. The analysis of both simulated and human resequencing data suggests that recent admixture does not result in an excess of false-positive results for neutrality tests based on the frequency spectrum after accounting for the population growth in the parental African population. Furthermore, when simulating positive selection, Tajima's D, Fu and Li's D, and haplotype homozygosity have lower power to detect population-specific selection using individuals sampled from the admixed population than from the nonadmixed population. Fay and Wu's H test, however, has more power to detect selection using individuals from the admixed population than from the nonadmixed population, especially when the selective sweep ended long ago. Our results have implications for interpreting recent genome-wide scans for positive selection in human populations.

  7. Detection of Intestinal Pathogens in River, Shore, and Drinking Water in Lima, Peru.

    PubMed

    Grothen, David C; Zach, Sydney J; Davis, Paul H

    2017-01-01

    Water quality management is an ongoing struggle for many locations worldwide. Current testing of water supplies can be time-consuming, expensive, and lack sensitivity. This study describes an alternative, easy-to-use, and inexpensive method to water sampling and testing at remote locations. This method was employed to detect a number of intestinal pathogens in various locations of Lima, Peru. A total of 34 PCR primer pairs were tested for specificity and high-yield amplification for 12 different pathogens using known DNA templates. Select primers for each pathogen were then tested for minimum detection limits of DNA. Water samples were collected from 22 locations. PCR was used to detect the presence of a pathogen, virulence factors, or differentiate between pathogenic species. In 22 water samples, cholera toxin gene was detected in 4.5% of samples, C. perfringens DNA was detected in 50% of samples, E. histolytica DNA was detected in 54.5% of samples, Giardia intestinalis DNA was detected in 4.5% of samples, Leptospira spp. DNA was detected in 29% of samples, and T. gondii DNA was detected in 31.8% of samples. DNA from three pathogens, C. perfringens, E. histolytica, and T. gondii, were found in residential samples, which accounted for 10 out of 22 samples.

  8. Detection of Intestinal Pathogens in River, Shore, and Drinking Water in Lima, Peru

    PubMed Central

    Grothen, David C.; Zach, Sydney J.; Davis, Paul H.

    2017-01-01

    Water quality management is an ongoing struggle for many locations worldwide. Current testing of water supplies can be time-consuming, expensive, and lack sensitivity. This study describes an alternative, easy-to-use, and inexpensive method to water sampling and testing at remote locations. This method was employed to detect a number of intestinal pathogens in various locations of Lima, Peru. A total of 34 PCR primer pairs were tested for specificity and high-yield amplification for 12 different pathogens using known DNA templates. Select primers for each pathogen were then tested for minimum detection limits of DNA. Water samples were collected from 22 locations. PCR was used to detect the presence of a pathogen, virulence factors, or differentiate between pathogenic species. In 22 water samples, cholera toxin gene was detected in 4.5% of samples, C. perfringens DNA was detected in 50% of samples, E. histolytica DNA was detected in 54.5% of samples, Giardia intestinalis DNA was detected in 4.5% of samples, Leptospira spp. DNA was detected in 29% of samples, and T. gondii DNA was detected in 31.8% of samples. DNA from three pathogens, C. perfringens, E. histolytica, and T. gondii, were found in residential samples, which accounted for 10 out of 22 samples. PMID:28138344

  9. Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins

    PubMed Central

    Byrne, Barry; Stack, Edwina; Gilmartin, Niamh; O'Kennedy, Richard

    2009-01-01

    Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided. PMID:22408533

  10. Using impedance measurements for detecting pathogens trapped in an electric field

    DOEpatents

    Miles, Robin R.

    2004-07-20

    Impedance measurements between the electrodes in an electric field is utilized to detect the presence of pathogens trapped in the electric field. Since particles trapped in a field using the dielectiphoretic force changes the impedance between the electrodes by changing the dielectric material between the electrodes, the degree of particle trapping can be determined by measuring the impedance. This measurement is used to determine if sufficient pathogen have been collected to analyze further or potentially to identify the pathogen.

  11. Nature-inspired magnetoelastic biosentinels for the detection of pathogenic bacteria in stagnant liquids

    NASA Astrophysics Data System (ADS)

    Horikawa, Shin; Chai, Yating; Wikle, Howard C.; Dai, Jing; Hu, Jiajia; Suh, Sang-Jin; Vodyanoy, Vitaly; Chin, Bryan A.

    2015-05-01

    This paper presents an investigation into magnetoelastic (ME) biosentinels that capture and detect low-concentration pathogenic bacteria in stagnant liquids. The ME biosentinels are designed to mimic a variety of white blood cell types, known as the main defensive mechanism in the human body against different pathogenic invaders. The ME biosentinels are composed of a freestanding ME resonator coated with an engineered phage that specifically binds with the pathogens of interest. These biosentinels are ferromagnetic and thus can be moved through a liquid by externally applied magnetic fields. In addition, when a time-varying magnetic field is applied, the ME biosentinels can be placed into mechanical resonance by magnetostriction. As soon as the biosentinels bind with the target pathogen through the phage-based biomolecular recognition, a change in the biosentinel's resonant frequency occurs, and thereby the presence of the target pathogen can be detected. Detection of Bacillus anthracis spores under stagnant flow conditions was demonstrated.

  12. [DNA microarrays and their application in detecting and identifying intestinal pathogens].

    PubMed

    Jin, Da-Zhi; Wen, Si-Yuan; Wang, Sheng-Qi

    2006-06-01

    DNA microarrays offer many advantages of high throughout, automation, rapid detection, and so on. Therefore, this technology had been used in many fields such as molecular epidemiology of bacteria, microbial gene identification, disease mechanism, gene mutation, gene expression identification, DNA sequencing and medicine screening etc. The assays for identifying pathogens using DNA microarrays reported aboard recently are introduced. The application of DNA microarrays in detecting and identifying intestinal pathogens mainly includes three aspects: the identification of toxin and characteristic genes of pathogens, the identification of bacterial DNA or RNA directly, the simultaneous detection of a large number of intestinal pathogens with the target - gene of ribosomal RNA. Because of its high efficiency, DNA microarrays is superior to other biological method. Obviously DNA microarrays technology may be useful in identifying intestinal pathogens and have a wide prospect.

  13. Validation of real-time PCR assays for bioforensic detection of model plant pathogens.

    PubMed

    James, Mindy; Blagden, Trenna; Moncrief, Ian; Burans, James P; Schneider, Katherine; Fletcher, Jacqueline

    2014-03-01

    The U.S. agricultural sector is vulnerable to intentionally introduced microbial threats because of its wide and open distribution and economic importance. To investigate such events, forensically valid assays for plant pathogen detection are needed. In this work, real-time PCR assays were developed for three model plant pathogens: Pseudomonas syringae pathovar tomato, Xylella fastidiosa, and Wheat streak mosaic virus. Validation included determination of the linearity and range, limit of detection, sensitivity, specificity, and exclusivity of each assay. Additionally, positive control plasmids, distinguishable from native signature by restriction enzyme digestion, were developed to support forensic application of the assays. Each assay displayed linear amplification of target nucleic acid, detected 100 fg or less of target nucleic acid, and was specific to its target pathogen. Results obtained with these model pathogens provide the framework for development and validation of similar assays for other plant pathogens of high consequence.

  14. Utilization of Molecular Detection Techniques to Find Soybean Pathogens

    USDA-ARS?s Scientific Manuscript database

    Soybeans continue to rise in prominence as a source of feed, food, oil, and renewable energy. Of many factors impacting yield, microbial pathogens alone can cause significant losses in production. Management of soybean diseases and pests involves many approaches including cultural aspects like crop ...

  15. Human pathogens in plant biofilms: Formation, physiology, and detection

    USDA-ARS?s Scientific Manuscript database

    Fresh produce, viewed as an essential part of a healthy life style is usually consumed in the form of raw or minimally processed fruits and vegetables, and is a potentially important source of food-borne human pathogenic bacteria and viruses. These are passed on to the consumer since the bacteria ca...

  16. Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit.

    PubMed

    Yanagihara, Katsunori; Kitagawa, Yuko; Tomonaga, Masao; Tsukasaki, Kunihiro; Kohno, Shigeru; Seki, Masafumi; Sugimoto, Hisashi; Shimazu, Takeshi; Tasaki, Osamu; Matsushima, Asako; Ikeda, Yasuo; Okamoto, Shinichiro; Aikawa, Naoki; Hori, Shingo; Obara, Hideaki; Ishizaka, Akitoshi; Hasegawa, Naoki; Takeda, Junzo; Kamihira, Shimeru; Sugahara, Kazuyuki; Asari, Seishi; Murata, Mitsuru; Kobayashi, Yoshio; Ginba, Hiroyuki; Sumiyama, Yoshinobu; Kitajima, Masaki

    2010-01-01

    Sepsis is a serious medical condition that requires rapidly administered, appropriate antibiotic treatment. Conventional methods take three or more days for final pathogen identification and antimicrobial susceptibility testing. We organized a prospective observational multicenter study in three study sites to evaluate the diagnostic accuracy and potential clinical utility of the SeptiFast system, a multiplex pathogen detection system used in the clinical setting to support early diagnosis of bloodstream infections. A total of 212 patients, suspected of having systemic inflammatory response syndrome (SIRS) caused by bacterial or fungal infection, were enrolled in the study. From these patients, 407 blood samples were taken and blood culture analysis was performed to identify pathogens. Whole blood was also collected for DNA Detection Kit analysis immediately after its collection for blood culture. The results of the DNA Detection Kit, blood culture and other culture tests were compared. The chosen antimicrobial treatment in patients whose samples tested positive in the DNA Detection Kit and/or blood culture analysis was examined to evaluate the effect of concomitant antibiotic exposure on the results of these analyses. SeptiFast analysis gave a positive result for 55 samples, while 43 samples were positive in blood culture analysis. The DNA Detection Kit identified a pathogen in 11.3% (45/400) of the samples, compared to 8.0% (32/400) by blood culture analysis. Twenty-three pathogens were detected by SeptiFast only; conversely, this system missed five episodes of clinically significant bacteremia (Methicillin-resistant Staphylococcus aureus (MRSA), 2; Pseudomonas aeruginosa, 1; Klebsiella spp, 1; Enterococcus faecium, 1). The number of samples that tested positive was significantly increased by combining the result of the blood culture analysis with those of the DNA Detection Kit analysis (P = 0.01). Among antibiotic pre-treated patients (prevalence, 72%), Septi

  17. Application of Fluorescence Spectroscopy for Rapid Detection of Pathogens in Food

    USDA-ARS?s Scientific Manuscript database

    The potential of fluorescence spectroscopy was investigated for the detection food bone pathogens. E coli, Salmonella and Campylobactor, the most commonly present in food, were selectively identified. Each pathogen, grown in agar plate, was diluted in saline and prepared in different concentrations....

  18. Pathogen detection in produce using applications of immunomagnetic beads and biosensors

    USDA-ARS?s Scientific Manuscript database

    The presence of pathogenic bacteria is of increasing public health concerns. Rapid and sensitive tests to detect pathogens are required to keep the safety of food supply. In this chapter we summarized our previous investigations where we applied immunomagnetic-bead (IMB) capture and various biosen...

  19. Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection

    USDA-ARS?s Scientific Manuscript database

    A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity o...

  20. Hyperspectral imaging using a color camera and its application for pathogen detection

    USDA-ARS?s Scientific Manuscript database

    This paper reports the results of a feasibility study for the development of a hyperspectral image recovery (reconstruction) technique using a RGB color camera and regression analysis in order to detect and classify colonies of foodborne pathogens. The target bacterial pathogens were the six represe...

  1. Pathogenic variants for Mendelian and complex traits in exomes of 6,517 European and African Americans: implications for the return of incidental results.

    PubMed

    Tabor, Holly K; Auer, Paul L; Jamal, Seema M; Chong, Jessica X; Yu, Joon-Ho; Gordon, Adam S; Graubert, Timothy A; O'Donnell, Christopher J; Rich, Stephen S; Nickerson, Deborah A; Bamshad, Michael J

    2014-08-07

    Exome sequencing (ES) is rapidly being deployed for use in clinical settings despite limited empirical data about the number and types of incidental results (with potential clinical utility) that could be offered for return to an individual. We analyzed deidentified ES data from 6,517 participants (2,204 African Americans and 4,313 European Americans) from the National Heart, Lung, and Blood Institute Exome Sequencing Project. We characterized the frequencies of pathogenic alleles in genes underlying Mendelian conditions commonly assessed by newborn-screening (NBS, n = 39) programs, genes associated with age-related macular degeneration (ARMD, n = 17), and genes known to influence drug response (PGx, n = 14). From these 70 genes, we identified 10,789 variants and curated them by manual review of OMIM, HGMD, locus-specific databases, or primary literature to a total of 399 validated pathogenic variants. The mean number of risk alleles per individual was 15.3. Every individual had at least five known PGx alleles, 99% of individuals had at least one ARMD risk allele, and 45% of individuals were carriers for at least one pathogenic NBS allele. The carrier burden for severe recessive childhood disorders was 0.57. Our results demonstrate that risk alleles of potential clinical utility for both Mendelian and complex traits are detectable in every individual. These findings highlight the necessity of developing guidelines and policies that consider the return of results to all individuals and underscore the need to develop innovative approaches and tools that enable individuals to exercise their choice about the return of incidental results.

  2. Fusarium agapanthi sp. nov, a novel bikaverin and fusarubin-producing leaf and stem spot pathogen of Agapanthus praecox (African lily) from Australia and Italy

    USDA-ARS?s Scientific Manuscript database

    This study was conducted to characterize a novel Fusarium species that caused leaf and stem spot on Agapanthus praecox (Agapanthus, African lily) in northern Italy and leaf rot and spot on the same host in Melbourne, Australia. Formally described as Fusarium agapanthi, this pathogen was analyzed usi...

  3. Metagenomic analysis for detecting pathogens in culture-negative infective endocarditis.

    PubMed

    Fukui, Yuto; Aoki, Kotaro; Okuma, Shinnosuke; Sato, Takahiro; Ishii, Yoshikazu; Tateda, Kazuhiro

    2015-12-01

    Pathogen identification is important for proper diagnosis and optimal treatment of infective endocarditis (IE). Blood and valve cultures are the gold standard for detecting pathogens responsible for IE. However, these tests only detect culturable pathogens, and have low sensitivity, especially in patients previously treated with antibiotics. Culture-negative IE is still a major clinical problem and a diagnostic challenge. Recently, metagenomic analysis using next generation sequencing has been used to detect pathogens directly from clinical samples. However, there are very few reports of the use of metagenomic analysis for pathogen identification in culture-negative IE cases and the usefulness of this new method is unknown. Here, we report a case of successful pathogen detection with metagenomic analysis in a patient of culture-negative IE. The patient underwent valve replacement surgery and received antibiotics for 5 weeks and survived. Using metagenomic analysis of resected vegetation, we detected Abiotrophia defectiva, which is often associated with culture-negative IE due to its fastidious growth. This method may be useful for pathogen identification in future cases of culture-negative IE.

  4. Detecting the emergence of novel, zoonotic viruses pathogenic to humans

    PubMed Central

    2015-01-01

    RNA viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. Despite great advances made in diagnostic technology since the 1950s, the annual rate at which novel virulent viruses have been found has remained at 2–3. Most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. An analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. Many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations. PMID:25416679

  5. Detecting the emergence of novel, zoonotic viruses pathogenic to humans.

    PubMed

    Rosenberg, Ronald

    2015-03-01

    RNA viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. Despite great advances made in diagnostic technology since the 1950s, the annual rate at which novel virulent viruses have been found has remained at 2-3. Most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. An analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. Many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations.

  6. Detection of Pathogenic Organisms in Food, Water and Body (Postprint)

    DTIC Science & Technology

    2002-06-27

    genes, lux C, D and E encode for a reductase, a transferase and a synthetase, respectively, that constitute the three enzymes from a multi-enzyme...reus, Listeria and E . coli are performed in this manner. ( 4, 14, 15). Bacteriophages are bacterial viruses, which are widespread in nature and often...bacteriophage specific for pathogenic bacteria. The bioreporter design will incorporate a lux gene from Vibrio fischeri into the genome of the

  7. A1C Combined With Glycated Albumin Improves Detection of Prediabetes in Africans: The Africans in America Study.

    PubMed

    Sumner, Anne E; Duong, Michelle T; Aldana, Paola C; Ricks, Madia; Tulloch-Reid, Marshall K; Lozier, Jay N; Chung, Stephanie T; Sacks, David B

    2016-02-01

    Slowing the diabetes epidemic in Africa requires improved detection of prediabetes. A1C, a form of glycated hemoglobin A, is recommended for diagnosing prediabetes. The glycated proteins, fructosamine and glycated albumin (GA), are hemoglobin-independent alternatives to A1C, but their efficacy in Africans is unknown. Our goals were to determine the ability of A1C, fructosamine, and GA to detect prediabetes in U.S.-based Africans and the value of combining A1C with either fructosamine or GA. Oral glucose tolerance tests (OGTT) were performed in 217 self-identified healthy African immigrants (69% male, age 39 ± 10 years [mean ± SD], BMI 27.6 ± 4.5 kg/m(2)). A1C, fructosamine, and GA were measured. Prediabetes was diagnosed by American Diabetes Association criteria for glucose obtained from a 2-h OGTT. The thresholds to diagnose prediabetes by A1C, fructosamine, and GA were the cutoff at the upper tertile for each variable: ≥5.7% (39 mmol/mol) (range 4.2-6.6% [22.4-48.6 mmol/mol]), ≥230 µmol/L (range 161-269 µmol/L), and ≥13.35% (range 10.20-16.07%), respectively. Prediabetes occurred in 34% (74 of 217). The diagnostic sensitivities of A1C, fructosamine, and GA were 50%, 41%, and 42%, respectively. The P values for comparison with A1C were both >0.3. Combining A1C with either fructosamine or GA increased sensitivities. However, the sensitivity of A1C combined with fructosamine was not better than for A1C alone (72% vs. 50%, P = 0.172). In contrast, the sensitivity of A1C combined with GA was higher than for A1C alone (78% vs. 50%, P < 0.001). As individual tests, A1C, fructosamine, and GA detected ≤50% of Africans with prediabetes. However, combining A1C with GA made it possible to identify nearly 80% of Africans with prediabetes. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  8. A1C Combined With Glycated Albumin Improves Detection of Prediabetes in Africans: The Africans in America Study

    PubMed Central

    Duong, Michelle T.; Aldana, Paola C.; Ricks, Madia; Tulloch-Reid, Marshall K.; Lozier, Jay N.; Chung, Stephanie T.; Sacks, David B.

    2016-01-01

    OBJECTIVE Slowing the diabetes epidemic in Africa requires improved detection of prediabetes. A1C, a form of glycated hemoglobin A, is recommended for diagnosing prediabetes. The glycated proteins, fructosamine and glycated albumin (GA), are hemoglobin-independent alternatives to A1C, but their efficacy in Africans is unknown. Our goals were to determine the ability of A1C, fructosamine, and GA to detect prediabetes in U.S.-based Africans and the value of combining A1C with either fructosamine or GA. RESEARCH DESIGN AND METHODS Oral glucose tolerance tests (OGTT) were performed in 217 self-identified healthy African immigrants (69% male, age 39 ± 10 years [mean ± SD], BMI 27.6 ± 4.5 kg/m2). A1C, fructosamine, and GA were measured. Prediabetes was diagnosed by American Diabetes Association criteria for glucose obtained from a 2-h OGTT. The thresholds to diagnose prediabetes by A1C, fructosamine, and GA were the cutoff at the upper tertile for each variable: ≥5.7% (39 mmol/mol) (range 4.2–6.6% [22.4–48.6 mmol/mol]), ≥230 µmol/L (range 161–269 µmol/L), and ≥13.35% (range 10.20–16.07%), respectively. RESULTS Prediabetes occurred in 34% (74 of 217). The diagnostic sensitivities of A1C, fructosamine, and GA were 50%, 41%, and 42%, respectively. The P values for comparison with A1C were both >0.3. Combining A1C with either fructosamine or GA increased sensitivities. However, the sensitivity of A1C combined with fructosamine was not better than for A1C alone (72% vs. 50%, P = 0.172). In contrast, the sensitivity of A1C combined with GA was higher than for A1C alone (78% vs. 50%, P < 0.001). CONCLUSIONS As individual tests, A1C, fructosamine, and GA detected ≤50% of Africans with prediabetes. However, combining A1C with GA made it possible to identify nearly 80% of Africans with prediabetes. PMID:26681716

  9. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    PubMed

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  10. DETECTION AND IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES IN WATER USING FLOW CYTOMETRY COUPLED WITH TAQMAN PCR

    EPA Science Inventory

    As the incidence of human fungal infection increases, the ability to detect and identify pathogenic fungi in potential environmental reservoirs becomes increasingly important for disease control. PCR based assays are widely used for diagnostic purposes, but may be inadequate for...

  11. DETECTION AND IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES IN WATER USING FLOW CYTOMETRY COUPLED WITH TAQMAN PCR

    EPA Science Inventory

    As the incidence of human fungal infection increases, the ability to detect and identify pathogenic fungi in potential environmental reservoirs becomes increasingly important for disease control. PCR based assays are widely used for diagnostic purposes, but may be inadequate for...

  12. Recommended advanced techniques for waterborne pathogen detection in developing countries.

    PubMed

    Alhamlan, Fatimah S; Al-Qahtani, Ahmed A; Al-Ahdal, Mohammed N

    2015-02-19

    The effect of human activities on water resources has expanded dramatically during the past few decades, leading to the spread of waterborne microbial pathogens. The total global health impact of human infectious diseases associated with pathogenic microorganisms from land-based wastewater pollution was estimated to be approximately three million disability-adjusted life years (DALY), with an estimated economic loss of nearly 12 billion US dollars per year. Although clean water is essential for healthy living, it is not equally granted to all humans. Indeed, people who live in developing countries are challenged every day by an inadequate supply of clean water. Polluted water can lead to health crises that in turn spread waterborne pathogens. Taking measures to assess the water quality can prevent these potential risks. Thus, a pressing need has emerged in developing countries for comprehensive and accurate assessments of water quality. This review presents current and emerging advanced techniques for assessing water quality that can be adopted by authorities in developing countries.

  13. Application of a Non-amplification based Technology to Detect Invasive Fungal Pathogens

    PubMed Central

    Hsu, Joe L.; Binkley, Jon; Clemons, Karl V.; Stevens, David A.; Nicolls, Mark R.; Holodniy, Mark

    2014-01-01

    Current diagnostic techniques for fungal diseases could be improved with respect to sensitivity, specificity and timeliness. To address this clinical need, we adapted a non-amplification based nucleic acid detection technology to identify fungal pathogens. We demonstrate a high-specificity, detection sensitivity, reproducibility and multiplex capacity for detecting fungal strains. PMID:24359934

  14. Immunomagnetic separation for MEMS-based biosensor of waterborne pathogens detection

    NASA Astrophysics Data System (ADS)

    Guo, Jianjiang; Zhang, Rongbiao

    2017-07-01

    Rapid isolation and detection of special pathogens present in environmental drinking water is critical for water quality monitoring. Numerical analysis and experimental investigations on immunomagnetic capture and isolation of waterborne pathogens with magnetic nanoparticles (MNPs) in microfluidic channel are performed. A finite-element COMSOL-based model is established to demonstrate the novel method of on-chip capturing pathogens using MNPs together with periodic pulse magnetic field. Simulation results determine the optimum magnetic pole current and switching frequency for magnetic separation. With the magnetic isolation experiment platform built up, as a pathogen example of Escherichia coli O157:H7, the performance of the method is experimentally verified. Both numerical and experimental results are found to agree reasonably well. Results of these investigations show that the capture efficiency of the immunomagnetic separation method is more than 92%, which could be encouraging for the design and optimization of MEMS-based biosensor of waterborne pathogen detection.

  15. A microfluidic nano-biosensor for the detection of pathogenic Salmonella.

    PubMed

    Kim, Giyoung; Moon, Ji-Hea; Moh, Chang-Yeon; Lim, Jong-guk

    2015-05-15

    Rapid detection of pathogenic Salmonella in food products is extremely important for protecting the public from salmonellosis. The objective of the present study was to explore the feasibility of using a microfluidic nano-biosensor to rapidly detect pathogenic Salmonella. Quantum dot nanoparticles were used to detect Salmonella cells. For selective detection of Salmonella, anti-Salmonella polyclonal antibodies were covalently immobilized onto the quantum dot surface. To separate and concentrate the cells from the sample, superparamagnetic particles and a microfluidic chip were used. A portable fluorometer was developed to measure the fluorescence signal from the quantum dot nanoparticles attached to Salmonella in the samples. The sensitivity for detection of pathogenic Salmonella was evaluated using serially diluted Salmonella Typhimurium in borate buffer and chicken extract. The fluorescence response of the nano-biosensor increased with increasing cell concentration. The detection limit of the sensor was 10(3) CFU/mL Salmonella in both borate buffer and food extract.

  16. A Quantitative Analysis of Complexity of Human Pathogen-Specific CD4 T Cell Responses in Healthy M. tuberculosis Infected South Africans

    PubMed Central

    Lindestam Arlehamn, Cecilia S.; McKinney, Denise M.; Carpenter, Chelsea; Paul, Sinu; Rozot, Virginie; Makgotlho, Edward; Gregg, Yolande; van Rooyen, Michele; Ernst, Joel D.; Hatherill, Mark; Hanekom, Willem A.; Peters, Bjoern; Scriba, Thomas J.; Sette, Alessandro

    2016-01-01

    We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb), in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50–75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total). The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive “megapool” of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response. PMID:27409590

  17. Phage-amplified bioluminescent bioreporters for the detection of foodborne pathogens

    NASA Astrophysics Data System (ADS)

    Ripp, Steven; Young, Jacque C.; Ozen, Aysu; Jegier, Patricia; Johnson, Courtney; Daumer, Kathleen; Garland, Jay; Sayler, Gary S.

    2004-06-01

    The objective of this investigation is to develop a bioluminescent bioreporter system for the detection and monitoring of pathogenic microbial species. Current detection methodologies typically rely on time-consuming sample pre-enrichment steps to elevate pathogen concentrations to detectable levels or DNA based polymerase chain reaction (PCR) techniques that require extensive user training and expensive instrumentation. Detection utilizing bioluminescent bioreporter organisms, however, can provide a simple and rapid means of monitoring foodborne pathogens. Bioluminescent bioreporters are engineered to produce light in response to specific environmental inducers. The light signal is then measured with photodetector devices to generate a quantitative assessment of inducer concentration. The immediate goal of this research effort is to integrate key quorum sensing signal transduction elements into pathogen specific bacteriophages. Upon infection of a unique pathogenic species by the bacteriophages, quorum sensing signals will be generated that will subsequently stimulate bioluminescence in neighboring bioluminescent bioreporter cells. Utilizing both bacteriophages and bioluminescent bioreporters, we realize exceptional pathogen specificity while attaining enhanced bioluminescence production. This integrative approach will lead to rapid pathogen identification without requisite sample pre-enrichment. Additionally, since the bioluminescent response is completely intrinsic to the bioreporter organism, no user interventions are required for generating light signals; the protocol requires only addition of the food sample with the bacteriophage/bioluminescent bioreporter system. Measurement of light responses can be achieved using high-throughput microtiter plate readers, hand-held photomultiplier units, or microchip luminometers.

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

    PubMed Central

    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

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

    PubMed

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

    2014-01-01

    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.

  20. Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

    PubMed Central

    Lui, Clarissa; Cady, Nathaniel C.; Batt, Carl A.

    2009-01-01

    The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform. PMID:22412335

  1. Functionalized Nanopipettes: A Sensitive Tool for Pathogen Detection

    NASA Astrophysics Data System (ADS)

    Actis, P.; Jejelowo, O.; Pourmand, N.

    2010-04-01

    Nanopipette technology is capable of detecting and functional analyzing biomolecules. Preliminary experiments are demonstrating the sensitivity and selectivity of the technique with specific proteins targeting environmental toxins.

  2. Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.

    PubMed Central

    Macek, M; Mackova, A; Hamosh, A; Hilman, B C; Selden, R F; Lucotte, G; Friedman, K J; Knowles, M R; Rosenstein, B J; Cutting, G R

    1997-01-01

    Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans. PMID:9150159

  3. Meeting current public health needs: optical biosensors for pathogen detection and analysis

    NASA Astrophysics Data System (ADS)

    Yang, Minghui; Sapsford, Kim E.; Sergeev, Nikolay; Sun, Steven; Rasooly, Avraham

    2009-02-01

    Pathogen detection and analysis is critical for medicine, food safety, agriculture, public health and biosecurity. Many current microbial detection approaches are based on century-old culturing methods which, while reliable, are slow, provide relatively little information about the pathogens and are not adaptable to high throughput operations. Optical biodetection represents a potential alternative. Most ELISA and chromatography systems are based on optical methods that are also used for analysis of molecular interactions, such as DNA hybridization and protein-protein interactions (e.g. microarrays or SPR biosensors). Various optical biosensor platforms have been developed that have many of the characteristics essential for modern pathogen molecular analysis including sensitivity, speed of analysis, multi-channel capability, relative simplicity and low cost. Here we provide several examples of the use of optical biosensor technology for pathogen detection and analysis including high throughput DNA microarray analysis, SPR-based rapid direct detection of bacterial toxins, CCD-based fluorescent activity analysis of microbial toxins and a simple ECL-based CCD detection system. However, while effective for molecular analysis, most of these technologies are not as sensitive as traditional culturing methods for detecting microorganisms. There is a need to combine optical biosensors with traditional methods to speed culture-based detection and to provide more information regarding the pathogens.

  4. Detection of Bartonella quintana in African Body and Head Lice

    PubMed Central

    Sangaré, Abdoul Karim; Boutellis, Amina; Drali, Rezak; Socolovschi, Cristina; Barker, Stephen C.; Diatta, Georges; Rogier, Christophe; Olive, Marie-Marie; Doumbo, Ogobara K.; Raoult, Didier

    2014-01-01

    Currently, the body louse is the only recognized vector of Bartonella quintana, an organism that causes trench fever. In this work, we investigated the prevalence of this bacterium in human lice in different African countries. We tested 616 head lice and 424 body lice from nine African countries using real-time polymerase chain reaction targeting intergenic spacer region 2 and specific B. quintana genes. Overall, B. quintana DNA was found in 54% and 2% of body and head lice, respectively. Our results also show that there are more body lice positive for B. quintana in poor countries, which was determined by the gross domestic product, than in wealthy areas (228/403 versus 0/21, P < 0.001). A similar finding was obtained for head lice (8/226 versus 2/390, P = 0.007). Our findings suggest that head lice in Africa may be infected by B. quintana when patients live in poor economic conditions and are also exposed to body lice. PMID:24935950

  5. Detection of Bartonella quintana in African body and head lice.

    PubMed

    Sangaré, Abdoul Karim; Boutellis, Amina; Drali, Rezak; Socolovschi, Cristina; Barker, Stephen C; Diatta, Georges; Rogier, Christophe; Olive, Marie-Marie; Doumbo, Ogobara K; Raoult, Didier

    2014-08-01

    Currently, the body louse is the only recognized vector of Bartonella quintana, an organism that causes trench fever. In this work, we investigated the prevalence of this bacterium in human lice in different African countries. We tested 616 head lice and 424 body lice from nine African countries using real-time polymerase chain reaction targeting intergenic spacer region 2 and specific B. quintana genes. Overall, B. quintana DNA was found in 54% and 2% of body and head lice, respectively. Our results also show that there are more body lice positive for B. quintana in poor countries, which was determined by the gross domestic product, than in wealthy areas (228/403 versus 0/21, P < 0.001). A similar finding was obtained for head lice (8/226 versus 2/390, P = 0.007). Our findings suggest that head lice in Africa may be infected by B. quintana when patients live in poor economic conditions and are also exposed to body lice.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Detection of Multiple Waterborne Pathogens Using Microsequencing Arrays

    EPA Science Inventory

    Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically dete...

  8. Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens

    USDA-ARS?s Scientific Manuscript database

    This study deals with the rapid detection and classification of three bacteria, Escherichia coli, Salmonella, and Campylobacter, using fluorescence spectroscopy and multivariative analysis. Each bacterial sample was diluted in physiologic saline for analysis. Fluoroscence spectra were collected ...

  9. Detection of Multiple Waterborne Pathogens Using Microsequencing Arrays

    EPA Science Inventory

    Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically dete...

  10. Bacteriophage Amplification-Coupled Detection and Identification of Bacterial Pathogens

    NASA Astrophysics Data System (ADS)

    Cox, Christopher R.; Voorhees, Kent J.

    Current methods of species-specific bacterial detection and identification are complex, time-consuming, and often require expensive specialized equipment and highly trained personnel. Numerous biochemical and genotypic identification methods have been applied to bacterial characterization, but all rely on tedious microbiological culturing practices and/or costly sequencing protocols which render them impractical for deployment as rapid, cost-effective point-of-care or field detection and identification methods. With a view towards addressing these shortcomings, we have exploited the evolutionarily conserved interactions between a bacteriophage (phage) and its bacterial host to develop species-specific detection methods. Phage amplification-coupled matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) was utilized to rapidly detect phage propagation resulting from species-specific in vitro bacterial infection. This novel signal amplification method allowed for bacterial detection and identification in as little as 2 h, and when combined with disulfide bond reduction methods developed in our laboratory to enhance MALDI-TOF-MS resolution, was observed to lower the limit of detection by several orders of magnitude over conventional spectroscopy and phage typing methods. Phage amplification has been combined with lateral flow immunochromatography (LFI) to develop rapid, easy-to-operate, portable, species-specific point-of-care (POC) detection devices. Prototype LFI detectors have been developed and characterized for Yersinia pestis and Bacillus anthracis, the etiologic agents of plague and anthrax, respectively. Comparable sensitivity and rapidity was observed when phage amplification was adapted to a species-specific handheld LFI detector, thus allowing for rapid, simple, POC bacterial detection and identification while eliminating the need for bacterial culturing or DNA isolation and amplification techniques.

  11. Lab-on-a-chip modules for detection of highly pathogenic bacteria: from sample preparation to detection

    NASA Astrophysics Data System (ADS)

    Julich, S.; Kopinč, R.; Hlawatsch, N.; Moche, C.; Lapanje, A.; Gärtner, C.; Tomaso, H.

    2014-05-01

    Lab-on-a-chip systems are innovative tools for the detection and identification of microbial pathogens in human and veterinary medicine. The major advantages are small sample volume and a compact design. Several fluidic modules have been developed to transform analytical procedures into miniaturized scale including sampling, sample preparation, target enrichment, and detection procedures. We present evaluation data for single modules that will be integrated in a chip system for the detection of pathogens. A microfluidic chip for purification of nucleic acids was established for cell lysis using magnetic beads. This assay was evaluated with spiked environmental aerosol and swab samples. Bacillus thuringiensis was used as simulant for Bacillus anthracis, which is closely related but non-pathogenic for humans. Stationary PCR and a flow-through PCR chip module were investigated for specific detection of six highly pathogenic bacteria. The conventional PCR assays could be transferred into miniaturized scale using the same temperature/time profile. We could demonstrate that the microfluidic chip modules are suitable for the respective purposes and are promising tools for the detection of bacterial pathogens. Future developments will focus on the integration of these separate modules to an entire lab-on-a-chip system.

  12. A novel sensitive pathogen detection system based on Microbead Quantum Dot System.

    PubMed

    Wu, Tzong-Yuan; Su, Yi-Yu; Shu, Wei-Hsien; Mercado, Augustus T; Wang, Shi-Kwun; Hsu, Ling-Yi; Tsai, Yow-Fu; Chen, Chung-Yung

    2016-04-15

    A fast and accurate detection system for pathogens can provide immediate measurements for the identification of infectious agents. Therefore, the Microbead Quantum-dots Detection System (MQDS) was developed to identify and measure target DNAs of pathogenic microorganisms and eliminated the need of PCR amplifications. This nanomaterial-based technique can detect different microorganisms by flow cytometry measurements. In MQDS, pathogen specific DNA probes were designed to form a hairpin structure and conjugated on microbeads. In the presence of the complementary target DNA sequence, the probes will compete for binding with the reporter probes but will not interfere with the binding between the probe and internal control DNA. To monitor the binding process by flow cytometry, both the reporter probes and internal control probes were conjugated with Quantum dots that fluoresce at different emission wavelengths using the click reaction. When MQDS was used to detect the pathogens in environmental samples, a high correlation coefficient (R=0.994) for Legionella spp., with a detection limit of 0.1 ng of the extracted DNAs and 10 CFU/test, can be achieved. Thus, this newly developed technique can also be applied to detect other pathogens, particularly viruses and other genetic diseases.

  13. Advanced Molecular Diagnostic Techniques for Detection of Food-borne Pathogens; Current Applications and Future Challenges.

    PubMed

    Umesha, S; Manukumar, H M

    2016-01-08

    The elimination of disease-causing microbes from the food supply is a primary goal and this review deals with the overall techniques availavle for detection of food-borne pathogens. Now-a-days conventional methods are replaced by advanced methods like Biosensors, Nucleic Acid-based Tests (NAT) and different PCR based techniques used in molecular biology to identify specific pathogens. Bacillus cereus, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Campylobacter, Listeria monocytogenes, Salmonella spp, Aspergillus spp. Fusarium spp. Penicillium spp., and pathogens are detected in contaminated food items which cause always diseases in human in any one or the other way. Identification of food-borne pathogens in a short period of time is still a challenge to the scientific field in general and food technology in particular. The low level of food contamination by major pathogens requires specific sensitive detection platforms and the present area of hot research looking forward to new nanomolecular techniques for nanomaterials, make them suitable for the development of assays with high sensitivity, response time and portability. With the sound of these we attemet to highlight a comprehensive overview about food-borne pathogen detection by rapid, sensitive, accurate and cost affordable in situ analytical methods from conventional methods to recent molecular approaches for advanced food and microbiology research.

  14. Detection of the emerging amphibian pathogens Batrachochytrium dendrobatidis and ranavirus in Russia.

    PubMed

    Reshetnikov, Andrey N; Chestnut, Tara; Brunner, Jesse L; Charles, Kaylene; Nebergall, Emily E; Olson, Deanna H

    2014-08-11

    In a population of the European common toad Bufo bufo from a rural pond in the region of Lake Glubokoe Regional Reserve in Moscow province, Russia, unexplained mass mortality events involving larvae and metamorphs have been observed over a monitoring period of >20 yr. We tested toads from this and a nearby site for the emerging amphibian pathogens Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv). Both pathogens were detected, and at the rural pond site, with the above-noted losses and decline in toad breeding success, 40% of B. bufo metamorphs were Bd positive, 46% were Rv positive and 20% were co-infected with both pathogens. Toad metamorphs from a neighbouring water body were also Bd and Rv positive (25 and 55%, respectively). This is the first confirmation of these pathogens in Russia. Questions remain as to the origins of these pathogens in Russia and their roles in documented mass mortality events.

  15. Detection of the emerging amphibian pathogens Batrachochytrium dendrobatidis and ranavirus in Russia

    USGS Publications Warehouse

    Reshetnikov, Andrey N.; Chestnut, Tara E.; Brunner, Jesse L.; Charles, Kaylene M.; Nebergall, Emily E.; Olson, Deanna H.

    2014-01-01

    In a population of the European common toad Bufo bufo from a rural pond in the region of Lake Glubokoe Regional Reserve in Moscow province, Russia, unexplained mass mortality events involving larvae and metamorphs have been observed over a monitoring period of >20 yr. We tested toads from this and a nearby site for the emerging amphibian pathogens Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv). Both pathogens were detected, and at the rural pond site, with the above-noted losses and decline in toad breeding success, 40% of B. bufo metamorphs were Bd positive, 46% were Rv positive and 20% were co-infected with both pathogens. Toad metamorphs from a neighbouring water body were also Bd and Rv positive (25 and 55%, respectively). This is the first confirmation of these pathogens in Russia. Questions remain as to the origins of these pathogens in Russia and their roles in documented mass mortality events.

  16. Surface plasmon resonance biosensors for detection of foodborne pathogens and toxins

    NASA Astrophysics Data System (ADS)

    Homola, Jiří; Hegnerová, Kateřina; Vala, Milan

    2009-02-01

    In the last decade surface plasmon resonance (SPR) biosensors have made great strides both in terms of technology and its applications. SPR biosensors have become a central tool for study of molecular interactions and have been widely used for detection of chemical and biological analytes. Food analysis belongs to major areas of potential applications of SPR biosensors. Therefore, numerous SPR biosensors for detection of analytes implicated in food safety (e.g. pathogens, toxins, drug residues, vitamins, hormones, chemical contaminants, and allergens) have been developed. This paper reviews recent developments in the field of SPR biosensors for food safety, in particular, for detection of foodborne pathogens and toxins.

  17. Periodontal profile and presence of periodontal pathogens in young African-Americans from Salvador, Ba, Brazil

    PubMed Central

    Victor, Ligia Valéria; Cortelli, Sheila Cavalca; Aquino, Davi Romeiro; de Carvalho Filho, Jonas; Cortelli, José Roberto

    2008-01-01

    This cross-sectional study evaluated the periodontal status and the presence of periodontopathogens in 132 young, black ethnic subjects who live in Salvador/Bahia-Brazil and have never smoked. Periodontal Probing Depth (PPD), Clinical Attachment Level (CAL), Plaque Index (PI) and Gingival Index (GI) were measured and analyzed by ANOVA and Wilcoxon tests (p<0.05) according to gender and age. The presence of A.actinomycetemcomitans, P.gingivalis, E.corrodens and F.nucleatum was determined by PCR and was analyzed by ANOVA, Wilcoxon, Student-t tests (p<0.05). Mean values of PPD and CAL were 2.18 and 1.0mm, respectively. Clinical parameters did not show differences between subjects of varying gender and age. The microbial prevalence was observed to be 95.45% for E.corrodens followed by F.nucleatum with 68.18%, A.actinomycetemcomitans with 45.45% and P gingivalis with 40.9%. An association between the presence of pathogens and gender and age was not observed (p<0.05). PPD, CAL and PI were not associated with P.gingivalis; however, GI appeared in higher frequencies among subjects without P.gingivalis. In this young, black ethnic, Brazilian population, a high percentage (96.96%) of subjects harbored at least one selected periodontal pathogen, but most subjects showed a healthy periodontal status. Further investigations are required to evaluate the actual influence of the presence of these bacterial species. PMID:24031206

  18. Development of a Cancer Prevention and Early Detection Program for Nurses Working with African Americans.

    ERIC Educational Resources Information Center

    Underwood, Sandra Millon

    1999-01-01

    Evaluation of a program for 250 nurses working with African Americans showed it enhanced their ability to provide cancer education, screening, and follow-up. It also heightened sensitivity to, and understanding of, issues and trends influencing prevention and early detection for this population. (SK)

  19. Biocontrol and Rapid Detection of Food-Borne Pathogens Using Bacteriophages and Endolysins

    PubMed Central

    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. PMID:27092128

  20. Molecular Detection and Characterization of Tick-borne Pathogens in Dogs and Ticks from Nigeria

    PubMed Central

    Kamani, Joshua; Baneth, Gad; Mumcuoglu, Kosta Y.; Waziri, Ndadilnasiya E.; Eyal, Osnat; Guthmann, Yifat; Harrus, Shimon

    2013-01-01

    Background Only limited information is currently available on the prevalence of vector borne and zoonotic pathogens in dogs and ticks in Nigeria. The aim of this study was to use molecular techniques to detect and characterize vector borne pathogens in dogs and ticks from Nigeria. Methodology/Principal Findings Blood samples and ticks (Rhipicephalus sanguineus, Rhipicephalus turanicus and Heamaphysalis leachi) collected from 181 dogs from Nigeria were molecularly screened for human and animal vector-borne pathogens by PCR and sequencing. DNA of Hepatozoon canis (41.4%), Ehrlichia canis (12.7%), Rickettsia spp. (8.8%), Babesia rossi (6.6%), Anaplasma platys (6.6%), Babesia vogeli (0.6%) and Theileria sp. (0.6%) was detected in the blood samples. DNA of E. canis (23.7%), H. canis (21.1%), Rickettsia spp. (10.5%), Candidatus Neoehrlichia mikurensis (5.3%) and A. platys (1.9%) was detected in 258 ticks collected from 42 of the 181 dogs. Co- infections with two pathogens were present in 37% of the dogs examined and one dog was co-infected with 3 pathogens. DNA of Rickettsia conorii israelensis was detected in one dog and Rhipicephalus sanguineus tick. DNA of another human pathogen, Candidatus N. mikurensis was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks, and is the first description of Candidatus N. mikurensis in Africa. The Theileria sp. DNA detected in a local dog in this study had 98% sequence identity to Theileria ovis from sheep. Conclusions/Significance The results of this study indicate that human and animal pathogens are abundant in dogs and their ticks in Nigeria and portray the potential high risk of human exposure to infection with these agents. PMID:23505591

  1. Clinical differences between respiratory viral and bacterial mono- and dual pathogen detected among Singapore military servicemen with febrile respiratory illness

    PubMed Central

    Ho, Zheng Jie Marc; Zhao, Xiahong; Cook, Alex R; Loh, Jin Phang; Ng, Sock Hoon; Tan, Boon Huan; Lee, Vernon J

    2015-01-01

    Background Although it is known that febrile respiratory illnesses (FRI) may be caused by multiple respiratory pathogens, there are no population-level studies describing its impact on clinical disease. Methods Between May 2009 and October 2012, 7733 FRI patients and controls in the Singapore military had clinical data and nasal wash samples collected prospectively and sent for PCR testing. Patients with one pathogen detected (mono-pathogen) were compared with those with two pathogens (dual pathogen) for differences in basic demographics and clinical presentation. Results In total, 45.8% had one pathogen detected, 20.2% had two pathogens detected, 30.9% had no pathogens detected, and 3.1% had more than two pathogens. Multiple pathogens were associated with recruits, those with asthma and non-smokers. Influenza A (80.0%), influenza B (73.0%) and mycoplasma (70.6%) were most commonly associated with mono-infections, while adenovirus was most commonly associated with dual infections (62.9%). Influenza A paired with S. pneumoniae had higher proportions of chills and rigors than their respective mono-pathogens (P = 0.03, P = 0.009). H. influenzae paired with either enterovirus or parainfluenzae had higher proportions of cough with phlegm than their respective mono-pathogens. Although there were observed differences in mean proportions of body temperature, nasal symptoms, sore throat, body aches and joint pains between viral and bacterial mono-pathogens, there were few differences between distinct dual-pathogen pairs and their respective mono-pathogen counterparts. Conclusion A substantial number of FRI patients have multiple pathogens detected. Observed clinical differences between patients of dual pathogen and mono-pathogen indicate the likely presence of complex microbial interactions between the various pathogens. PMID:25827870

  2. Polymerase Chain Reaction for Detection of Systemic Plant Pathogens

    USDA-ARS?s Scientific Manuscript database

    This chapter outlines the advances and application of the polymerase chain reaction (PCR) since its development in 1984 and its enhancements and applications to detection of viruses, viroids and phytoplasma in pome and stone fruits. PCR is probably the most rapidly and widely adopted technology eve...

  3. RESEARCH PROGRAM FOR ALERTING DETECTION AND IDENTIFICATION OF PATHOGENS.

    DTIC Science & Technology

    and Proteus vulgaris could be detected in growth medium within 2 hours incubation. The use of cometabilizable substrates, halogen substituted acids...examination of the sera of dogs infected with infectious hepatitus, herpes, and distemper viruses showed a specific response to each virus. The use of

  4. Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection.

    PubMed

    Oh, Seung Jun; Park, Byung Hyun; Jung, Jae Hwan; Choi, Goro; Lee, Doh C; Kim, Do Hyun; Seo, Tae Seok

    2016-01-15

    We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection.

  5. Genome-wide detection of natural selection in African Americans pre- and post-admixture

    PubMed Central

    Jin, Wenfei; Xu, Shuhua; Wang, Haifeng; Yu, Yongguo; Shen, Yiping; Wu, Bailin; Jin, Li

    2012-01-01

    It is particularly meaningful to investigate natural selection in African Americans (AfA) due to the high mortality their African ancestry has experienced in history. In this study, we examined 491,526 autosomal single nucleotide polymorphisms (SNPs) genotyped in 5210 individuals and conducted a genome-wide search for selection signals in 1890 AfA. Several genomic regions showing an excess of African or European ancestry, which were considered the footprints of selection since population admixture, were detected based on a commonly used approach. However, we also developed a new strategy to detect natural selection both pre- and post-admixture by reconstructing an ancestral African population (AAF) from inferred African components of ancestry in AfA and comparing it with indigenous African populations (IAF). Interestingly, many selection-candidate genes identified by the new approach were associated with AfA-specific high-risk diseases such as prostate cancer and hypertension, suggesting an important role these disease-related genes might have played in adapting to a new environment. CD36 and HBB, whose mutations confer a degree of protection against malaria, were also located in the highly differentiated regions between AAF and IAF. Further analysis showed that the frequencies of alleles protecting against malaria in AAF were lower than those in IAF, which is consistent with the relaxed selection pressure of malaria in the New World. There is no overlap between the top candidate genes detected by the two approaches, indicating the different environmental pressures AfA experienced pre- and post-population admixture. We suggest that the new approach is reasonably powerful and can also be applied to other admixed populations such as Latinos and Uyghurs. PMID:22128132

  6. Genome-wide detection of natural selection in African Americans pre- and post-admixture.

    PubMed

    Jin, Wenfei; Xu, Shuhua; Wang, Haifeng; Yu, Yongguo; Shen, Yiping; Wu, Bailin; Jin, Li

    2012-03-01

    It is particularly meaningful to investigate natural selection in African Americans (AfA) due to the high mortality their African ancestry has experienced in history. In this study, we examined 491,526 autosomal single nucleotide polymorphisms (SNPs) genotyped in 5210 individuals and conducted a genome-wide search for selection signals in 1890 AfA. Several genomic regions showing an excess of African or European ancestry, which were considered the footprints of selection since population admixture, were detected based on a commonly used approach. However, we also developed a new strategy to detect natural selection both pre- and post-admixture by reconstructing an ancestral African population (AAF) from inferred African components of ancestry in AfA and comparing it with indigenous African populations (IAF). Interestingly, many selection-candidate genes identified by the new approach were associated with AfA-specific high-risk diseases such as prostate cancer and hypertension, suggesting an important role these disease-related genes might have played in adapting to a new environment. CD36 and HBB, whose mutations confer a degree of protection against malaria, were also located in the highly differentiated regions between AAF and IAF. Further analysis showed that the frequencies of alleles protecting against malaria in AAF were lower than those in IAF, which is consistent with the relaxed selection pressure of malaria in the New World. There is no overlap between the top candidate genes detected by the two approaches, indicating the different environmental pressures AfA experienced pre- and post-population admixture. We suggest that the new approach is reasonably powerful and can also be applied to other admixed populations such as Latinos and Uyghurs.

  7. Evaluation of 3M Molecular Detection System and ANSR Pathogen Detection System for rapid detection of Salmonella from egg products.

    PubMed

    Hu, L; Ma, L M; Zheng, S; He, X; Wang, H; Brown, E W; Hammack, T S; Zhang, G

    2017-05-01

    Isothermal amplification assay is a novel simple detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to evaluate the effectiveness of the 3M Molecular Detection System (MDS) and ANSR Pathogen Detection System (PDS) for the detection of Salmonella in egg products as compared to the Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method and a modified culture method (3M MDS and ANSR PDS preferred method). Two Salmonella ser. Enteritidis (18579, PT4; CDC_2010K_1441, PT8), one Salmonella ser. Heidelberg (607310-1), and one Salmonella ser. Typhimurium (0723) isolates were used in this study. Seven wet egg products and 13 dry egg products were inoculated with these strains individually at 1 to 5 CFU/25 g. One set of test portions was prepared following FDA BAM procedures [with lactose broth (LB) as pre-enrichment broth]. Another set of test portions was prepared using buffered peptone water (BPW) as pre-enrichment broth, as instructed by the 2 detection systems. Results from 3M MDS and ANSR PDS were 100% in agreement with their BPW-based culture method results. When LB was used as pre-enrichment broth, the number of Salmonella positive test portions (80 tested), identified with the BAM, 3M MDS, and ANSR PDS, were 63, 61, and 60, respectively. In conclusion, both 3M MDS and ANSR PDS Salmonella assays were as effective as their BPW based culture methods and were equivalent to the BAM culture method for the detection of Salmonella in egg products. These sensitive isothermal assays can be used as rapid detection tools for Salmonella in egg products provided that BPW is used as pre-enrichment broth. Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  8. Multiplex Polymerase Chain Reaction for Detection of Gastrointestinal Pathogens in Migrant Workers in Qatar.

    PubMed

    Humphrey, John M; Ranbhise, Sanjay; Ibrahim, Emad; Al-Romaihi, Hamad E; Farag, Elmoubasher; Abu-Raddad, Laith J; Glesby, Marshall J

    2016-12-07

    The causes of infectious diarrhea among the migrant worker population in Qatar are not well understood. We conducted a prospective observational study to understand the demographic and clinical characteristics and infectious causes of diarrhea among migrant workers in Doha, Qatar. A total of 126 male workers presenting to the Qatar Red Crescent Worker's Health Center outpatient clinic or emergency department were studied over a 5-month period in 2015-2016. Epidemiologic surveys were administered to all subjects and the prevalence of 22 different stool pathogens was determined using multiplex polymerase chain reaction (PCR) (FilmArray(®) Gastrointestinal PCR). A target pathogen was identified in 62.7% of subjects. Enteropathogenic Escherichia coli was the most prevalent pathogen and was detected in 24.6% of subjects, followed by Salmonella (22.2%), enteroaggregative E. coli (15.1%), Giardia lamblia (9.5%), and enterotoxigenic E. coli (8.7%). Multiple pathogens were identified in 49.3% of positive stool samples. In a multivariable analysis, the presence of a heart rate ≥ 90 (adjusted odds ratio [OR] = 3.7, 95% confidence interval [CI] = 1.4-10.0) and > 5 fecal leukocytes/high-power field (adjusted OR = 2.8, 95% CI = 1.2-7.0) were significant predictors of detecting an acute inflammatory pathogen by PCR. Use of multiplex PCR enabled the detection of gastrointestinal pathogens in a high proportion of cases, illustrating the utility of this diagnostic tool in epidemiologic studies of infectious diarrhea.

  9. Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety.

    PubMed

    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.

  10. Recent Advances in Bacteriophage Based Biosensors for Food-Borne Pathogen Detection

    PubMed Central

    Singh, Amit; Poshtiban, Somayyeh; Evoy, Stephane

    2013-01-01

    Foodborne diseases are a major health concern that can have severe impact on society and can add tremendous financial burden to our health care systems. Rapid early detection of food contamination is therefore relevant for the containment of food-borne pathogens. Conventional pathogen detection methods, such as microbiological and biochemical identification are time-consuming and laborious, while immunological or nucleic acid-based techniques require extensive sample preparation and are not amenable to miniaturization for on-site detection. Biosensors have shown tremendous promise to overcome these limitations and are being aggressively studied to provide rapid, reliable and sensitive detection platforms for such applications. Novel biological recognition elements are studied to improve the selectivity and facilitate integration on the transduction platform for sensitive detection. Bacteriophages are one such unique biological entity that show excellent host selectivity and have been actively used as recognition probes for pathogen detection. This review summarizes the extensive literature search on the application of bacteriophages (and recently their receptor binding proteins) as probes for sensitive and selective detection of foodborne pathogens, and critically outlines their advantages and disadvantages over other recognition elements. PMID:23364199

  11. New method of antibody detection by indirect immunoperoxidase plaque staining for serodiagnosis of African swine fever.

    PubMed Central

    Pan, I C; Huang, T S; Hess, W R

    1982-01-01

    An indirect immunoperoxidase plaque-staining method was developed for detecting antibody to African swine fever virus infection. In both sensitivity and specificity, the test was comparable to indirect immunofluorescence. Because it has all of the desirable features of the indirect immunofluorescence test and may also be readily used for testing large numbers of sera, the indirect immunoperoxidase plaque-staining method can be used as a single and final serodiagnostic test in a large-scale survey of African swine fever. The indirect immunoperoxidase plaque-staining method should be applicable to other viruses that can be adapted to and grown in cell cultures. Images PMID:6185528

  12. Pathogen detection, testing, and control in fresh broccoli sprouts

    PubMed Central

    Fahey, Jed W; Ourisson, Philippe J; Degnan, Frederick H

    2006-01-01

    Background The recent increased interest in consuming green vegetable sprouts has been tempered by the fact that fresh sprouts can in some cases be vehicles for food-borne illnesses. They must be grown according to proper conditions of sanitation and handled as a food product rather than as an agricultural commodity. When sprouts are grown in accordance with the criteria proposed from within the sprout industry, developed by regulatory agencies, and adhered to by many sprouters, green sprouts can be produced with very low risk. Contamination may occur when these guidelines are not followed. Methods A one year program of microbial hold-and-release testing, conducted in concert with strict seed and facility cleaning procedures by 13 U.S. broccoli sprout growers was evaluated. Microbial contamination tests were performed on 6839 drums of sprouts, equivalent to about 5 million consumer packages of fresh green sprouts. Results Only 24 (0.75%) of the 3191 sprout samples gave an initial positive test for Escherichia coli O157:H7 or Salmonella spp., and when re-tested, 3 drums again tested positive. Composite testing (e.g., pooling up to 7 drums for pathogen testing) was equally sensitive to single drum testing. Conclusion By using a "test-and-re-test" protocol, growers were able to minimize crop destruction. By pooling drums for testing, they were also able to reduce testing costs which now represent a substantial portion of the costs associated with sprout growing. The test-and-hold scheme described herein allowed those few batches of contaminated sprouts to be found prior to packaging and shipping. These events were isolated, and only safe sprouts entered the food supply. PMID:16630354

  13. Detection and removal of pathogenic biofilms on medical implant surfaces.

    PubMed

    Dunlop, Patrick; Oliver, Louise; Byrne, Tony; McAdams, Eric

    2005-01-01

    Advances in sensor technology have had a significant impact in medical research and practice in the last decade. However, within the hospital environment problems still exist where the application of sensing technology could provide the solution. The presence of antibiotic resistant bacteria within hospitals and the risk of serious infection that they pose is a cause for concern. This paper describes a research project that has recently started at the University of Ulster investigating the potential of "Sense and Destroy" tactics to reduce the spread of medical device related infections. It is proposed that Electrical Impedance Spectroscopy (EIS) probes implanted within a catheter may be used to detect subclinical biofilm formation. Furthermore, if the presence of a biofilm is detected, activation of a photocatalytic coating on the catheter wall may be used to inactivate the responsible microorganisms.

  14. Porous silicon-based biosensor for pathogen detection.

    PubMed

    Mathew, Finny P; Alocilja, Evangelyn C

    2005-02-15

    A porous silicon-based biosensor for rapid detection of bacteria was fabricated. Silicon (0.01 ohmcm, p-type) was anodized electrochemically in an electrochemical Teflon cell containing ethanoic hydrofluoric acid solution to produce sponge-like porous layer of silicon. Anodizing conditions of 5 mA/cm2 for 85 min proved best for biosensor fabrication. A single-tube chemiluminescence-based assay, previously developed, was adapted to the biosensor for detection of Escherichia coli. Porous silicon chips were functionalized with a dioxetane-Polymyxin B (cell wall permeabilizer) mixture by diffusion and adsorption on to the porous surface. The reaction of beta-galactosidase enzyme from E. coli with the dioxetane substrate generated light at 530 nm. Light emission for the porous silicon biosensor chip with E. coli was significantly greater than that of the control and planar silicon chip with E. coli (P<0.01). Sensitivity of the porous silicon biosensor was determined to be 101-102 colony forming units (CFU) of E. coli. The porous silicon-based biosensor was fabricated and functionalized to successfully detect E. coli and has potential applications in food and environmental testing.

  15. Identification of fungal pathogens in a patient with acute myelogenic leukemia using a pathogen detection array technology

    PubMed Central

    Banerjee, Sagarika; Peck, Kristen N.; Feldman, Michael D.; Schuster, Mindy G.; Alwine, James C.; Robertson, Erle S.

    2016-01-01

    ABSTRACT Invasive zygomycosis in immunocompromised patients results in a high mortality rate, and early identification is crucial to optimize therapy and to reduce morbidity. However, diagnosing specific species of zygomycetes fungi possess challenge in the clinical laboratories. A need for a rapid and sensitive diagnostic tool for early recognition of a zygomycetes fungus in clinical samples to the species level will lead to prompt and accurate therapy and the PathoChip provides one such platform. We utilized a pathogen array technology referred to as PathoChip, comprised of oligonucleotide probes that can detect all the sequenced viruses as well as known pathogenic bacteria, fungi and parasites and family-specific conserved probes, thus providing a means for detecting previously uncharacterized members of a family. We rapidly identified a zygomycetous fungus, Rhizomucor pusillus, an otherwise challenge for the clinical laboratories, predominantly in a patient with acute myelogenous leukemia. This report highlights the value of PathoChip as a diagnostic tool to identify micro-organisms to the species level, especially for those difficult to identify in most clinical laboratories. It will also help clinicians to obtain a critical snapshot of the infection profile of a patient to plan treatment strategies. PMID:26619325

  16. Development of a Multiplex PCR Method to Detect Fungal Pathogens for Quarantine on Exported Cacti.

    PubMed

    Cho, Hyun Ji; Hong, Seong Won; Kim, Hyun-Ju; Kwak, Youn-Sig

    2016-02-01

    Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B. cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum), 510-bp (B. cactivora), 313-bp (P. nicotinae), and 447-bp (P. cactorum). The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti.

  17. Development of a Multiplex PCR Method to Detect Fungal Pathogens for Quarantine on Exported Cacti

    PubMed Central

    Cho, Hyun ji; Hong, Seong Won; Kim, Hyun-ju; Kwak, Youn-Sig

    2016-01-01

    Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B. cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum), 510-bp (B. cactivora), 313-bp (P. nicotinae), and 447-bp (P. cactorum). The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti. PMID:26889115

  18. A handheld real time thermal cycler for bacterial pathogen detection.

    PubMed

    Higgins, James A; Nasarabadi, Shanavaz; Karns, Jeffrey S; Shelton, Daniel R; Cooper, Mary; Gbakima, Aiah; Koopman, Ronald P

    2003-08-15

    The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.

  19. Molecular detection of plant pathogenic bacteria using polymerase chain reaction single-strand conformation polymorphism.

    PubMed

    Srinivasa, Chandrashekar; Sharanaiah, Umesha; Shivamallu, Chandan

    2012-03-01

    The application of polymerase chain reaction (PCR) technology to molecular diagnostics holds great promise for the early identification of agriculturally important plant pathogens. Ralstonia solanacearum, Xanthomoans axonopodis pv. vesicatoria, and Xanthomonas oryzae pv. oryzae are phytopathogenic bacteria, which can infect vegetables, cause severe yield loss. PCR-single-strand conformation polymorphism (PCR-SSCP) is a simple and powerful technique for identifying sequence changes in amplified DNA. The technique of PCR-SSCP is being exploited so far, only to detect and diagnose human bacterial pathogens in addition to plant pathogenic fungi. Selective media and serology are the commonly used methods for the detection of plant pathogens in infected plant materials. In this study, we developed PCR-SSCP technique to identify phytopathogenic bacteria. The PCR product was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids. We tested over 56 isolates of R. solanacearum, 44 isolates of X. axonopodis pv. vesicatoria, and 20 isolates of X. oryzae pv. oryzae. With the use of universal primer 16S rRNA, we could discriminate such species at the genus and species levels. Species-specific patterns were obtained for bacteria R. solanacearum, X. axonopodis pv. vesicatoria, and X. oryzae pv. oryzae. The potential use of PCR-SSCP technique for the detection and diagnosis of phytobacterial pathogens is discussed in the present paper.

  20. A microfluidic system integrated with buried optical fibers for detection of Phalaenopsis orchid pathogens.

    PubMed

    Lin, Chih-Lin; Chang, Wen-Hsin; Wang, Chih-Hung; Lee, Chia-Hwa; Chen, Tzong-Yueh; Jan, Fuh-Jyh; Lee, Gwo-Bin

    2015-01-15

    Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Fast, accurate, and on-site detection of pathogens in these orchids is therefore of critical importance in order to prevent or suppress costly disease outbreaks. Traditional pathogen detection methods are time-consuming, require well-equipped laboratories with highly trained personnel, and cannot be conducted in situ. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by a pre-treatment incubation with magnetic beads, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. A buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip itself by measuring the change in the optical signals caused by the turbidity change associated with a positive amplification. The limit of detection for this system was found to be 25 fg, which is of similar sensitivity to existing, more laborious methods. Therefore, by using the integrated microfluidic system, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65 min.

  1. Evaluation of detection methods for screening meat and poultry products for the presence of foodborne pathogens.

    PubMed

    Bohaychuk, Valerie M; Gensler, Gary E; King, Robin K; Wu, John T; McMullen, Lynn M

    2005-12-01

    Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.

  2. Evaluation of Statens Serum Institut Enteric Medium for Detection of Enteric Pathogens

    PubMed Central

    Blom, Marianne; Meyer, Aase; Gerner-Smidt, Peter; Gaarslev, Knud; Espersen, Frank

    1999-01-01

    The efficacy of the Statens Serum Institut (SSI) enteric medium for isolation and direct identification of enteric pathogens was evaluated. Six different biochemical reactions can be read by using the SSI enteric medium, allowing direct identification of a range of enteric pathogens. All 248 gram-negative bacterial species that were tested grew on the SSI enteric medium. Only 10 of 248 bacteria (4%) showed discrepant results in the biochemical reactions, and none of these were enteric pathogens. Forty-three of 47 enteric pathogens (92%) produced identical rates of semiquantitative growth on the SSI enteric medium and 5% blood agar, whereas three Vibrio spp. and one Aeromonas spp. showed reduced growth. Gram-positive bacteria did not grow on the SSI enteric medium. Most enteric pathogens had a detection limit of 50 bacteria per ml of feces, but higher numbers of Vibrio spp. and some Shigella spp. were required for detection. The growth rates of 125 enteric pathogens and 12 Yersinia spp. on the SSI enteric medium, xylose lysine deoxycholate (XLD), Hektoen enteric (HE), Salmonella-Shigella (SS), and cefsulodin-irgasan-novobiocin (CIN) agar were compared. Detection rates after application of 200 CFU were 99% for SSI enteric medium, 92% for XLD, 88% for HE, and 82% for SS agar. The 12 Yersinia spp. grew excellently on both the SSI enteric medium and CIN agar. We conclude that the performance of the SSI enteric medium compares favorably to those of other media tested. Its ability to detect Yersinia spp. may limit the number of media needed in the typical laboratory. The direct identification of enteric pathogens on the medium may also provide a more rapid diagnosis. PMID:10364603

  3. Rapid, Portable, Multiplexed Detection of Bacterial Pathogens Directly from Clinical Sample Matrices

    PubMed Central

    Phaneuf, Christopher R.; Mangadu, Betty; Piccini, Matthew E.; Singh, Anup K.; Koh, Chung-Yan

    2016-01-01

    Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. This platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min. PMID:27669320

  4. Photoluminescent lateral-flow immunoassay revealed by graphene oxide: highly sensitive paper-based pathogen detection.

    PubMed

    Morales-Narváez, Eden; Naghdi, Tina; Zor, Erhan; Merkoçi, Arben

    2015-08-18

    A paper-based lateral flow immunoassay for pathogen detection that avoids the use of secondary antibodies and is revealed by the photoluminescence quenching ability of graphene oxide is reported. Escherichia coli has been selected as a model pathogen. The proposed device is able to display a highly specific and sensitive performance with a limit of detection of 10 CFU mL(-1) in standard buffer and 100 CFU mL(-1) in bottled water and milk. This low-cost disposable and easy-to-use device will prove valuable for portable and automated diagnostics applications.

  5. Rapid, portable, multiplexed detection of bacterial pathogens directly from clinical sample matrices

    SciTech Connect

    Phaneuf, Christopher R.; Mangadu, Betty Lou Bosano; Piccini, Matthew E.; Singh, Anup K.; Koh, Chung -Yan

    2016-09-23

    Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. Furthermore, this platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min.

  6. Detection of pathogens in foods: the current state-of-the-art and future directions.

    PubMed

    Dwivedi, Hari P; Jaykus, Lee-Ann

    2011-02-01

    Over the last fifty years, microbiologists have developed reliable culture-based techniques to detect food borne pathogens. Although these are considered to be the "gold-standard," they remain cumbersome and time consuming. Despite the advent of rapid detection methods such as ELISA and PCR, it is clear that reduction and/or elimination of cultural enrichment will be essential in the quest for truly real-time detection methods. As such, there is an important role for bacterial concentration and purification from the sample matrix as a step preceding detection, so-called pre-analytical sample processing. This article reviews recent advancements in food borne pathogen detection and discusses future methods with a focus on pre-analytical sample processing, culture independent methods, and biosensors.

  7. Luciferase-Zinc-Finger System for the Rapid Detection of Pathogenic Bacteria.

    PubMed

    Shi, Chu; Xu, Qing; Ge, Yue; Jiang, Ling; Huang, He

    2017-08-09

    Rapid and reliable detection of pathogenic bacteria is crucial for food safety control. Here, we present a novel luciferase-zinc finger system for the detection of pathogens that offers rapid and specific profiling. The system, which uses a zinc-finger protein domain to probe zinc finger recognition sites, was designed to bind the amplified conserved regions of 16S rDNA, and the obtained products were detected using a modified luciferase. The luciferase-zinc finger system not only maintained luciferase activity but also allowed the specific detection of different bacterial species, with a sensitivity as low as 10 copies and a linear range from 10 to 10(4) copies per microliter of the specific PCR product. Moreover, the system is robust and rapid, enabling the simultaneous detection of 6 species of bacteria in artificially contaminated samples with excellent accuracy. Thus, we envision that our luciferase-zinc finger system will have far-reaching applications.

  8. Rapid, electrical impedance detection of bacterial pathogens using immobilized antimicrobial peptides.

    PubMed

    Lillehoj, Peter B; Kaplan, Christopher W; He, Jian; Shi, Wenyuan; Ho, Chih-Ming

    2014-02-01

    The detection of bacterial pathogens plays an important role in many biomedical applications, including clinical diagnostics, food and water safety, and biosecurity. Most current bacterial detection technologies, however, are unsuitable for use in resource-limited settings where the highest disease burdens often exist. Thus, there is an urgent need to develop portable, user-friendly biosensors capable of rapid detection of multiple pathogens in situ. We report a microfluidic chip for multiplexed detection of bacterial cells that uses antimicrobial peptides (AMPs) with species-specific targeting and binding capabilities. The AMPs are immobilized onto an electrical impedance microsensor array and serve as biorecognition elements for bacterial cell detection. Characterization of peptide immobilization on the sensors revealed robust surface binding via cysteine-gold interactions and vertical alignment relative to the sensor surface. Samples containing Streptococcus mutans and Pseudomonas aeruginosa were loaded in the chip, and both microorganisms were detected at minimum concentrations of 10⁵ cfu/mL within 25 min. Measurements performed in a variety of solutions revealed that high-conductivity solutions produced the largest impedance values. By integrating a highly specific bacterial cell capture scheme with rapid electrical detection, this device demonstrates great potential as a next-generation, point-of-care diagnostic platform for the detection of disease-causing pathogenic agents.

  9. A legacy of low-impact logging does not elevate prevalence of potentially pathogenic protozoa in free-ranging gorillas and chimpanzees in the Republic of Congo: logging and parasitism in African apes.

    PubMed

    Gillespie, Thomas R; Morgan, David; Deutsch, J Charlie; Kuhlenschmidt, Mark S; Salzer, Johanna S; Cameron, Kenneth; Reed, Trish; Sanz, Crickette

    2009-12-01

    Many studies have examined the long-term effects of selective logging on the abundance and diversity of free-ranging primates. Logging is known to reduce the abundance of some primate species through associated hunting and the loss of food trees for frugivores; however, the potential role of pathogens in such primate population declines is largely unexplored. Selective logging results in a suite of alterations in host ecology and forest structure that may alter pathogen dynamics in resident wildlife populations. In addition, environmental pollution with human fecal material may present a risk for wildlife infections with zoonotic protozoa, such as Cryptosporidium and Giardia. To better understand this interplay, we compared patterns of infection with these potentially pathogenic protozoa in sympatric western lowland gorillas (Gorilla gorilla gorilla) and chimpanzees (Pan troglodytes troglodytes) in the undisturbed Goualougo Triangle of Nouabalé-Ndoki National Park and the adjacent previously logged Kabo Concession in northern Republic of Congo. No Cryptosporidium infections were detected in any of the apes examined and prevalence of infection with Giardia was low (3.73% overall) and did not differ between logged and undisturbed forest for chimpanzees or gorillas. These results provide a baseline for prevalence of these protozoa in forest-dwelling African apes and suggest that low-intensity logging may not result in long-term elevated prevalence of potentially pathogenic protozoa.

  10. A Legacy of Low-Impact Logging does not Elevate Prevalence of Potentially Pathogenic Protozoa in Free-Ranging Gorillas and Chimpanzees in the Republic of Congo: Logging and Parasitism in African Apes

    PubMed Central

    Morgan, David; Deutsch, J. Charlie; Kuhlenschmidt, Mark S.; Salzer, Johanna S.; Cameron, Kenneth; Reed, Trish; Sanz, Crickette

    2010-01-01

    Many studies have examined the long-term effects of selective logging on the abundance and diversity of free-ranging primates. Logging is known to reduce the abundance of some primate species through associated hunting and the loss of food trees for frugivores; however, the potential role of pathogens in such primate population declines is largely unexplored. Selective logging results in a suite of alterations in host ecology and forest structure that may alter pathogen dynamics in resident wildlife populations. In addition, environmental pollution with human fecal material may present a risk for wildlife infections with zoonotic protozoa, such as Cryptosporidium and Giardia. To better understand this interplay, we compared patterns of infection with these potentially pathogenic protozoa in sympatric western lowland gorillas (Gorilla gorilla gorilla) and chimpanzees (Pan troglodytes troglodytes) in the undisturbed Goualougo Triangle of Nouabalé-Ndoki National Park and the adjacent previously logged Kabo Concession in northern Republic of Congo. No Cryptosporidium infections were detected in any of the apes examined and prevalence of infection with Giardia was low (3.73% overall) and did not differ between logged and undisturbed forest for chimpanzees or gorillas. These results provide a baseline for prevalence of these protozoa in forest-dwelling African apes and suggest that low-intensity logging may not result in long-term elevated prevalence of potentially pathogenic protozoa. PMID:20238141

  11. Innovations in air sampling to detect plant pathogens.

    PubMed

    West, Js; Kimber, Rbe

    2015-01-01

    Many innovations in the development and use of air sampling devices have occurred in plant pathology since the first description of the Hirst spore trap. These include improvements in capture efficiency at relatively high air-volume collection rates, methods to enhance the ease of sample processing with downstream diagnostic methods and even full automation of sampling, diagnosis and wireless reporting of results. Other innovations have been to mount air samplers on mobile platforms such as UAVs and ground vehicles to allow sampling at different altitudes and locations in a short space of time to identify potential sources and population structure. Geographical Information Systems and the application to a network of samplers can allow a greater prediction of airborne inoculum and dispersal dynamics. This field of technology is now developing quickly as novel diagnostic methods allow increasingly rapid and accurate quantifications of airborne species and genetic traits. Sampling and interpretation of results, particularly action-thresholds, is improved by understanding components of air dispersal and dilution processes and can add greater precision in the application of crop protection products as part of integrated pest and disease management decisions. The applications of air samplers are likely to increase, with much greater adoption by growers or industry support workers to aid in crop protection decisions. The same devices are likely to improve information available for detection of allergens causing hay fever and asthma or provide valuable metadata for regional plant disease dynamics.

  12. Detection of respiratory tract pathogens with molecular biology methods.

    PubMed

    Wozniak-Kosek, A; Kosek, J; Zielnik-Jurkiewicz, B

    2015-01-01

    This paper describes the use in routine diagnosis of virological kit, which was designed to identify the 15 most common respiratory viruses in clinical specimens of nasopharyngeal aspirates, swabs, and bronchoalveolar lavage. It is a one-step multiplex RT-PCR system for the detection of influenza virus type A and type B, human respiratory syncytial virus type A, B; human adenovirus, human metapneumovirus, human coronaviruses 229E/NL63 and OC43, human parainfluenza type 1, 2, 3, human rhinovirus type A, B, human enterovirus, and bocavirus 1, 2, 3, 4. The article presents research conducted on the basis of swabs collected from patients who came to the Ear, Nose, and Throat Emergency Care Unit at the Department of Otolaryngology, Military Medical Institute in Warsaw, in February 2013. Due to the nature of work in an laryngological emergency ward, the material was collected only from those patients who reported problems associated with rhinitis or any dysfunction of the upper respiratory tract. The study shows that patients who came to seek laryngological assistance were usually infected with viruses having affinity for the airway epithelium.

  13. Innovations in air sampling to detect plant pathogens

    PubMed Central

    West, JS; Kimber, RBE

    2015-01-01

    Many innovations in the development and use of air sampling devices have occurred in plant pathology since the first description of the Hirst spore trap. These include improvements in capture efficiency at relatively high air-volume collection rates, methods to enhance the ease of sample processing with downstream diagnostic methods and even full automation of sampling, diagnosis and wireless reporting of results. Other innovations have been to mount air samplers on mobile platforms such as UAVs and ground vehicles to allow sampling at different altitudes and locations in a short space of time to identify potential sources and population structure. Geographical Information Systems and the application to a network of samplers can allow a greater prediction of airborne inoculum and dispersal dynamics. This field of technology is now developing quickly as novel diagnostic methods allow increasingly rapid and accurate quantifications of airborne species and genetic traits. Sampling and interpretation of results, particularly action-thresholds, is improved by understanding components of air dispersal and dilution processes and can add greater precision in the application of crop protection products as part of integrated pest and disease management decisions. The applications of air samplers are likely to increase, with much greater adoption by growers or industry support workers to aid in crop protection decisions. The same devices are likely to improve information available for detection of allergens causing hay fever and asthma or provide valuable metadata for regional plant disease dynamics. PMID:25745191

  14. Multiplex PCR assay for simultaneous detection of six major bacterial pathogens of rice.

    PubMed

    Cui, Z; Ojaghian, M R; Tao, Z; Kakar, K U; Zeng, J; Zhao, W; Duan, Y; Vera Cruz, C M; Li, B; Zhu, B; Xie, G

    2016-05-01

    The aim of this study was to develop a multiplex PCR (mPCR) assay for rapid, sensitive and simultaneous detection of six important rice pathogens: Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae. Specific primers were designed through a bioinformatics pipeline. Sensitivity of detection was established using both traditional PCR and quantitative real-time PCR on isolated DNA and on bacterial cells both in vitro and in simulated diseased seeds and the parameters were optimized for an mPCR assay. A total of 150 bacterial strains were tested for specificity. The mPCR assay accurately predicted the presence of pathogens among 44 symptomatic and asymptomatic rice seed, sheath and leaf samples. This study confirmed that this mPCR assay is a rapid, reliable and simple tool for the simultaneous detection of six important rice bacterial pathogens. This study is the first report of a method allowing simultaneous detection of six major rice pathogens. The ability to use crude extracts from plants without bacterial isolation or DNA extraction enhances the value of this mPCR technology for rapid detection and aetiological/epidemiological studies. © 2016 The Society for Applied Microbiology.

  15. RCA-Based Biosensor for Electrical and Colorimetric Detection of Pathogen DNA

    NASA Astrophysics Data System (ADS)

    Jeong, Jaepil; Kim, Hyejin; Lee, Dong Jun; Jung, Byung Jun; Lee, Jong Bum

    2016-05-01

    For the diagnosis and prevention of diseases, a range of strategies for the detection of pathogens have been developed. In this study, we synthesized the rolling circle amplification (RCA)-based biosensor that enables detection of pathogen DNA in two analytical modes. Only in the presence of the target DNA, the template DNA can be continuously polymerized by simply carrying out RCA, which gives rise to a change of surface structure of Au electrodes and the gap between the electrodes. Electrical signal was generated after introducing hydrogen tetrachloroaurate (HAuCl4) to the DNA-coated biosensor for the improvement of the conductivity of DNA, which indicates that the presence of the pathogen DNA can be detected in an electrical approach. Furthermore, the existence of the target DNA was readily detected by the naked eyes through change in colors of the electrodes from bright yellow to orange-red after RCA reaction. The RCA-based biosensor offers a new platform for monitoring of pathogenic DNA with two different detection modes in one system.

  16. Molecular detection of avian pathogens in poultry red mite (Dermanyssus gallinae) collected in chicken farms.

    PubMed

    Huong, Chu Thi Thanh; Murano, Takako; Uno, Yukiko; Usui, Tatsufumi; Yamaguchi, Tsuyoshi

    2014-12-01

    Poultry red mite (PRM, Dermanyssus gallinae) is a blood-sucking ectoparasite as well as a possible vector of several avian pathogens. In this study, to define the role of PRM in the prevalence of avian infectious agents, we used polymerase chain reaction (PCR) to check for the presence of seven pathogens: Avipox virus (APV), Fowl Adenovirus (FAdV), Marek's disease virus (MDV), Erysipelothrix rhusiopathiae (ER), Salmonella enterica (SE), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). A total of 159 PRM samples collected between 2004 and 2012 from 142 chicken farms in 38 prefectures in Japan were examined. APV DNA was detected in 22 samples (13.8%), 19 of which were wild-type APV. 16S ribosomal RNA (16S rRNA) of MS was detected in 15 samples (9.4%), and the mgc2 gene of MG was detected in 2 samples (1.3%). Eight of 15 MS 16S rRNA sequences differed from the vaccine sequence, indicating they were wild-type strains, while both of the MG mgc2 gene sequences detected were identical to the vaccine sequences. Of these avian pathogen-positive mite samples, three were positive for both wild-types of APV and MS. On the other hand, the DNAs of ER, SE, FAdV and MDV were not detected in any samples. These findings indicated that PRM can harbor the wild-type pathogens and might play a role as a vector in spreading these diseases in farms.

  17. Multiplexed bead-based mesofluidic system for detection of food-borne pathogenic bacteria.

    PubMed

    Jin, Sheng-Quan; Yin, Bin-Cheng; Ye, Bang-Ce

    2009-11-01

    In the present study, a simple and rapid multiplexed bead-based mesofluidic system (BMS) was developed for simultaneous detection of food-borne pathogenic bacteria, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazakii, Shigella, Escherichia coli O157:H7, and Campylobacter jejuni. This system is based on utilization of isothiocyanate-modified microbeads that are 250 mum in diameter, which were immobilized with specific amino-modified oligonucleotide probes and placed in polydimethylsiloxane microchannels. PCR products from the pathogens studied were pumped into microchannels to hybridize with the oligonucleotide-modified beads, and hybridization signals were detected using a conventional microarray scanner. The short sequences of nucleic acids (21 bases) and PCR products characteristic of bacterial pathogens could be detected at concentrations of 1 pM and 10 nM, respectively. The detection procedure could be performed in less than 30 min with high sensitivity and specificity. The assay was simple and fast, and the limits of quantification were in the range from 500 to 6,000 CFU/ml for the bacterial species studied. The feasibility of identification of food-borne bacteria was investigated with samples contaminated with bacteria, including milk, egg, and meat samples. The results demonstrated that the BMS method can be used for effective detection of multiple pathogens in different foodstuffs.

  18. Spatial sampling to detect an invasive pathogen outside of an eradication zone.

    PubMed

    Demon, I; Cunniffe, N J; Marchant, B P; Gilligan, C A; van den Bosch, F

    2011-06-01

    Invasive pathogens are known to cause major damage to the environments they invade. Effective control of such invasive pathogens depends on early detection. In this paper we focus on sampling with the aim of detecting an invasive pathogen. To that end, we introduce the concept of optimized spatial sampling, using spatial simulated annealing, to plant pathology. It has been mathematically proven (15) that this optimization method converges to the optimum allocation of sampling points that give the largest detection probability. We show the benefits of the method to plant pathology by (i) first illustrating that optimized spatial sampling can easily be applied for disease detection, and then we show that (ii) combining it with a spatially explicit epidemic model, we can develop optimum sample schemes, i.e., optimum locations to sample that maximize the probability of detecting an invasive pathogen. This method is then used as baseline against which other sampling methods can be tested for their accuracy. For the specific example case of this paper, we test (i) random sampling, (ii) stratified sampling as well as (iii) sampling based on the output of the simulation model (using the most frequently infected hosts as sample points), and (iv) sampling the hosts closest to the outbreak point.

  19. A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment.

    PubMed

    Vutukuru, Manjula Ramya; Sharma, Divya Khandige; Ragavendar, M S; Schmolke, Susanne; Huang, Yiwei; Gumbrecht, Walter; Mitra, Nivedita

    2016-12-01

    Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. New chromogenic plating media for detection and enumeration of pathogenic Listeria spp.--an overview.

    PubMed

    Reissbrodt, Rolf

    2004-08-15

    In recent years a number of selective chromogenic plating media for pathogenic Listeria spp. have been developed and marketed. Their advantages are direct detection and enumeration of pathogenic Listeria spp. utilizing cleavage of substrates by the virulence factor phosphatidylinositol-phospholipase C (PI-PLC) and, to a lesser extent, by phosphatidylcholin-phospholipase C (PC-PLC). There are two groups of such media: the first utilizes cleavage by PI-PLC of L-alpha-phosphatidyl-inositol, forming a white precipitation zone around the colony, combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside for detection of beta-d-glucosidase, which occurs in all Listeria spp. All Listeria spp. produce turquoise colonies on these media which include ALOA , CHROMagar Listeria, BBL CHROMagar Listeria, and OCLA. The second group of media utilizes 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, forming blue-turquoise colonies of pathogenic Listeria spp. and white colonies of non-pathogenic Listeria spp. BCM trade mark Listeria monocytogenes plating medium, Rapid'L.mono and LIMONO-Ident-Agar belong to this group. Selective chromogenic L. monocytogenes plating media offer the attraction of rapid economic detection and enumeration of pathogenic Listeria spp. within 24 or 48 h of incubation at 36+/-1 degrees C. This overview summarises the characteristics of these chromogenic plating media, reviews important evaluations, and focuses on replacement of conventional by these chromogenic plating media, particularly for applications in the food industry.

  1. Detection of pathogens in food using a SERS-based assay in just a few hours

    NASA Astrophysics Data System (ADS)

    Shende, Chetan; Sengupta, Atanu; Huang, Hermes; Farquharson, Stuart

    2014-05-01

    In 2011 Escherichia, Listeria, and Salmonella species infected over 1.2 million people in the United States, resulting in over 23,000 hospitalizations and 650 deaths. In January 2013 President Obama signed into law the Food and Drug Administration (FDA) Food Safety Modernization Act (FSMA), which requires constant microbial testing of food processing equipment and food to minimize contamination and distribution of food tainted with pathogens. The challenge to preventing distribution and consumption of contaminated foods lies in the fact that just a few bacterial cells can rapidly multiply to millions, reaching infectious doses within a few days. Unfortunately, current methods used to detect these few cells rely on similar growth steps to multiply the cells to the point of detection, which also takes a few days. Consequently, there is a critical need for an analyzer that can rapidly extract and detect foodborne pathogens at 1000 colony forming units per gram of food in 1-2 hours (not days), and with a specificity that differentiates from indigenous microflora, so that false alarms are eliminated. In an effort to meet this need, we have been developing an assay that extracts such pathogens from food, selectively binds these pathogens, and produces surface-enhanced Raman spectra (SERS) when read by a Raman analyzer. Here we present SERS measurements of these pathogens in actual food samples using this assay.

  2. Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray.

    PubMed

    Chang, Chin-I; Hung, Pei-Hsin; Wu, Chia-Che; Cheng, Ta Chih; Tsai, Jyh-Ming; Lin, King-Jung; Lin, Chung-Yen

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10(3) CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.

  3. Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray

    PubMed Central

    Chang, Chin-I; Hung, Pei-Hsin; Wu, Chia-Che; Cheng, Ta Chih; Tsai, Jyh-Ming; Lin, King-Jung; Lin, Chung-Yen

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens. PMID:22736973

  4. Marburg Virus Infection Detected in a Common African Bat

    PubMed Central

    Towner, Jonathan S.; Pourrut, Xavier; Albariño, César G.; Nkogue, Chimène Nze; Bird, Brian H.; Grard, Gilda; Ksiazek, Thomas G.; Gonzalez, Jean-Paul; Nichol, Stuart T.; Leroy, Eric M.

    2007-01-01

    Marburg and Ebola viruses can cause large hemorrhagic fever (HF) outbreaks with high case fatality (80–90%) in human and great apes. Identification of the natural reservoir of these viruses is one of the most important topics in this field and a fundamental key to understanding their natural history. Despite the discovery of this virus family almost 40 years ago, the search for the natural reservoir of these lethal pathogens remains an enigma despite numerous ecological studies. Here, we report the discovery of Marburg virus in a common species of fruit bat (Rousettus aegyptiacus) in Gabon as shown by finding virus-specific RNA and IgG antibody in individual bats. These Marburg virus positive bats represent the first naturally infected non-primate animals identified. Furthermore, this is the first report of Marburg virus being present in this area of Africa, thus extending the known range of the virus. These data imply that more areas are at risk for MHF outbreaks than previously realized and correspond well with a recently published report in which three species of fruit bats were demonstrated to be likely reservoirs for Ebola virus. PMID:17712412

  5. Marburg virus infection detected in a common African bat.

    PubMed

    Towner, Jonathan S; Pourrut, Xavier; Albariño, César G; Nkogue, Chimène Nze; Bird, Brian H; Grard, Gilda; Ksiazek, Thomas G; Gonzalez, Jean-Paul; Nichol, Stuart T; Leroy, Eric M

    2007-08-22

    Marburg and Ebola viruses can cause large hemorrhagic fever (HF) outbreaks with high case fatality (80-90%) in human and great apes. Identification of the natural reservoir of these viruses is one of the most important topics in this field and a fundamental key to understanding their natural history. Despite the discovery of this virus family almost 40 years ago, the search for the natural reservoir of these lethal pathogens remains an enigma despite numerous ecological studies. Here, we report the discovery of Marburg virus in a common species of fruit bat (Rousettus aegyptiacus) in Gabon as shown by finding virus-specific RNA and IgG antibody in individual bats. These Marburg virus positive bats represent the first naturally infected non-primate animals identified. Furthermore, this is the first report of Marburg virus being present in this area of Africa, thus extending the known range of the virus. These data imply that more areas are at risk for MHF outbreaks than previously realized and correspond well with a recently published report in which three species of fruit bats were demonstrated to be likely reservoirs for Ebola virus.

  6. Using Giant African Pouched Rats ("Cricetomys Gambianus") to Detect Landmines

    ERIC Educational Resources Information Center

    Poling, Alan; Weetjens, Bart J.; Cox, Christophe; Beyene, Negussie W.; Sully, Andrew

    2010-01-01

    Within the past decade, giant pouched rats have been used successfully to detect landmines. This manuscript summarizes how these rats are trained and used operationally. The information provided is intended to be of practical value toward strengthening best practices in using "Cricetomys" for humanitarian purposes while simultaneously…

  7. Detecting animals in African Savanna with UAVs and the crowds

    NASA Astrophysics Data System (ADS)

    Rey, Nicolas; Volpi, Michele; Joost, Stéphane; Tuia, Devis

    2017-10-01

    Unmanned aerial vehicles (UAVs) offer new opportunities for wildlife monitoring, with several advantages over traditional field-based methods. They have readily been used to count birds, marine mammals and large herbivores in different environments, tasks which are routinely performed through manual counting in large collections of images. In this paper, we propose a semi-automatic system able to detect large mammals in semi-arid Savanna. It relies on an animal-detection system based on machine learning, trained with crowd-sourced annotations provided by volunteers who manually interpreted sub-decimeter resolution color images. The system achieves a high recall rate and a human operator can then eliminate false detections with limited effort. Our system provides good perspectives for the development of data-driven management practices in wildlife conservation. It shows that the detection of large mammals in semi-arid Savanna can be approached by processing data provided by standard RGB cameras mounted on affordable fixed wings UAVs.

  8. Using Giant African Pouched Rats ("Cricetomys Gambianus") to Detect Landmines

    ERIC Educational Resources Information Center

    Poling, Alan; Weetjens, Bart J.; Cox, Christophe; Beyene, Negussie W.; Sully, Andrew

    2010-01-01

    Within the past decade, giant pouched rats have been used successfully to detect landmines. This manuscript summarizes how these rats are trained and used operationally. The information provided is intended to be of practical value toward strengthening best practices in using "Cricetomys" for humanitarian purposes while simultaneously…

  9. Compatible immuno-NASBA LOC device for quantitative detection of waterborne pathogens: design and validation.

    PubMed

    Zhao, Xinyan; Dong, Tao; Yang, Zhaochu; Pires, Nuno; Høivik, Nils

    2012-02-07

    Waterborne pathogens usually pose a global threat to animals and human beings. There has been a growing demand for convenient and sensitive tools to detect the potential emerging pathogens in water. In this study, a lab-on-a-chip (LOC) device based on the real-time immuno-NASBA (immuno-nucleic acid sequence-based amplification) assay was designed, fabricated and verified. The disposable immuno-NASBA chip is modelled on a 96-well ELISA microplate, which contains 43 reaction chambers inside the bionic channel networks. All valves are designed outside the chip and are reusable. The sample and reagent solutions were pushed into each chamber in turn, which was controlled by the valve system. Notably, the immuno-NASBA chip is completely compatible with common microplate readers in a biological laboratory, and can distinguish multiple waterborne pathogens in water samples quantitatively and simultaneously. The performance of the LOC device was demonstrated by detecting the presence of a synthetic peptide, ACTH (adrenocorticotropic hormone) and two common waterborne pathogens, Escherichia coli (E. coli) and rotavirus, in artificial samples. The results indicated that the LOC device has the potential to quantify traces of waterborne pathogens at femtomolar levels with high specificity, although the detection process was still subject to some factors, such as ribonuclease (RNase) contamination and non-specific adsorption. As an ultra-sensitive tool to quantify waterborne pathogens, the LOC device can be used to monitor water quality in the drinking water system. Furthermore, a series of compatible high-throughput LOC devices for monitoring waterborne pathogens could be derived from this prototype with the same design idea, which may render the complicated immuno-NASBA assays convenient to common users without special training.

  10. Bacteriophages for detection and control of bacterial pathogens in food and food-processing environment.

    PubMed

    Brovko, Lubov Y; Anany, Hany; Griffiths, Mansel W

    2012-01-01

    This chapter presents recent advances in bacteriophage research and their application in the area of food safety. Section 1 describes general facts on phage biology that are relevant to their application for control and detection of bacterial pathogens in food and environmental samples. Section 2 summarizes the recently acquired data on application of bacteriophages to control growth of bacterial pathogens and spoilage organisms in food and food-processing environment. Section 3 deals with application of bacteriophages for detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed. The chapter is intended for food scientist and food product developers, and people in food inspection and health agencies with the ultimate goal to attract their attention to the new developing technology that has a tremendous potential in providing means for producing wholesome and safe food.

  11. Extraction of total nucleic acids from ticks for the detection of bacterial and viral pathogens.

    PubMed

    Crowder, Chris D; Rounds, Megan A; Phillipson, Curtis A; Picuri, John M; Matthews, Heather E; Halverson, Justina; Schutzer, Steven E; Ecker, David J; Eshoo, Mark W

    2010-01-01

    Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot.

  12. Extraction of Total Nucleic Acids From Ticks for the Detection of Bacterial and Viral Pathogens

    PubMed Central

    Crowder, Chris D.; Rounds, Megan A.; Phillipson, Curtis A.; Picuri, John M.; Matthews, Heather E.; Halverson, Justina; Schutzer, Steven E.; Ecker, David J.; Eshoo, Mark W.

    2010-01-01

    Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot. PMID:20180313

  13. Bacterial and viral pathogens detected in sea turtles stranded along the coast of Tuscany, Italy.

    PubMed

    Fichi, G; Cardeti, G; Cersini, A; Mancusi, C; Guarducci, M; Di Guardo, G; Terracciano, G

    2016-03-15

    During 2014, six loggerhead turtles, Caretta caretta and one green turtle, Chelonia mydas, found stranded on the Tuscany coast of Italy, were examined for the presence of specific bacterial and viral agents, along with their role as carriers of fish and human pathogens. Thirteen different species of bacteria, 10 Gram negative and 3 Gram positive, were identified. Among them, two strains of Vibrio parahaemolyticus and one strain of Lactococcus garviae were recovered and confirmed by specific PCR protocols. No trh and tdh genes were detected in V. parahaemolyticus. The first isolation of L. garviae and the first detection of Betanodavirus in sea turtles indicate the possibility for sea turtles to act as carriers of fish pathogens. Furthermore, the isolation of two strains of V. parahaemolyticus highlights the possible role of these animals in human pathogens' diffusion.

  14. HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

    PubMed Central

    Noda, Angel Alberto; Rodríguez, Islay; Rodríguez, Yaindrys; Govín, Anamays; Fernández, Carmen; Obregón, Ana Margarita

    2014-01-01

    This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries. PMID:25229221

  15. Improved PCR Methods for Detection of African Rabies and Rabies-Related Lyssaviruses ▿

    PubMed Central

    Coertse, Jessica; Weyer, Jacqueline; Nel, Louis H.; Markotter, Wanda

    2010-01-01

    Eleven different lyssavirus species, four of which occur on the African continent, are presently recognized. These viruses cause rabies, the burden of which is highest in the developing world, where routine laboratory diagnosis is often not available. From an epidemiological and control perspective, it is necessary that diagnostic methods detect the diversity of lyssaviruses present in different regions of the world. A published and widely used heminested reverse transcription-PCR (hnRT-PCR) was evaluated for its ability to detect a panel of diverse African lyssaviruses. Due to the limitations experienced for this assay, an alternative hnRT-PCR was developed. The new assay was found to be accurate and sensitive in the detection of African lyssavirus RNA in a variety of clinical specimens. The assay was further adapted to a real-time PCR platform to allow rapid, one-step, quantitative, and single-probe detection, and an internal control for the verification of sample preparation was included. The limit of detection of the real-time PCR assay was 10 RNA copies per reaction, with inter- and intra-assay variability below 4%. Subsequently, in demonstrating utility, both assays were successfully applied to antemortem rabies diagnosis in humans. We believe that the quantitative real-time PCR assay could find application as a routine confirmatory test for rabies diagnosis in the future and that it will serve as a valuable research tool in the biology of African lyssaviruses. Alternatively, the hnRT-PCR assay can be used in laboratories that do not have access to expensive real-time PCR equipment for sensitive diagnosis of lyssaviruses. PMID:20810772

  16. The detection of African horse sickness virus antigens and antibodies in young Equidae.

    PubMed Central

    Hamblin, C.; Anderson, E. C.; Mellor, P. S.; Graham, S. D.; Mertens, P. P.; Burroughs, J. N.

    1992-01-01

    Four ponies were each inoculated with a different serotype of African horse sickness virus (AHSV) which had been passaged through cell culture in order to achieve attenuation. Three of the ponies died suddenly after showing mild clinical signs, the fourth pony remained clinically normal and was killed at day 38. Infectious AHSV was isolated from blood samples collected at intervals from all four ponies. Positive antigen ELISA reactions were only observed with blood samples from two of the ponies on the two days preceding death. Specific AHSV antibodies were detected by ELISA in serum samples from the other two ponies although one eventually died. African horse sickness viral antigens were detected by ELISA in post-mortem tissue samples collected from all four ponies. No infectious virus could be detected in tissue samples taken post-mortem from the pony which survived African horse sickness (AHS) infection. In the event of a suspected outbreak of AHS it is recommended that sera and heparinized blood should be tested for specific antibodies and AHSV antigen respectively. When available, post-mortem tissues, including spleen, heart, lung and liver, should also be tested for AHSV antigen. Although the ELISA used for the detection of AHSV antigen is highly sensitive and specific, negative ELISA results should be confirmed by virus isolation attempts. PMID:1547837

  17. Development of an Automated Microfluidic System for DNA Collection, Amplification, and Detection of Pathogens

    SciTech Connect

    Hagan, Bethany S.; Bruckner-Lea, Cynthia J.

    2002-12-01

    This project was focused on developing and testing automated routines for a microfluidic Pathogen Detection System. The basic pathogen detection routine has three primary components; cell concentration, DNA amplification, and detection. In cell concentration, magnetic beads are held in a flow cell by an electromagnet. Sample liquid is passed through the flow cell and bacterial cells attach to the beads. These beads are then released into a small volume of fluid and delivered to the peltier device for cell lysis and DNA amplification. The cells are lysed during initial heating in the peltier device, and the released DNA is amplified using polymerase chain reaction (PCR) or strand displacement amplification (SDA). Once amplified, the DNA is then delivered to a laser induced fluorescence detection unit in which the sample is detected. These three components create a flexible platform that can be used for pathogen detection in liquid and sediment samples. Future developments of the system will include on-line DNA detection during DNA amplification and improved capture and release methods for the magnetic beads during cell concentration.

  18. Detection of Microbial Water Quality Indicators and Fecal Waterborne Pathogens in Environmental Waters: A Review of Methods, Applications, and Limitations

    EPA Science Inventory

    Environmental waters are important reservoirs of pathogenic microorganisms, many of which are of fecal origin. In most cases, the presence of pathogens is determined using surrogate bacterial indicators. In other cases, direct detection of the pathogen in question is required. M...

  19. Biomarkers in the Detection of Prostate Cancer in African Americans

    DTIC Science & Technology

    2015-09-01

    effect” tests that can be used to detect occult high grade cancer in patients who are considering active surveillance. This reporting period also...During this last reporting period, we identified cut-off values that optimize this prototype biomarker test for predicting a low risk of occult high...will test the hypothesis that a molecular test for cancer field effects may help differentiate MRI suspicious regions that are contain a an occult

  20. Biomarkers in the Detection of Prostate Cancer in African Americans

    DTIC Science & Technology

    2015-09-01

    that can be used to detect occult high grade cancer in patients who are considering active surveillance. This reporting period also covers a...reporting period, we identified cut-off values that optimize this prototype biomarker test for predicting a low risk of occult high grade cancer. This...hypothesis that a molecular test for cancer field effects may help differentiate MRI suspicious regions that are contain a an occult prostate cancer

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

    PubMed

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

    2017-02-28

    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.

  2. Electroanalytical biosensors and their potential for food pathogen and toxin detection.

    PubMed

    Palchetti, Ilaria; Mascini, Marco

    2008-05-01

    The detection and identification of foodborne pathogens continue to rely on conventional culturing techniques. These are very elaborate, time-consuming, and have to be completed in a microbiology laboratory and are therefore not suitable for on-site monitoring. The need for a more rapid, reliable, specific, and sensitive method of detecting a target analyte, at low cost, is the focus of a great deal of research. Biosensor technology has the potential to speed up the detection, increase specificity and sensitivity, enable high-throughput analysis, and to be used for monitoring of critical control points in food production. This article reviews food pathogen detection methods based on electrochemical biosensors, specifically amperometric, potentiometric, and impedimetric biosensors. The underlying principles and application of these biosensors are discussed with special emphasis on new biorecognition elements, nanomaterials, and lab on a chip technology.

  3. MOLECULAR DETECTION OF INFECTIOUS PATHOGENS OF THE UPPER RESPIRATORY TRACT IN CAPTIVE NONDOMESTIC FELIDS.

    PubMed

    Lara, Valéria M; Benassi, Julia C; Bisetto, Shayne P; de Oliveira, Trícia M; Ferreira, Helena L; Júnior, João P Araújo; Carregaro, Adriano B

    2017-06-01

    Upper respiratory tract disease is a complex infectious disease process with multiple pathogens involved. Identification of infectious agents in wild animals is of great importance for wildlife conservation. The aim of this study was to evaluate the molecular detection of feline herpesvirus type 1, feline calicivirus (FCV), Bordetella bronchiseptica , Chlamydophila felis , and Mycoplasma felis using ocular and nasal swabs in three species of captive nondomestic felids. Mycoplasma felis was detected in two ocular samples of Puma concolor and in one nasal sample of one Panthera onca . FCV was detected in association with M. felis in one P. concolor . The other pathogens tested were not detected. To the authors' knowledge, this is the first report of M. felis in nondomestic felids from Brazil.

  4. Efficiency of Airborne Sample Analysis Platform (ASAP) bioaerosol sampler for pathogen detection

    PubMed Central

    Sharma, Anurag; Clark, Elizabeth; McGlothlin, James D.; Mittal, Suresh K.

    2015-01-01

    The threat of bioterrorism and pandemics has highlighted the urgency for rapid and reliable bioaerosol detection in different environments. Safeguarding against such threats requires continuous sampling of the ambient air for pathogen detection. In this study we investigated the efficacy of the Airborne Sample Analysis Platform (ASAP) 2800 bioaerosol sampler to collect representative samples of air and identify specific viruses suspended as bioaerosols. To test this concept, we aerosolized an innocuous replication-defective bovine adenovirus serotype 3 (BAdV3) in a controlled laboratory environment. The ASAP efficiently trapped the surrogate virus at 5 × 103 plaque-forming units (p.f.u.) [2 × 105 genome copy equivalent] concentrations or more resulting in the successful detection of the virus using quantitative PCR. These results support the further development of ASAP for bioaerosol pathogen detection. PMID:26074900

  5. Electroanalytical Sensors and Devices for Multiplexed Detection of Foodborne Pathogen Microorganisms

    PubMed Central

    Pedrero, María; Campuzano, Susana; Pingarrón, José M.

    2009-01-01

    The detection and identification of pathogen microorganisms still rely on conventional culturing techniques, which are not suitable for on-site monitoring. Therefore, a great research challenge in this field is focused on the need to develop rapid, reliable, specific, and sensitive methods to detect these bacteria at low cost. Moreover, the growing interest in biochip development for large scale screening analysis implies improved miniaturization, reduction of analysis time and cost, and multi-analyte detection, which has nowadays become a crucial challenge. This paper reviews multiplexed foodborne pathogen microorganisms detection methods based on electrochemical sensors incorporating microarrays and other platforms. These devices usually involve antibody-antigen and DNA hybridization specific interactions, although other approaches such as the monitoring of oxygen consumption are also considered. PMID:22346711

  6. Transmutation of Personal Glucose Meters into Portable and Highly Sensitive Microbial Pathogen Detection Platform.

    PubMed

    Wang, Zhenzhen; Chen, Zhaowei; Gao, Nan; Ren, Jinsong; Qu, Xiaogang

    2015-10-07

    Herein, for the first time, we presented a simple and general approach by using personal glucose meters (PGM) for portable and ultrasensitive detection of microbial pathogens. Upon addition of pathogenic bacteria, glucoamylase-quaternized magnetic nanoparticles (GA-QMNPS) conjugates were disrupted by the competitive multivalent interactions between bacteria and QMNPS, resulting in the release of GA. After magnetic separation, the free GA could catalyze the hydrolysis of amylose into glucose for quantitative readout by PGM. In such way, PGM was transmuted into a bacterial detection device and extremely low detection limits down to 20 cells mL(-1) was achieved. More importantly, QMNPS could inhibit the growth of the bacteria and destroy its cellular structure, which enabled bacteria detection and inhibition simultaneously. The simplicity, portability, sensitivity and low cost of presented work make it attractive for clinical applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Utility of multiplex PCR in detecting the causative pathogens for pediatric febrile neutropenia.

    PubMed

    Mitsuda, Yoshihiro; Takeshima, Yasuhiro; Mori, Takeshi; Yanai, Tomoko; Hayakawa, Akira; Matsuo, Masafumi

    2011-08-17

    Febrile neutropenia (FN) is a life-threatening complication, and the primary cause of FN is considered to be microbial infection. Therefore, prompt and appropriate antimicrobial therapy is crucial. Clinicians usually prescribe antimicrobial therapy on the basis of presumptive and empirical data. This is because the causative pathogen for FN in blood culture (BC) analysis is detected several days after sampling. Polymerase chain reaction (PCR) analysis has been used for detecting the causative bacteria of infections. Here, we examined whether multiplex PCR is useful for detecting the causative pathogens for FN patients. We extracted DNA from the patients' whole blood and performed multiplex PCR. In total, 128 samples of 40 patients clinically diagnosed with FN were used in this study. Multiplex PCR analysis revealed the causative pathogen in 3 patients with FN; the DNA fragments amplified were those of Pseudomonas aeruginosa in 2 cases and Psedomonas putida in 1 case. These patients could be started on appropriate antimicrobial therapy a few hours after sampling. However, the DNA fragment of the causative pathogen could not be amplified by PCR in 2 patients, although BC analysis did detect the causative bacteria. Thus, we conclude that multiplex PCR is serviceable in case of FN because of its rapidness. However, BC is also indispensable to treating FN owing to its high sensitivity.

  8. 9 CFR 113.37 - Detection of pathogens by the chicken embryo inoculation test.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... embryo inoculation test. 113.37 Section 113.37 Animals and Animal Products ANIMAL AND PLANT HEALTH... VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.37 Detection of pathogens by the chicken embryo...-serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos. (1)...

  9. 9 CFR 113.37 - Detection of pathogens by the chicken embryo inoculation test.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... embryo inoculation test. 113.37 Section 113.37 Animals and Animal Products ANIMAL AND PLANT HEALTH... VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.37 Detection of pathogens by the chicken embryo...-serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos. (1)...

  10. 9 CFR 113.37 - Detection of pathogens by the chicken embryo inoculation test.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... embryo inoculation test. 113.37 Section 113.37 Animals and Animal Products ANIMAL AND PLANT HEALTH... VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.37 Detection of pathogens by the chicken embryo...-serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos. (1)...

  11. 9 CFR 113.37 - Detection of pathogens by the chicken embryo inoculation test.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... embryo inoculation test. 113.37 Section 113.37 Animals and Animal Products ANIMAL AND PLANT HEALTH... VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.37 Detection of pathogens by the chicken embryo...-serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos. (1)...

  12. 9 CFR 113.37 - Detection of pathogens by the chicken embryo inoculation test.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... embryo inoculation test. 113.37 Section 113.37 Animals and Animal Products ANIMAL AND PLANT HEALTH... VECTORS STANDARD REQUIREMENTS Standard Procedures § 113.37 Detection of pathogens by the chicken embryo...-serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos. (1)...

  13. An enrichment, amplification, and sequence-based typing (EAST) approach for foodborne pathogen detection and surveillance

    USDA-ARS?s Scientific Manuscript database

    Introduction: Detection of foodborne pathogens typically involves microbiological enrichment with subsequent isolation and identification of a pure culture. This is typically followed by strain typing, which provides information critical to outbreak and source investigations. In the early 1990’s pul...

  14. Preclinical Assessment of a Fully Automated Multiplex PCR Panel for Detection of Central Nervous System Pathogens

    PubMed Central

    Slechta, E. S.; Killpack, J. A.; Heyrend, C.; Lunt, T.; Daly, J. A; Hemmert, A. C.

    2015-01-01

    We evaluated a multiplexed PCR panel for the detection of 16 bacterial, viral, and fungal pathogens in cerebrospinal fluid. Panel results were compared to routine testing, and discrepancies were resolved by additional nucleic acid amplification tests or sequencing. Overall, the positive and negative agreements across methods were 92.9% and 91.9%, respectively. PMID:26719436

  15. Ultrasensitive detection and rapid identification of multiple foodborne pathogens with the naked eyes.

    PubMed

    Zhang, Heng; Zhang, Yali; Lin, Yankui; Liang, Tongwen; Chen, Zhihua; Li, Jinfeng; Yue, Zhenfeng; Lv, Jingzhang; Jiang, Qing; Yi, Changqing

    2015-09-15

    In this study, a novel approach for ultrasensitive detection and rapid high-throughput identification of a panel of common foodborne pathogens with the naked eyes is presented. As a proof-of-concept application, a multiple pathogen analysis array is fabricated through immobilizing three specific polyT-capture probes which can respectively recognize rfbE gene (Escherichia coli O157:H7), invA gene (Salmonella enterica), inlA gene (Listeria monocytogenes) on the plastic substrates. PCR has been developed for amplification and labeling target genes of rfbE, invA, inlA with biotin. The biotinated target DNA is then captured onto the surface of plastic strips through specific DNA hybridization. The succeeding staining of biotinated DNA duplexes with avidin-horseradish peroxidise (AV-HRP) and biotinated anti-HRP antibody greatly amplifies the detectable signal through the multiple cycle signal amplification strategy, and thus realizing ultrasensitive and specific detection of the above three pathogens in food samples with the naked eyes. Results showed approximately 5 copies target pathogenic DNA could be detected with the naked eyes. This simple but very efficient colorimetric assay also show excellent anti-interference capability and good stability, and can be readily applied to point-of-care diagnosis.

  16. Hyperspectral image reconstruction using RGB color for foodborne pathogen detection on agar plates

    USDA-ARS?s Scientific Manuscript database

    This paper reports the latest development of a color vision technique for detecting colonies of foodborne pathogens grown on agar plates with a hyperspectral image classification model that was developed using full hyperspectral data. The hyperspectral classification model depended on reflectance sp...

  17. Food pathogen detection using Ag nanorod-based surface plasmon resonance sensor

    USDA-ARS?s Scientific Manuscript database

    Food safety is world-wide issue for protecting public health. Many researchers have been working on development of biosensors for pathogenic bacteria detection. However, current biosensing methods and techniques do not meet the requirement of demanding as a biosensor in terms of sensitivity, speci...

  18. Recent Advancements in Nanobioassays and Nanobiosensors for Foodborne Pathogenic Bacteria Detection

    USDA-ARS?s Scientific Manuscript database

    Bacterial pathogens are one of the leading causes of food safety incidents and product recalls worldwide. Timely detection and identification of microbial contamination in agricultural and food products is crucial for disease prevention and outbreak investigation. Current gold standards are specific...

  19. Molecular versus conventional culture for detection of respiratory bacterial pathogens in poultry.

    PubMed

    Ammar, A M; Abd El-Aziz, N K; Abd El Wanis, S; Bakry, N R

    2016-02-29

    Acute respiratory tract infections are leading causes of morbidity in poultry farms allover the world. Six pathogens; Escherichia coli, Mycoplasma gallisepticum, Staphylococcus aureus, Pasteurella multocida, Mannheimia haemolytica and Pseudomonas aeruginosa were involved in respiratory infections in poultry. Herein, conventional identification procedures and polymerase chain reaction (PCR) were applied for detection of the most common respiratory bacterial pathogens in clinical specimens of poultry obtained from 53 Egyptian farms with various respiratory problems and the results were compared statistically. The analyzed data demonstrated a significantly higher rate of detection of the most recovered microorganisms (P<0.05) by PCR comparing to classical culture procedures. Further, multiplex PCR could detect E. coli, M. gallisepticum, S. aureus and Ps. aeruginosa in a single reaction, however, M. haemolytica was reported in a uinplex system. According to PCR results, the most commonly recorded bacterial pathogens in examined poultry farms were E. coli and Ps. aeruginosa (54.71% each), followed by M. haemolylica (35.85%) and M. gallisepticum (20.75%). In conclusion, PCR assay offered an effective alternative to traditional typing methods for the identification and simultaneous detection of the most clinically relevant respiratory pathogens in poultry.

  20. 9 CFR 113.36 - Detection of pathogens by the chicken inoculation test.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Detection of pathogens by the chicken inoculation test. 113.36 Section 113.36 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS...

  1. Surface plasmon resonance imaging for label-free detection of foodborne pathogens and toxins

    USDA-ARS?s Scientific Manuscript database

    More rapid and efficient detection methods for foodborne pathogenic bacteria and toxins are needed to address the long assay time and limitations in multiplex capacity. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multi...

  2. Reference-compensated surface plasmon resonance biosensor for detection of foodborne pathogens

    NASA Astrophysics Data System (ADS)

    Homola, Jiri; Dostalek, Jakub; Chen, Shengfu; Rasooly, Avraham; Jiang, Shaoyi; Yee, Sinclair S.

    2001-05-01

    We present a dual-channel surface plasmon resonance (SPR) biosensor and demonstrate its applicability to detection of foodborne pathogens such as Staphylococcal enterotoxin B (SEB). Experimental results indicate that the SPR biosensor can detect SEB at very low concentrations: 5 ng/ml in pure samples directly, 0.5 ng/ml in both pure samples and in milk using a sandwich assay.

  3. Evolution of Drosophila resistance against different pathogens and infection routes entails no detectable maintenance costs.

    PubMed

    Faria, Vítor G; Martins, Nelson E; Paulo, Tânia; Teixeira, Luís; Sucena, Élio; Magalhães, Sara

    2015-11-01

    Pathogens exert a strong selective pressure on hosts, entailing host adaptation to infection. This adaptation often affects negatively other fitness-related traits. Such trade-offs may underlie the maintenance of genetic diversity for pathogen resistance. Trade-offs can be tested with experimental evolution of host populations adapting to parasites, using two approaches: (1) measuring changes in immunocompetence in relaxed-selection lines and (2) comparing life-history traits of evolved and control lines in pathogen-free environments. Here, we used both approaches to examine trade-offs in Drosophila melanogaster populations evolving for over 30 generations under infection with Drosophila C Virus or the bacterium Pseudomonas entomophila, the latter through different routes. We find that resistance is maintained after up to 30 generations of relaxed selection. Moreover, no differences in several classical life-history traits between control and evolved populations were found in pathogen-free environments, even under stresses such as desiccation, nutrient limitation, and high densities. Hence, we did not detect any maintenance costs associated with resistance to pathogens. We hypothesize that extremely high selection pressures commonly used lead to the disproportionate expression of costs relative to their actual occurrence in natural systems. Still, the maintenance of genetic variation for pathogen resistance calls for an explanation.

  4. Development of a DNA-based microarray for the detection of zoonotic pathogens in rodent species.

    PubMed

    Giles, Timothy; Yon, Lisa; Hannant, Duncan; Barrow, Paul; Abu-Median, Abu-Bakr

    2015-12-01

    The demand for diagnostic tools that allow simultaneous screening of samples for multiple pathogens is increasing because they overcome the limitations of other methods, which can only screen for a single or a few pathogens at a time. Microarrays offer the advantages of being capable to test a large number of samples simultaneously, screening for multiple pathogen types per sample and having comparable sensitivity to existing methods such as PCR. Array design is often considered the most important process in any microarray experiment and can be the deciding factor in the success of a study. There are currently no microarrays for simultaneous detection of rodent-borne pathogens. The aim of this report is to explicate the design, development and evaluation of a microarray platform for use as a screening tool that combines ease of use and rapid identification of a number of rodent-borne pathogens of zoonotic importance. Nucleic acid was amplified by multiplex biotinylation PCR prior to hybridisation onto microarrays. The array sensitivity was comparable to standard PCR, though less sensitive than real-time PCR. The array presented here is a prototype microarray identification system for zoonotic pathogens that can infect rodent species.

  5. Bioelectronic DNA detection of human papillomaviruses using eSensor™: a model system for detection of multiple pathogens

    PubMed Central

    Vernon, Suzanne D; Farkas, Daniel H; Unger, Elizabeth R; Chan, Vivian; Miller, Donna L; Chen, Yin-Peng; Blackburn, Gary F; Reeves, William C

    2003-01-01

    Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor™) in identifying multiple pathogens. Methods Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences specific for HPV types. Electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1 region that was amplified by using consensus primers served as target DNA. The amplified target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA chip to allow for hybridization with complementary capture probes. Results Two bioelectric chips were designed and successfully detected 86% of the HPV types contained in clinical samples. Conclusions This model system demonstrates the potential of the eSensor platform for rapid and integrated detection of multiple pathogens. PMID:12814521

  6. Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus

    PubMed Central

    Ogata, Hiroyuki; Toyoda, Kensuke; Tomaru, Yuji; Nakayama, Natsuko; Shirai, Yoko; Claverie, Jean-Michel; Nagasaki, Keizo

    2009-01-01

    Heterocapsa circularisquama DNA virus (HcDNAV; previously designated as HcV) is a giant virus (girus) with a ~356-kbp double-stranded DNA (dsDNA) genome. HcDNAV lytically infects the bivalve-killing marine dinoflagellate H. circularisquama, and currently represents the sole DNA virus isolated from dinoflagellates, one of the most abundant protists in marine ecosystems. Its morphological features, genome type, and host range previously suggested that HcDNAV might be a member of the family Phycodnaviridae of Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), though no supporting sequence data was available. NCLDVs currently include two families found in aquatic environments (Phycodnaviridae, Mimiviridae), one mostly infecting terrestrial animals (Poxviridae), another isolated from fish, amphibians and insects (Iridoviridae), and the last one (Asfarviridae) exclusively represented by the animal pathogen African swine fever virus (ASFV), the agent of a fatal hemorrhagic disease in domestic swine. In this study, we determined the complete sequence of the type B DNA polymerase (PolB) gene of HcDNAV. The viral PolB was transcribed at least from 6 h post inoculation (hpi), suggesting its crucial function for viral replication. Most unexpectedly, the HcDNAV PolB sequence was found to be closely related to the PolB sequence of ASFV. In addition, the amino acid sequence of HcDNAV PolB showed a rare amino acid substitution within a motif containing highly conserved motif: YSDTDS was found in HcDNAV PolB instead of YGDTDS in most dsDNA viruses. Together with the previous observation of ASFV-like sequences in the Sorcerer II Global Ocean Sampling metagenomic datasets, our results further reinforce the ideas that the terrestrial ASFV has its evolutionary origin in marine environments. PMID:19860921

  7. Simultaneous, specific and real-time detection of biothreat and frequently encountered food-borne pathogens

    PubMed Central

    Woubit, Abdela Salah; Yehualaeshet, Teshome; Habtemariam, Tsegaye; Samuel, Temesgen

    2012-01-01

    The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia and Francisella include important food safety and biothreat agents causing food-related and other human illnesses worldwide. We aimed to develop rapid methods with the capability to simultaneously and differentially detect all six pathogens in one run. Our initial experiments to use previously reported sets of primers revealed non-specificity of some of the sequences when tested against a broader array of pathogens, or proved not optimal for simultaneous detection parameters. By extensive mining of the whole genome and protein databases of diverse closely and distantly related bacterial species and strains, we have identified unique genome regions, which we utilized to develop a detection platform. Twelve of the specific genomic targets we have identified to design the primers in F. tularensis ssp. tularensis, F. tularensis ssp. novicida, S. dysentriae, S. typhimurium, V. cholera, Y. pestis, and Y. pseudotuberculosis contained either hypothetical or putative proteins, the functions of which have not been clearly defined. Corresponding primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in-silico PCR against whole genome sequences of different species, sub-species, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (E.coli O157:H7 strain EDL 933, Shigella dysentriae, Salmonella typhi, Francisella tularensis ssp. tularensis, Vibrio cholera, and Yersinia pestis) and six foodborne pathogens (Salmonella typhimurium, Salmonella saintpaul, Shigella sonnei, Francisella novicida, Vibrio parahemolytica and Yersinia pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed using purified DNA showed the lowest detection limit of 640 fg

  8. Rapid detection and identification of infectious pathogens based on high-throughput sequencing.

    PubMed

    Ni, Pei-Xiang; Ding, Xin; Zhang, Yin-Xin; Yao, Xue; Sun, Rui-Xue; Wang, Peng; Gong, Yan-Ping; Zhou, Jia-Li; Li, Dong-Fang; Wu, Hong-Long; Yi, Xin; Yang, Ling; Long, Yun

    2015-04-05

    The dilemma of pathogens identification in patients with unidentified clinical symptoms such as fever of unknown origin exists, which not only poses a challenge to both the diagnostic and therapeutic process by itself, but also to expert physicians. In this report, we have attempted to increase the awareness of unidentified pathogens by developing a method to investigate hitherto unidentified infectious pathogens based on unbiased high-throughput sequencing. Our observations show that this method supplements current diagnostic technology that predominantly relies on information derived five cases from the intensive care unit. This methodological approach detects viruses and corrects the incidence of false positive detection rates of pathogens in a much shorter period. Through our method is followed by polymerase chain reaction validation, we could identify infection with Epstein-Barr virus, and in another case, we could identify infection with Streptococcus viridians based on the culture, which was false positive. This technology is a promising approach to revolutionize rapid diagnosis of infectious pathogens and to guide therapy that might result in the improvement of personalized medicine.

  9. Simultaneous Detection of Nine Key Bacterial Respiratory Pathogens Using Luminex xTAG® Technology

    PubMed Central

    Jiang, Luxi; Ren, Hongyu; Zhou, Haijian; Qin, Tian; Chen, Yu

    2017-01-01

    Early diagnosis and treatment are crucial to the outcome of lower respiratory tract infections (LRTIs). In this study, we developed an assay combining multiplex PCR and Luminex technology (MPLT) for the detection of nine important respiratory bacterial pathogens, which frequently cause LRTIs. These were Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae, Mycoplasma pneumoniae, Legionella spp., Pseudomonas aeruginosa, and Klebsiella pneumoniae. Through the hybridization reaction between two new synthesized multiplex PCR products and MagPlex-TAG Microspheres, we demonstrate that the detection limits for these nine pathogens were as low as 102–103 CFU/mL. Furthermore, 86 clinical bronchoalveolar lavage fluid specimens were used to evaluate this method. Compared with the results of nine simplex real-time PCR reactions targeting these nine pathogens, this MPLT assay demonstrated a high diagnostic accuracy for Streptococcus pneumoniae (sensitivity, 87.5% and specificity, 100%). Furthermore, sensitivity and specificity for the other eight pathogens all attained 100% diagnostic accuracy. In addition, the consistency between MPLT and the nine real-time PCR reactions exceeded 98.8%. In conclusion, MPLT is a high-throughput, labor-saving and reliable method with high sensitivity and specificity for identifying nine respiratory pathogens responsible for LRTIs. Indeed, this assay may be a promising supplement to conventional methods used to diagnose LRTIs. PMID:28241513

  10. Simplified detection of food-borne pathogens: an in situ high affinity capture and staining concept.

    PubMed

    Junillon, Thomas; Vimont, Antoine; Mosticone, David; Mallen, Benoît; Baril, Florent; Rozand, Christine; Flandrois, Jean-Pierre

    2012-12-01

    Food industries need simple, rapid and cost-effective solutions for pathogen detection in food and environmental samples. In this paper, we describe a simple but novel detection concept combining an affinity capture surface and intracellular metabolic marker to visualize the bacterial presence on the affinity surface. The surface of a Solid Phase Support (SPS) is functionalized with specific phage tail proteins targeted to the bacterial pathogen of interest. The SPS is placed directly into the primary food enrichment bag after stomaching. Following incubation, the captured bacteria are visually detected in situ as a result of the bacterial reduction of the colorless soluble substrate triphenyltetrazolium chloride (TTC) (present in the primary culture medium) to an intracellular red insoluble formazan product. Detection on the SPS is observed as an intense red color after 22 to 40 hours of enrichment. This is not impaired by the presence of food particles and the natural background microflora. The in situ method significantly simplifies pathogen detection by eliminating any post-enrichment intervention that is necessary in the traditional methods of analysis. We have demonstrated the application of this new approach for the detection of Escherichia coli O157: H7, Listeria spp. and Salmonella spp. in artificially contaminated food samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Glycated Albumin Identifies Prediabetes Not Detected by Hemoglobin A1c: The Africans in America Study.

    PubMed

    Sumner, Anne E; Duong, Michelle T; Bingham, Brianna A; Aldana, Paola C; Ricks, Madia; Mabundo, Lilian S; Tulloch-Reid, Marshall K; Chung, Stephanie T; Sacks, David B

    2016-11-01

    Following immigration to the US, many Africans transition from a low-normal to a high-normal or overweight body mass index (BMI). This weight change is associated with a high rate of prediabetes in the nonobese. Studies in East Asians reveal that glycated albumin is effective in identifying prediabetes in nonobese Asians. Whether this is true in African immigrants is unknown. Therefore, we evaluated the ability of hemoglobin A1c (Hb A1c) and glycated albumin to detect prediabetes in nonobese (BMI <30 kg/m(2)) and obese (BMI ≥30 kg/m(2)) African immigrants. Oral glucose tolerance tests (OGTTs) were performed in 236 self-identified healthy African immigrants [mean (SD) BMI 27.6 (4.4) kg/m(2)]. Prediabetes diagnosis was based on glucose criteria for the OGTT. Diagnostic sensitivity of Hb A1c and glycated albumin was determined by thresholds at the upper quartile for each [Hb A1c ≥5.7% (39 mmol/mol), glycated albumin ≥13.77%]. Based on glucose criteria for the OGTT, prediabetes was detected in 36% (85/236). BMI and Hb A1c were positively correlated (r = 0.22, P < 0.001), whereas BMI and glycated albumin were negatively correlated (r = -0.24, P < 0.001). Although the sensitivities of Hb A1c and glycated albumin were similar in nonobese immigrants (37% vs 42%, P = 0.75), prediabetes was detected in 21 nonobese Africans by glycated albumin alone, in 18 by Hb A1c alone, and in 4 by both tests. Therefore, sensitivity of the combined tests was better than for Hb A1c alone(72% vs 37%, P < 0.01). In the obese, Hb A1c was a much better diagnostic test than glycated albumin (64% vs 16%, P < 0.01) and combining the tests did not improve sensitivity (72% vs 64%, P = 0.50). Glycated albumin contributes by identifying prediabetes not detected by Hb A1c in nonobese African immigrants. ClinicalTrials.gov Identifier: NCT00001853. © 2016 American Association for Clinical Chemistry.

  12. Detection of Emerging and Re-Emerging Pathogens in Surface Waters Close to an Urban Area

    PubMed Central

    Marcheggiani, Stefania; D’Ugo, Emilo; Puccinelli, Camilla; Giuseppetti, Roberto; D’Angelo, Anna Maria; Gualerzi, Claudio Orlando; Spurio, Roberto; Medlin, Linda K.; Guillebault, Delphine; Weigel, Wilfried; Helmi, Karim; Mancini, Laura

    2015-01-01

    Current knowledge about the spread of pathogens in aquatic environments is scarce probably because bacteria, viruses, algae and their toxins tend to occur at low concentrations in water, making them very difficult to measure directly. The purpose of this study was the development and validation of tools to detect pathogens in freshwater systems close to an urban area. In order to evaluate anthropogenic impacts on water microbiological quality, a phylogenetic microarray was developed in the context of the EU project µAQUA to detect simultaneously numerous pathogens and applied to samples from two different locations close to an urban area located upstream and downstream of Rome in the Tiber River. Furthermore, human enteric viruses were also detected. Fifty liters of water were collected and concentrated using a hollow-fiber ultrafiltration approach. The resultant concentrate was further size-fractionated through a series of decreasing pore size filters. RNA was extracted from pooled filters and hybridized to the newly designed microarray to detect pathogenic bacteria, protozoa and toxic cyanobacteria. Diatoms as indicators of the water quality status, were also included in the microarray to evaluate water quality. The microarray results gave positive signals for bacteria, diatoms, cyanobacteria and protozoa. Cross validation of the microarray was performed using standard microbiological methods for the bacteria. The presence of oral-fecal transmitted human enteric-viruses were detected using q-PCR. Significant concentrations of Salmonella, Clostridium, Campylobacter and Staphylococcus as well as Hepatitis E Virus (HEV), noroviruses GI (NoGGI) and GII (NoGII) and human adenovirus 41 (ADV 41) were found in the Mezzocammino site, whereas lower concentrations of other bacteria and only the ADV41 virus was recovered at the Castel Giubileo site. This study revealed that the pollution level in the Tiber River was considerably higher downstream rather than upstream of

  13. Detection of hepatitis E virus and other livestock-related pathogens in Iowa streams

    USGS Publications Warehouse

    Givens, Carrie E.; Kolpin, Dana W.; Borchardt, Mark A.; Duris, Joseph; Moorman, Thomas B.; Spencer, Susan K.

    2016-01-01

    Manure application is a source of pathogens to the environment. Through overland runoff and tile drainage, zoonotic pathogens can contaminate surface water and streambed sediment and could affect both wildlife and human health. This study examined the environmental occurrence of gene markers for livestock-related bacterial, protozoan, and viral pathogens and antibiotic resistance in surface waters within the South Fork Iowa River basin before and after periods of swine manure application on agricultural land. Increased concentrations of indicator bacteria after manure application exceeding Iowa's state bacteria water quality standards suggest that swine manure contributes to diminished water quality and may pose a risk to human health. Additionally, the occurrence of HEV and numerous bacterial pathogen genes for Escherichia coli, Enterococcus spp., Salmonella sp., and Staphylococcus aureus in both manure samples and in corresponding surface water following periods of manure application suggests a potential role for swine in the spreading of zoonotic pathogens to the surrounding environment. During this study, several zoonotic pathogens were detected including Shiga-toxin producing E. coli, Campylobacter jejuni, pathogenic enterococci, and S. aureus; all of which can pose mild to serious health risks to swine, humans, and other wildlife. This research provides the foundational understanding required for future assessment of the risk to environmental health from livestock-related zoonotic pathogen exposures in this region. This information could also be important for maintaining swine herd biosecurity and protecting the health of wildlife near swine facilities.

  14. Multiplex Polymerase Chain Reaction for Detection of Gastrointestinal Pathogens in Migrant Workers in Qatar

    PubMed Central

    Humphrey, John M.; Ranbhise, Sanjay; Ibrahim, Emad; Al-Romaihi, Hamad E.; Farag, Elmoubasher; Abu-Raddad, Laith J.; Glesby, Marshall J.

    2016-01-01

    The causes of infectious diarrhea among the migrant worker population in Qatar are not well understood. We conducted a prospective observational study to understand the demographic and clinical characteristics and infectious causes of diarrhea among migrant workers in Doha, Qatar. A total of 126 male workers presenting to the Qatar Red Crescent Worker's Health Center outpatient clinic or emergency department were studied over a 5-month period in 2015–2016. Epidemiologic surveys were administered to all subjects and the prevalence of 22 different stool pathogens was determined using multiplex polymerase chain reaction (PCR) (FilmArray® Gastrointestinal PCR). A target pathogen was identified in 62.7% of subjects. Enteropathogenic Escherichia coli was the most prevalent pathogen and was detected in 24.6% of subjects, followed by Salmonella (22.2%), enteroaggregative E. coli (15.1%), Giardia lamblia (9.5%), and enterotoxigenic E. coli (8.7%). Multiple pathogens were identified in 49.3% of positive stool samples. In a multivariable analysis, the presence of a heart rate ≥ 90 (adjusted odds ratio [OR] = 3.7, 95% confidence interval [CI] = 1.4–10.0) and > 5 fecal leukocytes/high-power field (adjusted OR = 2.8, 95% CI = 1.2–7.0) were significant predictors of detecting an acute inflammatory pathogen by PCR. Use of multiplex PCR enabled the detection of gastrointestinal pathogens in a high proportion of cases, illustrating the utility of this diagnostic tool in epidemiologic studies of infectious diarrhea. PMID:27928081

  15. Molecular detection of pathogens in water--the pros and cons of molecular techniques.

    PubMed

    Girones, Rosina; Ferrús, Maria Antonia; Alonso, José Luis; Rodriguez-Manzano, Jesus; Calgua, Byron; Corrêa, Adriana de Abreu; Hundesa, Ayalkibet; Carratala, Anna; Bofill-Mas, Sílvia

    2010-08-01

    Pollution of water by sewage and run-off from farms produces a serious public health problem in many countries. Viruses, along with bacteria and protozoa in the intestine or in urine are shed and transported through the sewer system. Even in highly industrialized countries, pathogens, including viruses, are prevalent throughout the environment. Molecular methods are used to monitor viral, bacterial, and protozoan pathogens, and to track pathogen- and source-specific markers in the environment. Molecular techniques, specifically polymerase chain reaction-based methods, provide sensitive, rapid, and quantitative analytical tools with which to study such pathogens, including new or emerging strains. These techniques are used to evaluate the microbiological quality of food and water, and to assess the efficiency of virus removal in drinking and wastewater treatment plants. The range of methods available for the application of molecular techniques has increased, and the costs involved have fallen. These developments have allowed the potential standardization and automation of certain techniques. In some cases they facilitate the identification, genotyping, enumeration, viability assessment, and source-tracking of human and animal contamination. Additionally, recent improvements in detection technologies have allowed the simultaneous detection of multiple targets in a single assay. However, the molecular techniques available today and those under development require further refinement in order to be standardized and applicable to a diversity of matrices. Water disinfection treatments may have an effect on the viability of pathogens and the numbers obtained by molecular techniques may overestimate the quantification of infectious microorganisms. The pros and cons of molecular techniques for the detection and quantification of pathogens in water are discussed. (c) 2010 Elsevier Ltd. All rights reserved.

  16. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

    PubMed

    Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

    2004-12-01

    This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

  17. Development and validation of an arthropod maceration protocol for zoonotic pathogen detection in mosquitoes and fleas.

    PubMed

    Harrison, Genelle F; Scheirer, Jessica L; Melanson, Vanessa R

    2015-06-01

    Arthropod-borne diseases remain a pressing international public health concern. While progress has been made in the rapid detection of arthropod-borne pathogens via quantitative real-time (qPCR), or even hand-held detection devices, a simple and robust maceration and nucleic acid extraction method is necessary to implement biosurveillance capabilities. In this study, a comparison of maceration techniques using five types of beads followed by nucleic acid extraction and detection were tested using two morphologically disparate arthropods, the Aedes aegypti mosquito and Xenopsylla spp. flea, to detect the zoonotic diseases dengue virus serotype-1 and Yersinia pestis. Post-maceration nucleic acid extraction was carried out using the 1-2-3 Platinum-Path-Sample-Purification (PPSP) kit followed by qPCR detection using the Joint Biological Agent Identification and Diagnostic System (JBAIDS). We found that the 5mm stainless steel beads added to the beads provided in the PPSP kit were successful in macerating the exoskeleton for both Ae. aegypti and Xenopsylla spp. Replicates in the maceration/extraction/detection protocol were increased in a stepwise fashion until a final 128 replicates were obtained. For dengue virus detection there was a 99% positivity rate and for Y. pestis detection there was a 95% positive detection rate. In the examination of both pathogens, there were no significant differences between qPCR instruments, days ran, time of day ran, or operators. © 2015 The Society for Vector Ecology.

  18. Detection of pathogens in Boidae and Pythonidae with and without respiratory disease.

    PubMed

    Schmidt, V; Marschang, R E; Abbas, M D; Ball, I; Szabo, I; Helmuth, R; Plenz, B; Spergser, J; Pees, M

    2013-03-02

    Respiratory diseases in boid snakes are common in captivity, but little information is available on their aetiology. This study was carried out to determine the occurrence of lung associated pathogens in boid snakes with and without respiratory signs and/or pneumonia. In total, 80 boid snakes of the families Boidae (n = 30) and Pythonidae (n = 50) from 48 private and zoo collections were included in this survey. Husbandry conditions were evaluated using a detailed questionnaire. All snakes were examined clinically and grouped into snakes with or without respiratory signs. Tracheal wash samples from all snakes were examined bacteriologically as well as virologically. All snakes were euthanased, and a complete pathological examination was performed. Respiratory signs and pneumonia were detected more often in pythons than in boas. An acute catarrhal pneumonia was diagnosed more often in snakes without respiratory signs than in snakes with respiratory signs, which revealed fibrinous and fibrous pneumonia. Poor husbandry conditions are an important trigger for the development of respiratory signs and pneumonia. Different bacterial pathogens were isolated in almost all snakes with pneumonia, with Salmonella species being the most common. Ferlavirus (formerly known as ophidian paramyxovirus)-RNA was detected only in pythons. Inclusion body disease was rarely seen in pythons but often in boas. Adenovirus and Mycoplasma were other pathogens that were diagnosed in single snakes with pneumonia. In living boid snakes with respiratory signs, tracheal wash samples were found to be a useful diagnostic tool for the detection of viral and bacterial pathogens.

  19. Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens.

    PubMed

    Li, Yanmei; Fan, Penghui; Zhou, Shishui; Zhang, Li

    2017-03-18

    Foodborne bacterial infections and diseases have been considered to be a major threat for public health in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP) has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been applied to detection and identification on pathogens from microbial diseases, as it showed significant advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming sample preparation. An overview of LAMP mainly containing the development history, reaction principle and its application to four kind of foodborne pathogens detection are presented in this paper. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to provide theoretical basis for the clinical application of LAMP technology.

  20. Label-Free Detection and Discrimination of Bacterial Pathogens Based on Hemin Recognition.

    PubMed

    Maltais, Thora R; Adak, Avijit K; Younis, Waleed; Seleem, Mohamed N; Wei, Alexander

    2016-07-20

    Hemin linked to hexa(ethylene glycol)bishydrazide was patterned by inkjet printing into periodic microarrays, and evaluated for their ability to capture bacterial pathogens expressing various hemin receptors. Bacterial adhesion was imaged under darkfield conditions with Fourier analysis, supporting a label-free method of pathogen detection. Hemin microarrays were screened against a panel of 16 bacteria and found capable of capturing multiple species, some with limits of detection as low as 10(3) cfu/mL. Several Gram-positive strains including Staphylococcus aureus and Bacillus anthracis also exhibited rapid adhesion, enabling pattern recognition within minutes of exposure. This can be attributed to differences in hemin acquisition systems: aggressively adherent bacteria express cell-surface hemin receptors (CSHRs) that enable direct hemin binding and uptake, whereas other types of bacteria including most Gram-negative strains rely on the secretion and recapture of soluble proteins (hemophores) for hemin acquisition, with consequently longer times for ligand binding and detection.

  1. A new pentaplex-nested PCR to detect five pathogenic bacteria in free living amoebae.

    PubMed

    Calvo, L; Gregorio, I; García, A; Fernández, M T; Goñi, P; Clavel, A; Peleato, M L; Fillat, M F

    2013-02-01

    Changes in water use and anthropogenic activity have major impacts on the quality of natural aquatic ecosystems, water distribution and wastewater plants. One of the main problems is the presence of some pathogenic microorganisms that are resistant to disinfection procedures when they are hosted by free living amoeba and that in many cases are hardly detectable by culture-based procedures. In this work we report a sensitive, low-cost procedure consisting of a pentaplex-nested PCR that allows simultaneous detection of Legionella pneumophila, Mycobacterium spp., Pseudomonas spp., Vibrio cholerae and the microcystin-producing cyanobacteria Microcystis aeruginosa. The method has been used to detect the presence of these pathogenic bacteria in water and inside free living amoeba. Its validation in 72 samples obtained from different water sources from Aragon (Spain) evidences that Mycobacterium and Pseudomonas spp are prevailing as amoeba-resistant bacteria.

  2. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    SciTech Connect

    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.

  3. Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria.

    PubMed

    Yang, Liju; Bashir, Rashid

    2008-01-01

    The realization of rapid, sensitive, and specific methods to detect foodborne pathogenic bacteria is central to implementing effective practice to ensure food safety and security. As a principle of transduction, the impedance technique has been applied in the field of microbiology as a means to detect and/or quantify foodborne pathogenic bacteria. The integration of impedance with biological recognition technology for detection of bacteria has led to the development of impedance biosensors that are finding wide-spread use in the recent years. This paper reviews the progress and applications of impedance microbiology for foodborne pathogenic bacteria detection, particularly the new aspects that have been added to this subject in the past few years, including the use of interdigitated microelectrodes, the development of chip-based impedance microbiology, and the use of equivalent circuits for analysis of the impedance systems. This paper also reviews the significant developments of impedance biosensors for bacteria detection in the past 5 years, focusing on microfabricated microelectrodes-based and microfluidic-based Faradaic electrochemical impedance biosensors, non-Faradaic impedance biosensors, and the integration of impedance biosensors with other techniques such as dielectrophoresis and electropermeabilization.

  4. Label-free detection of pathogenic bacteria via immobilized antimicrobial peptides.

    PubMed

    Dong, Zong-Mu; Zhao, Guang-Chao

    2015-05-01

    A novel label-free strategy for the detection of bacteria was developed by using a specific antimicrobial peptide (AMP)-functionalized quartz crystal microbalance (QCM) electrode. This electrode interface was successfully applied to detect pathogenic Escherichia coli O157:H7 based on the specific affinity between the small synthetic antimicrobial peptide and the bacterial cell of pathogenic E. coli O157:H7. The concentrations of pathogenic E. coli O157:H7 were sensitively measured by the frequency response of the QCM with a detection limit of 0.4 cfu μL(-1). The detection can be fulfilled within 10 min because it does not require germiculture process. On the other hand, if the specific antimicrobial peptides were immobilized on a gold electrode, this label-free strategy can also be performed by electrochemical impedance spectroscopy (EIS). Compared with QCM technique, the EIS measurement gives a lower sensitivity and needs a longer assay time. The combination of antimicrobial peptides with the real-time responses of QCM, as well as electronic read-out monitoring of EIS, may open a new way for the direct detection of bacteria.

  5. Rapid detection of pathogens using antibody-coated microbeads with bioluminescence in microfluidic chips.

    PubMed

    Guan, Xiao; Zhang, Hui-jing; Bi, Yin-nan; Zhang, Li; Hao, Dun-ling

    2010-08-01

    Detection of pathogens was demonstrated in a polydimethylsiloxane (PDMS)/glass microfluidic chip with which microbead-based immunoseparation platform and the bioluminescence technology were integrated. Escherichia coli (E. coli) O157:H7 was used as the model bacteria. The microchamber in microfluidic chip was filled with glass beads coated with antibodies which could capture specific organism, and the capture efficiency of the chip for the bacteria was about 91.75% approximately 95.62%. Then the concentration of bacteria was determined by detecting adenosine triphosphate (ATP) employing bioluminescence reaction of firefly luciferin-lucifera-ATP on chip. The method allowed reliable detection of E. coli O157:H7 concentrations from 3.2 x 10(1) cfu/microL to 3.2 x 10(5) cfu/microL within 20 min. This research demonstrated excellent reproducibility, stability, and specificity, and could accurately detect the pathogenic bacteria in food samples. The microfluidic chip and the equipments used in this method are easy to miniaturize, thus the method has great potential to be developed to a portable device for rapid detection of pathogens.

  6. Multiplex detection and identification of viral, bacterial, and protozoan pathogens in human blood and plasma using a high-density resequencing pathogen microarray platform.

    PubMed

    Kourout, Moussa; Fisher, Carolyn; Purkayastha, Anjan; Tibbetts, Clark; Winkelman, Valerie; Williamson, Phillip; Nakhasi, Hira L; Duncan, Robert

    2016-06-01

    The implementation of nucleic acid-based tests for blood donor screening has improved the safety of the blood supply; however, the increasing number of emerging pathogen tests is burdensome. Development of multiplex testing platforms that allow simultaneous screening for different pathogens is a potential solution. The TessArray resequencing microarray is a platform that allows multiplex detection and identification of 97 different blood-borne pathogens in one single test. The objective was to evaluate the lowest concentration detected in blood or plasma, species discrimination, and applicability of the TessArray microarray platform for testing blood donors. Human blood or plasma spiked with selected pathogens (10,000, 1000, or 100 cells or copies/mL), including three viral, four bacterial, and four protozoan pathogens were each tested on this platform. The nucleic acids were extracted, amplified using multiplexed sets of pooled specific primers, fragmented, labeled, and hybridized to a microarray. Finally, the detected sequences were identified using an automated genomic database alignment algorithm. The performance of this platform demonstrated detection for spiked bacterial and protozoan pathogens of 100 cells/mL and viral pathogens as low as 100 copies/mL. Coded specimens, including spiked and negative controls, were identified correctly for blood specimens (31/32, 97%) and for plasma specimens (20/22, 91%) demonstrating the effectiveness of the platform. These results indicated that the TessArray microarray platform could be employed for multiplex detection and identification, with a high level of discriminatory power for numerous blood-borne pathogen targets with potential for use in blood safety. © 2016 AABB.

  7. Novel aptamer-linked nanoconjugate approach for detection of waterborne bacterial pathogens: an update

    NASA Astrophysics Data System (ADS)

    Singh, Gulshan; Manohar, Murli; Adegoke, Anthony Ayodeji; Stenström, Thor Axel; Shanker, Rishi

    2017-01-01

    The lack of microbiologically safe water in underdeveloped nations is the prime cause of infectious disease outbreaks. The need for the specific identification and detection of microorganisms encourages the development of advanced, rapid, sensitive and highly specific methods for the monitoring of pathogens and management of potential risk to human health. The rapid molecular assays based on detection of specific molecular signatures offer advantages over conventional methods in terms of specificity and sensitivity but require complex instrumentation and skilled personnel. Nanotechnology is an emerging area and provides a robust approach for the identification of pathogenic microorganism utilizing the peculiar properties of nanomaterials, i.e. small size (1-100 nm) and large surface area. This emerging technology promises to fulfill the urgent need of a novel strategy to enhance the bacterial identification and quantitation in the environment. In this context, the peculiar properties of gold nanoparticles, their plasmonic shifts, and changes in magnetic properties have been utilized for the simple and cost-effective detection of bacterial nucleic acids, antigens and toxins with quite improved sensitivity. One of the promising leads to develop an advance detection method might be the coupling of nucleic acid aptamers (capable of interacting specifically with bacteria, protozoa, and viruses) with nanomaterials. Such aptamer-nano conjugate can be used for the specific recognition of infectious agents in different environmental matrices. This review summarizes the application of nanotechnology in the area of pathogen detection and discusses the prospects of coupling nucleic acid aptamers with nanoparticles for the specific detection of targeted pathogens.

  8. Selective detection of live pathogens via surface-confined electric field perturbation on interdigitated silicon transducers.

    PubMed

    de la Rica, Roberto; Baldi, Antonio; Fernández-Sánchez, César; Matsui, Hiroshi

    2009-05-15

    Detection of physical changes of cells is emerging as a new diagnostic approach to determine their phenotypical features. One of such changes is related to their viability; live (viable) cells are more voluminous than the dead ones, and monitoring this parameter in tissue cells becomes essential in fields such as drug discovery and hazard evaluation. In the area of pathogen detection, an analytical system capable of specifically detecting viable cells with the simple sample preparation and detection process would be highly desirable since live microorganisms can rapidly increase their numbers even at extremely low concentration and become a severe health risk. However, current sensing strategies cannot clearly determine the viability of cells, and hence they are susceptible to false-positive signals from harmless dead pathogens. Here we developed a robust electronic immunoassay that uses a pair of polycrystalline silicon interdigitated electrodes for the rapid detection of pathogens with high specificity for live cells. After bacterial cells were specifically anchored to the surface of the antibody-modified electrode, the characteristic geometry of the transducer enables the selective detection of viable cells with a limit of detection of 3 x 10(2) cfu/mL and an incubation time of only 1 h. The CMOS compatible fabrication process of the chip along with the label-free, reagent-less electronic detection and the easy electrode regeneration to recycle for another impedance measurement make this approach an excellent candidate for oncoming economical in-field viable-cell detection systems, fully integrable with sophisticated signal processing circuits.

  9. Non-tuberculous Mycobacteria in South African Wildlife: Neglected Pathogens and Potential Impediments for Bovine Tuberculosis Diagnosis

    PubMed Central

    Gcebe, Nomakorinte; Hlokwe, Tiny M.

    2017-01-01

    subsp. bolletti was the most frequently detected NTM identified in 37 of the 102 isolates. Other NTM species were also isolated from animals not showing any pathological changes. Knowledge gained in this study contribute to the understanding of NTM species circulating in wild animals in South Africa and the pathogenic potential of certain species, whose role in disease causation need to be examined, as well as to a certain extent the potential of M. simiae to hamper the diagnosis of bTB. PMID:28194371

  10. Non-tuberculous Mycobacteria in South African Wildlife: Neglected Pathogens and Potential Impediments for Bovine Tuberculosis Diagnosis.

    PubMed

    Gcebe, Nomakorinte; Hlokwe, Tiny M

    2017-01-01

    subsp. bolletti was the most frequently detected NTM identified in 37 of the 102 isolates. Other NTM species were also isolated from animals not showing any pathological changes. Knowledge gained in this study contribute to the understanding of NTM species circulating in wild animals in South Africa and the pathogenic potential of certain species, whose role in disease causation need to be examined, as well as to a certain extent the potential of M. simiae to hamper the diagnosis of bTB.

  11. Genomic analysis of three African strains of Bacillus anthracis demonstrates that they are part of the clonal expansion of an exclusively pathogenic bacterium

    PubMed Central

    Rouli, L; MBengue, M; Robert, C; Ndiaye, M; La Scola, B; Raoult, D

    2014-01-01

    Bacillus anthracis is the causative agent of anthrax and is classified as a ‘Category A’ biological weapon. Six complete genomes of B. anthracis (A0248, Ames, Ames Ancestor, CDC684, H0491, and Sterne) are currently available. In this report, we add three African strain genomes: Sen2Col2, Sen3 and Gmb1. To study the pan-genome of B. anthracis, we used bioinformatics tools, such as Cluster of Orthologous Groups, and performed phylogenetic analysis. We found that the three African strains contained the pX01 and pX02 plasmids, the nonsense mutation in the plcR gene and the four known prophages. These strains are most similar to the CDC684 strain and belong to the A cluster. We estimated that the B. anthracis pan-genome has 2893 core genes (99% of the genome size) and 85 accessory genes. We validated the hypothesis that B. anthracis has a closed pan-genome and found that the three African strains carry the two plasmids associated with bacterial virulence. The pan-genome nature of B. anthracis confirms its lack of exchange (similar to Clostridium tetani) and supports its exclusively pathogenic role, despite its survival in the environment. Moreover, thanks to the study of the core content single nucleotide polymorphisms, we can see that our three African strains diverged very recently from the other B. anthracis strains. PMID:25566394

  12. Parallel detection of ancient pathogens via array-based DNA capture

    PubMed Central

    Bos, Kirsten I.; Jäger, Günter; Schuenemann, Verena J.; Vågene, Åshild J.; Spyrou, Maria A.; Herbig, Alexander; Nieselt, Kay; Krause, Johannes

    2015-01-01

    DNA capture coupled with next generation sequencing is highly suitable for the study of ancient pathogens. Screening for pathogens can, however, be meticulous when assays are restricted to the enrichment of single organisms, which is common practice. Here, we report on an array-based DNA capture screening technique for the parallel detection of nearly 100 pathogens that could have potentially left behind molecular signatures in preserved ancient tissues. We demonstrate the sensitivity of our method through evaluation of its performance with a library known to harbour ancient Mycobacterium leprae DNA. This rapid and economical technique will be highly useful for the identification of historical diseases that are difficult to characterize based on archaeological information alone. PMID:25487327

  13. High-Throughput Biosensors for Multiplexed Food-Borne Pathogen Detection

    NASA Astrophysics Data System (ADS)

    Gehring, Andrew G.; Tu, Shu-I.

    2011-07-01

    Incidental contamination of foods by pathogenic bacteria and/or their toxins is a serious threat to public health and the global economy. The presence of food-borne pathogens and toxins must be rapidly determined at various stages of food production, processing, and distribution. Producers, processors, regulators, retailers, and public health professionals need simple and cost-effective methods to detect different species or serotypes of bacteria and associated toxins in large numbers of food samples. This review addresses the desire to replace traditional microbiological plate culture with more timely and less cumbersome rapid, biosensor-based methods. Emphasis focuses on high-throughput, multiplexed techniques that allow for simultaneous testing of numerous samples, in rapid succession, for multiple food-borne analytes (primarily pathogenic bacteria and/or toxins).

  14. Africanized honey bees are efficient at detecting, uncapping and removing dead brood.

    PubMed

    Morais, M M; Francoy, T M; Pereira, R A; De Jong, D; Gonçalves, L S

    2009-01-01

    The hygienic behavior of honey bees is based on a two-step process, including uncapping and removing diseased, dead, damaged, or parasitized brood inside the cell. We evaluated during periods of 1 h the time that hygienic and non-hygienic colonies of Africanized honey bees spend to detect, uncap and remove pin-killed brood using comb inserts with transparent walls placed in observation hives. We observed that hygienic colonies are significantly faster in detecting, uncapping and removing dead brood in the cells (P < 0.001).

  15. DNA microarray-based detection of multiple pathogens: Mycoplasma spp. and Chlamydia spp.

    PubMed

    Schnee, Christiane; Sachse, Konrad

    2015-01-01

    Rapid detection of slow-growing or non-culturable microorganisms, such as Mycoplasma spp. and Chlamydia spp., is still a challenge to diagnosticians in the veterinary field. In addition, as epidemiological evidence on the frequency of mixed infections involving two and more bacterial species has been emerging, detection methods allowing simultaneous identification of different pathogens are required. In the present chapter, we describe DNA microarray-based procedures for the detection of 83 Mollicutes species (Mycoplasma assay) and 11 Chlamydia spp. (Chlamydia assay). The assays are suitable for use in a routine diagnostic environment, as well as in microbiological research.

  16. Multiplex detection of plant pathogens through the Luminex MagPlex bead system.

    PubMed

    van der Vlugt, René A A; van Raaij, Henry; de Weerdt, Marjanne; Bergervoet, Jan H W

    2015-01-01

    Here we describe a versatile multiplex method for both the serological and molecular detection of plant pathogens. The Luminex MagPlex bead system uses small paramagnetic microspheres ("beads"), either coated with specific antibodies or oligonucleotides, which capture respectively viruses and/or bacteria or PCR products obtained from their genetic material. The Luminex MagPlex bead system allows true multiplex detection of up to 500 targets in a single sample on a routine basis. The liquid suspension nature of the method significantly improves (1) assay speed, (2) detection limits and (3) dynamic range. It can also considerably reduce labor and consumables costs.

  17. A new approach for detecting fungal and oomycete plant pathogens in next generation sequencing metagenome data utilising electronic probes.

    PubMed

    Espindola, Andres; Schneider, William; Hoyt, Peter R; Marek, Stephen M; Garzon, Carla

    2015-01-01

    Early stage infections caused by fungal/oomycete spores may not be detected until signs or symptoms develop. Serological and molecular techniques are currently used for detecting these pathogens. Next-generation sequencing (NGS) has potential as a diagnostic tool, due to the capacity to target multiple unique signature loci of pathogens in an infected plant metagenome. NGS has significant potential for diagnosis of important eukaryotic plant pathogens. However, the assembly and analysis of huge amounts of sequence is laborious, time consuming, and not necessary for diagnostic purposes. Previous work demonstrated that a bioinformatic tool termed Electronic probe Diagnostic Nucleic acid Analysis (EDNA) had potential for greatly simplifying detecting fungal and oomycete plant pathogens in simulated metagenomes. The initial study demonstrated limitations for detection accuracy related to the analysis of matches between queries and metagenome reads. This study is a modification of EDNA demonstrating a better accuracy for detecting fungal and oomycete plant pathogens.

  18. Progress in rapid detection and identification of unknown human and agricultural pathogens

    SciTech Connect

    Barnes, T; Holzrichter, J F; Milanovich, F P

    1999-08-13

    The medical industry is driving pathogen detection technology from its present characteristics of $50/sample, 100 sample capability systems, with several day time responses, having several percent error rates in reported outcomes. The systems described above are capable of providing samples at < $5/test, managing several million samples, < 1-hour cycle times, (or just minutes in some cases) and < 0.1% error rates. Because of their importance to the medical and agricultural communities, all ''important'' pathogens will have detection kits available (within air transport times, anywhere in the world) by 2020, and the most well known pathogens will have kits available within a few years. Many are available now. Because of the importance of the food supply to modern nations, these technologies will be employed everywhere in this industry. For example, the United States imports 30 B tons of food a year, but inspects < 1%. Portable inspection systems will make it possible to test for dangerous pathogens in feed lots, food processing plants, markets, and points of use. Outbreaks of animal or plant disease will be immediately detectable using field instrumentation, and more complex samples can be sent to central testing laboratories where more sophisticated test systems will be available. Unusual pathogens either naturally or purposefully selected or developed, will require special attention because there is not a commercial economic driver for the development of detection systems and curative agents. Their development, and production for sufficient availability, will require significant investments by the world community. The strategy and costs for developing vaccines or curative drugs will be very expensive and will need special attention. However it is important that attention be directed to these problems because such attention has a strong deterrent effect on potential developers or users. The capacity to use the full information content contained in pathogen systems

  19. Low-cost, real-time, continuous flow PCR system for pathogen detection.

    PubMed

    Fernández-Carballo, B Leticia; McGuiness, Ian; McBeth, Christine; Kalashnikov, Maxim; Borrós, Salvador; Sharon, Andre; Sauer-Budge, Alexis F

    2016-04-01

    In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device.

  20. Detection of viral respiratory pathogens in mild and severe acute respiratory infections in Singapore

    PubMed Central

    Jiang, Lili; Lee, Vernon Jian Ming; Cui, Lin; Lin, Raymond; Tan, Chyi Lin; Tan, Linda Wei Lin; Lim, Wei-yen; Leo, Yee-Sin; Low, Louie; Hibberd, Martin; Chen, Mark I-Cheng

    2017-01-01

    To investigate the performance of laboratory methods and clinical case definitions in detecting the viral pathogens for acute respiratory infections (ARIs) from a prospective community cohort and hospital inpatients, nasopharyngeal swabs from cohort members reporting ARIs (community-ARI) and inpatients admitted with ARIs (inpatient-ARI) were tested by Singleplex Real Time-Polymerase Chain Reaction (SRT-PCR), multiplex RT-PCR (MRT-PCR) and pathogen-chip system (PathChip) between April 2012 and December 2013. Community-ARI and inpatient-ARI was also combined with mild and severe cases of influenza from a historical prospective study as mild-ARI and severe-ARI respectively to evaluate the performance of clinical case definitions. We analysed 130 community-ARI and 140 inpatient-ARI episodes (5 inpatient-ARI excluded because multiple pathogens were detected), involving 138 and 207 samples respectively. Detection by PCR declined with days post-onset for influenza virus; decrease was faster for community-ARI than for inpatient-ARI. No such patterns were observed for non-influenza respiratory virus infections. PathChip added substantially to viruses detected for community-ARI only. Clinical case definitions discriminated influenza from other mild-ARI but performed poorly for severe-ARI and for older participants. Rational strategies for diagnosis and surveillance of influenza and other respiratory virus must acknowledge the differences between ARIs presenting in community and hospital settings. PMID:28218288

  1. Unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach.

    PubMed

    Yang, Jian; Yang, Fan; Ren, Lili; Xiong, Zhaohui; Wu, Zhiqiang; Dong, Jie; Sun, Lilian; Zhang, Ting; Hu, Yongfeng; Du, Jiang; Wang, Jianwei; Jin, Qi

    2011-10-01

    Viral infectious diseases represent a major threat to public health and are among the greatest disease burdens worldwide. Rapid and accurate identification of viral agents is crucial for both outbreak control and estimating regional disease burdens. Recently developed metagenomic methods have proven to be powerful tools for simultaneous pathogen detection. Here, we performed a systematic study of the capability of the short-read-based metagenomic approach in the molecular detection of viral pathogens in nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections (n = 16). Using the high-throughput capacity of ultradeep sequencing and a dedicated data interpretation method, we successfully identified seven species of known respiratory viral agents from 15 samples, a result that was consistent with results of conventional PCR assays. We also detected a coinfected case that was missed by regular PCR testing. Using the metagenomic data, 11 draft genomes of the abundantly detected viruses in the samples were reconstructed with 21.84% to 98.53% coverage. Our results show the power of the short-read-based metagenomic approach for accurate and parallel screening of viral pathogens. Although there are some inherent difficulties in applying this approach to clinical samples, including a lack of controls, limited specimen quantity, and high contamination rate, our work will facilitate further application of this unprecedented high-throughput method to clinical samples.

  2. In-situ detection of multiple pathogenic bacteria on food surfaces

    NASA Astrophysics Data System (ADS)

    Chai, Yating; Horikawa, Shin; Hu, Jiajia; Chen, I.-Hsuan; Hu, Jing; Barbaree, James M.; Chin, Bryan A.

    2015-05-01

    Real-time in-situ detection of pathogenic bacteria on fresh food surfaces was accomplished with phage-based magnetoelastic (ME) biosensors. The ME biosensor is constructed of a small rectangular strip of ME material that is coated with a biomolecular recognition element (phage, antibodies or proteins, etc.) that is specific to the target pathogen. This mass-sensitive ME biosensor is wirelessly actuated into mechanical resonance by an externally applied time-varying magnetic field. When the biosensor binds with target bacteria, the mass of the sensor increases, resulting in a decrease in the sensor's resonant frequency. In order to compensate for nonspecific binding, control biosensors without phage were used in this experiment. In previous research, the biosensors were measured one by one. However, the simultaneous measurement of multiple sensors was accomplished in this research, and promises to greatly shorten the analysis time for bacterial detection. Additionally, the use of multiple biosensors enables the possibility of simultaneous detection of different pathogenic bacteria. This paper presents results of experiments in which multiple phage-based ME biosensors were simultaneously monitored. The E2 phage and JRB7 phage from a landscape phage library served as the bio-recognition element that have the capability of binding specifically with Salmonella typhimurium and B. anthracis spores, respectively. Real-time in-situ detection of Salmonella typhimurium and B. anthracis spores on food surfaces are presented.

  3. An Improved Detection and Quantification Method for the Coral Pathogen Vibrio coralliilyticus

    PubMed Central

    Wilson, Bryan; Muirhead, Andrew; Bazanella, Monika; Huete-Stauffer, Carla; Vezzulli, Luigi; Bourne, David G.

    2013-01-01

    DNA- and RNA-based PCR and reverse-transcription real-time PCR assays were developed for diagnostic detection of the vcpA zinc-metalloprotease implicated in the virulence of the coral pathogen Vibrio coralliilyticus. Both PCR methods were highly specific for V. coralliilyticus and failed to amplify strains of closely-related Vibrio species. The assays correctly detected all globally occurring V. coralliilyticus isolates including a newly-described isolate [TAV24] infecting gorgonians in the Mediterranean Sea and highlighted those isolates that had been potentially misidentified, in particular V. tubiashii strains ATCC 19105 and RE22, historically described as important oyster pathogens. The real-time assay is sensitive, detecting 10 gene copies and the relationships between gene copy number and cycle threshold (CT) were highly linear (R2≥99.7). The real-time assay was also not affected by interference from non-target DNA. These assays are useful for rapid detection of V. coralliilyticus and monitoring of virulence levels in environmental samples, allowing for implementation of timely management steps to limit and possibly prevent losses due to V. coralliilyticus infection, as well as furthering investigations of factors affecting pathogenesis of this important marine pathogen. PMID:24339968

  4. Biosensors and bio-based methods for the separation and detection of foodborne pathogens.

    PubMed

    Bhunia, Arun K

    2008-01-01

    The safety of our food supply is always a major concern to consumers, food producers, and regulatory agencies. A safer food supply improves consumer confidence and brings economic stability. The safety of foods from farm-to-fork through the supply chain continuum must be established to protect consumers from debilitating, sometimes fatal episodes of pathogen outbreaks. The implementation of preventive strategies like hazard analysis critical control points (HACCP) assures safety but its full utility will not be realized unless supportive tools are fully developed. Rapid, sensitive, and accurate detection methods are such essential tools that, when integrated with HACCP, will improve safety of products. Traditional microbiological methods are powerful, error-proof, and dependable but these lengthy, cumbersome methods are often ineffective because they are not compatible with the speed at which the products are manufactured and the short shelf life of products. Automation in detection methods is highly desirable, but is not achievable with traditional methods. Therefore, biosensor-based tools offer the most promising solutions and address some of the modern-day needs for fast and sensitive detection of pathogens in real time or near real time. The application of several biosensor tools belonging to the categories of optical, electrochemical, and mass-based tools for detection of foodborne pathogens is reviewed in this chapter. Ironically, geometric growth in biosensor technology is fueled by the imminent threat of bioterrorism through food, water, and air and by the funding through various governmental agencies.

  5. Electrochemical Biosensor for Rapid and Sensitive Detection of Magnetically Extracted Bacterial Pathogens

    PubMed Central

    Setterington, Emma B.; Alocilja, Evangelyn C.

    2012-01-01

    Biological defense and security applications demand rapid, sensitive detection of bacterial pathogens. This work presents a novel qualitative electrochemical detection technique which is applied to two representative bacterial pathogens, Bacillus cereus (as a surrogate for B. anthracis) and Escherichia coli O157:H7, resulting in detection limits of 40 CFU/mL and 6 CFU/mL, respectively, from pure culture. Cyclic voltammetry is combined with immunomagnetic separation in a rapid method requiring approximately 1 h for presumptive positive/negative results. An immunofunctionalized magnetic/polyaniline core/shell nano-particle (c/sNP) is employed to extract target cells from the sample solution and magnetically position them on a screen-printed carbon electrode (SPCE) sensor. The presence of target cells significantly inhibits current flow between the electrically active c/sNPs and SPCE. This method has the potential to be adapted for a wide variety of target organisms and sample matrices, and to become a fully portable system for routine monitoring or emergency detection of bacterial pathogens. PMID:25585629

  6. FISHing for bacteria in food--a promising tool for the reliable detection of pathogenic bacteria?

    PubMed

    Rohde, Alexander; Hammerl, Jens Andre; Appel, Bernd; Dieckmann, Ralf; Al Dahouk, Sascha

    2015-04-01

    Foodborne pathogens cause millions of infections every year and are responsible for considerable economic losses worldwide. The current gold standard for the detection of bacterial pathogens in food is still the conventional cultivation following standardized and generally accepted protocols. However, these methods are time-consuming and do not provide fast information about food contaminations and thus are limited in their ability to protect consumers in time from potential microbial hazards. Fluorescence in situ hybridization (FISH) represents a rapid and highly specific technique for whole-cell detection. This review aims to summarize the current data on FISH-testing for the detection of pathogenic bacteria in different food matrices and to evaluate its suitability for the implementation in routine testing. In this context, the use of FISH in different matrices and their pretreatment will be presented, the sensitivity and specificity of FISH tests will be considered and the need for automation shall be discussed as well as the use of technological improvements to overcome current hurdles for a broad application in monitoring food safety. In addition, the overall economical feasibility will be assessed in a rough calculation of costs, and strengths and weaknesses of FISH are considered in comparison with traditional and well-established detection methods. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Next generation sequencing-based multigene panel for high throughput detection of food-borne pathogens.

    PubMed

    Ferrario, Chiara; Lugli, Gabriele Andrea; Ossiprandi, Maria Cristina; Turroni, Francesca; Milani, Christian; Duranti, Sabrina; Mancabelli, Leonardo; Mangifesta, Marta; Alessandri, Giulia; van Sinderen, Douwe; Ventura, Marco

    2017-09-01

    Contamination of food by chemicals or pathogenic bacteria may cause particular illnesses that are linked to food consumption, commonly referred to as foodborne diseases. Bacteria are present in/on various foods products, such as fruits, vegetables and ready-to-eat products. Bacteria that cause foodborne diseases are known as foodborne pathogens (FBPs). Accurate detection methods that are able to reveal the presence of FBPs in food matrices are in constant demand, in order to ensure safe foods with a minimal risk of causing foodborne diseases. Here, a multiplex PCR-based Illumina sequencing method for FBP detection in food matrices was developed. Starting from 25 bacterial targets and 49 selected PCR primer pairs, a primer collection called foodborne pathogen - panel (FPP) consisting of 12 oligonucleotide pairs was developed. The FPP allows a more rapid and reliable identification of FBPs compared to classical cultivation methods. Furthermore, FPP permits sensitive and specific FBP detection in about two days from food sample acquisition to bioinformatics-based identification. The FPP is able to simultaneously identify eight different bacterial pathogens, i.e. Listeria monocytogenes, Campylobacter jejuni, Campylobacter coli, Salmonella enterica subsp. enterica serovar enteritidis, Escherichia coli, Shigella sonnei, Staphylococcus aureus and Yersinia enterocolitica, in a given food matrix at a threshold contamination level of 10(1)cell/g. Moreover, this novel detection method may represent an alternative and/or a complementary approach to PCR-based techniques, which are routinely used for FBP detection, and could be implemented in (parts of) the food chain as a quality check. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Molecular Detection of Avian Pathogens in Poultry Red Mite (Dermanyssus gallinae) Collected in Chicken Farms

    PubMed Central

    HUONG, Chu Thi Thanh; MURANO, Takako; UNO, Yukiko; USUI, Tatsufumi; YAMAGUCHI, Tsuyoshi

    2014-01-01

    Poultry red mite (PRM, Dermanyssus gallinae) is a blood-sucking ectoparasite as well as a possible vector of several avian pathogens. In this study, to define the role of PRM in the prevalence of avian infectious agents, we used polymerase chain reaction (PCR) to check for the presence of seven pathogens: Avipox virus (APV), Fowl Adenovirus (FAdV), Marek’s disease virus (MDV), Erysipelothrix rhusiopathiae (ER), Salmonella enterica (SE), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). A total of 159 PRM samples collected between 2004 and 2012 from 142 chicken farms in 38 prefectures in Japan were examined. APV DNA was detected in 22 samples (13.8%), 19 of which were wild-type APV. 16S ribosomal RNA (16S rRNA) of MS was detected in 15 samples (9.4%), and the mgc2 gene of MG was detected in 2 samples (1.3%). Eight of 15 MS 16S rRNA sequences differed from the vaccine sequence, indicating they were wild-type strains, while both of the MG mgc2 gene sequences detected were identical to the vaccine sequences. Of these avian pathogen-positive mite samples, three were positive for both wild-types of APV and MS. On the other hand, the DNAs of ER, SE, FAdV and MDV were not detected in any samples. These findings indicated that PRM can harbor the wild-type pathogens and might play a role as a vector in spreading these diseases in farms. PMID:25649939

  9. Current Technical Approaches for the Early Detection of Foodborne Pathogens: Challenges and Opportunities.

    PubMed

    Cho, Il-Hoon; Ku, Seockmo

    2017-09-30

    The development of novel and high-tech solutions for rapid, accurate, and non-laborious microbial detection methods is imperative to improve the global food supply. Such solutions have begun to address the need for microbial detection that is faster and more sensitive than existing methodologies (e.g., classic culture enrichment methods). Multiple reviews report the technical functions and structures of conventional microbial detection tools. These tools, used to detect pathogens in food and food homogenates, were designed via qualitative analysis methods. The inherent disadvantage of these analytical methods is the necessity for specimen preparation, which is a time-consuming process. While some literature describes the challenges and opportunities to overcome the technical issues related to food industry legal guidelines, there is a lack of reviews of the current trials to overcome technological limitations related to sample preparation and microbial detection via nano and micro technologies. In this review, we primarily explore current analytical technologies, including metallic and magnetic nanomaterials, optics, electrochemistry, and spectroscopy. These techniques rely on the early detection of pathogens via enhanced analytical sensitivity and specificity. In order to introduce the potential combination and comparative analysis of various advanced methods, we also reference a novel sample preparation protocol that uses microbial concentration and recovery technologies. This technology has the potential to expedite the pre-enrichment step that precedes the detection process.

  10. A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection.

    PubMed

    Yu, Xu; Xia, Yiqiu; Tang, Yi; Zhang, Wen-Long; Yeh, Yin-Ting; Lu, Huaguang; Zheng, Si-Yang

    2017-06-01

    Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 10(3) EID50 mL(-1) (EID50 : 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme-linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off-chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low-cost and convenient biomarker detection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks

    PubMed Central

    Hellmér, Maria; Paxéus, Nicklas; Magnius, Lars; Enache, Lucica; Arnholm, Birgitta; Johansson, Annette; Bergström, Tomas

    2014-01-01

    Most persons infected with enterically transmitted viruses shed large amounts of virus in feces for days or weeks, both before and after onset of symptoms. Therefore, viruses causing gastroenteritis may be detected in wastewater, even if only a few persons are infected. In this study, the presence of eight pathogenic viruses (norovirus, astrovirus, rotavirus, adenovirus, Aichi virus, parechovirus, hepatitis A virus [HAV], and hepatitis E virus) was investigated in sewage to explore whether their identification could be used as an early warning of outbreaks. Samples of the untreated sewage were collected in proportion to flow at Ryaverket, Gothenburg, Sweden. Daily samples collected during every second week between January and May 2013 were pooled and analyzed for detection of viruses by concentration through adsorption to milk proteins and PCR. The largest amount of noroviruses was detected in sewage 2 to 3 weeks before most patients were diagnosed with this infection in Gothenburg. The other viruses were detected at lower levels. HAV was detected between weeks 5 and 13, and partial sequencing of the structural VP1protein identified three different strains. Two strains were involved in an ongoing outbreak in Scandinavia and were also identified in samples from patients with acute hepatitis A in Gothenburg during spring of 2013. The third strain was unique and was not detected in any patient sample. The method used may thus be a tool to detect incipient outbreaks of these viruses and provide early warning before the causative pathogens have been recognized in health care. PMID:25172863

  12. Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

    PubMed

    Alcaine, S D; Tilton, L; Serrano, M A C; Wang, M; Vachet, R W; Nugen, S R

    2015-10-01

    Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

  13. Advanced biosensors for detection of pathogens related to livestock and poultry.

    PubMed

    Vidic, Jasmina; Manzano, Marisa; Chang, Chung-Ming; Jaffrezic-Renault, Nicole

    2017-02-21

    Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health and well-being of wildlife, livestock, and human populations, limit productivity and increase significantly economic losses to each sector. The pathogen detection is an important step for the diagnostics, successful treatment of animal infection diseases and control management in farms and field conditions. Current techniques employed to diagnose pathogens in livestock and poultry include classical plate-based methods and conventional biochemical methods as enzyme-linked immunosorbent assays (ELISA). These methods are time-consuming and frequently incapable to distinguish between low and highly pathogenic strains. Molecular techniques such as polymerase chain reaction (PCR) and real time PCR (RT-PCR) have also been proposed to be used to diagnose and identify relevant infectious disease in animals. However these DNA-based methodologies need isolated genetic materials and sophisticated instruments, being not suitable for in field analysis. Consequently, there is strong interest for developing new swift point-of-care biosensing systems for early detection of animal diseases with high sensitivity and specificity. In this review, we provide an overview of the innovative biosensing systems that can be applied for livestock pathogen detection. Different sensing strategies based on DNA receptors, glycan, aptamers and antibodies are presented. Besides devices still at development level some are validated according to standards of the World Organization for Animal Health and are commercially available. Especially, paper-based platforms proposed as an affordable, rapid and easy to perform sensing systems for implementation in field condition are included in this review.

  14. Rapid detection of pathogenic bacteria by volatile organic compound (VOC) analysis

    NASA Astrophysics Data System (ADS)

    Senecal, Andre G.; Magnone, Joshua; Yeomans, Walter; Powers, Edmund M.

    2002-02-01

    Developments in rapid detection technologies have made countless improvements over the years. However, because of the limited sample that these technologies can process in a single run, the chance of capturing and identifying a small amount of pathogens is difficult. The problem is further magnified by the natural random distribution of pathogens in foods. Methods to simplify pathogenic detection through the identification of bacteria specific VOC were studied. E. coli O157:H7 and Salmonella typhimurium were grown on selected agar medium to model protein, and carbohydrate based foods. Pathogenic and common spoilage bacteria (Pseudomonas and Morexella) were screened for unique VOC production. Bacteria were grown on agar slants in closed vials. Headspace sampling was performed at intervals up to 24 hours using Solid Phase Micro-Extraction (SPME) techniques followed by GC/MS analysis. Development of unique volatiles was followed to establish sensitivity of detection. E. coli produced VOC not found in either Trypticase Soy Yeast (TSY) agar blanks or spoilage organism samples were - indole, 1-decanol, and 2-nonanone. Salmonella specific VOC grown on TSY were 3-methyl-1-butanol, dimethyl sulfide, 2-undecanol, 2-pentadecanol and 1-octanol. Trials on potato dextrose agar (PDA) slants indicated VOC specific for E. coli and Salmonella when compared to PDA blanks and Pseudomonas samples. However, these VOC peaks were similar for both pathogens. Morexella did not grow on PDA slants. Work will continue with model growth mediums at various temperatures, and mixed flora inoculums. As well as, VOC production based on the dynamics of bacterial growth.

  15. Detection of Gastrointestinal Pathogens from Stool Samples on Hemoccult Cards by Multiplex PCR

    PubMed Central

    Schlenker, Nicklas; Bauer, Malkin; Helfrich, Kerstin; Mengele, Carolin; Löscher, Thomas; Nothdurft, Hans Dieter; Bretzel, Gisela; Beissner, Marcus

    2017-01-01

    Purpose. Up to 30% of international travelers are affected by travelers' diarrhea (TD). Reliable data on the etiology of TD is lacking. Sufficient laboratory capacity at travel destinations is often unavailable and transporting conventional stool samples to the home country is inconvenient. We evaluated the use of Hemoccult cards for stool sampling combined with a multiplex PCR for the detection of model viral, bacterial, and protozoal TD pathogens. Methods. Following the creation of serial dilutions for each model pathogen, last positive dilution steps (LPDs) and thereof calculated last positive sample concentrations (LPCs) were compared between conventional stool samples and card samples. Furthermore, card samples were tested after a prolonged time interval simulating storage during a travel duration of up to 6 weeks. Results. The LPDs/LPCs were comparable to testing of conventional stool samples. After storage on Hemoccult cards, the recovery rate was 97.6% for C. jejuni, 100% for E. histolytica, 97.6% for norovirus GI, and 100% for GII. Detection of expected pathogens was possible at weekly intervals up to 42 days. Conclusion. Stool samples on Hemoccult cards stored at room temperature can be used in combination with a multiplex PCR as a reliable tool for testing of TD pathogens. PMID:28408937

  16. A novel microbial source tracking microarray for pathogen detection and fecal source identification in environmental systems.

    PubMed

    Li, Xiang; Harwood, Valerie J; Nayak, Bina; Staley, Christopher; Sadowsky, Michael J; Weidhaas, Jennifer

    2015-06-16

    Pathogen detection and the identification of fecal contamination sources are challenging in environmental waters. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. A custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, and antibiotic resistance genes was tested against DNA obtained from whole genome amplification (WGA) of RNA and DNA from sewage and animal (avian, cattle, poultry, and swine) feces. Perfect and mismatch probes established the specificity of the microarray in sewage, and fluorescence decrease of positive probes over a 1:10 dilution series demonstrated semiquantitative measurement. Pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21-33%) was lower than specificity (83-90%, percentage of true negatives). Next generation DNA sequencing revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources. Phyla and families not represented on the microarray are possible candidates for inclusion in subsequent array designs.

  17. Detection of Gastrointestinal Pathogens from Stool Samples on Hemoccult Cards by Multiplex PCR.

    PubMed

    Alberer, Martin; Schlenker, Nicklas; Bauer, Malkin; Helfrich, Kerstin; Mengele, Carolin; Löscher, Thomas; Nothdurft, Hans Dieter; Bretzel, Gisela; Beissner, Marcus

    2017-01-01

    Purpose. Up to 30% of international travelers are affected by travelers' diarrhea (TD). Reliable data on the etiology of TD is lacking. Sufficient laboratory capacity at travel destinations is often unavailable and transporting conventional stool samples to the home country is inconvenient. We evaluated the use of Hemoccult cards for stool sampling combined with a multiplex PCR for the detection of model viral, bacterial, and protozoal TD pathogens. Methods. Following the creation of serial dilutions for each model pathogen, last positive dilution steps (LPDs) and thereof calculated last positive sample concentrations (LPCs) were compared between conventional stool samples and card samples. Furthermore, card samples were tested after a prolonged time interval simulating storage during a travel duration of up to 6 weeks. Results. The LPDs/LPCs were comparable to testing of conventional stool samples. After storage on Hemoccult cards, the recovery rate was 97.6% for C. jejuni, 100% for E. histolytica, 97.6% for norovirus GI, and 100% for GII. Detection of expected pathogens was possible at weekly intervals up to 42 days. Conclusion. Stool samples on Hemoccult cards stored at room temperature can be used in combination with a multiplex PCR as a reliable tool for testing of TD pathogens.

  18. Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

    PubMed Central

    Lodes, Michael J.; Suciu, Dominic; Wilmoth, Jodi L.; Ross, Marty; Munro, Sandra; Dix, Kim; Bernards, Karen; Stöver, Axel G.; Quintana, Miguel; Iihoshi, Naomi; Lyon, Wanda J.; Danley, David L.; McShea, Andrew

    2007-01-01

    Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format. PMID:17895966

  19. Analysis of bacterial metagenomes from the Southwestern Gulf of Mexico for pathogens detection.

    PubMed

    Escobedo-Hinojosa, Wendy; Pardo-López, Liliana

    2017-07-31

    Little is known about the diversity of bacteria in the Southwestern Gulf of Mexico. The aim of the study illustrated in this perspective was to search for the presence of bacterial pathogens in this ecosystem, using metagenomic data recently generated by the Mexican research group known as the Gulf of Mexico Research Consortium. Several genera of bacteria annotated as pathogens were detected in water and sediment marine samples. As expected, native and ubiquitous pathogenic bacteria genera such as Burkolderia, Halomonas, Pseudomonas, Shewanella and Vibrio were highly represented. Surprisingly, non-native genera of public health concern were also detected, including Borrelia, Ehrlichia, Leptospira, Mycobacterium, Mycoplasma, Salmonella, Staphylococcus, Streptococcus and Treponema. While there are no previous metagenomics studies of this environment, the potential influences of natural, anthropogenic and ecological factors on the diversity of putative pathogenic bacteria found in it are reviewed. The taxonomic annotation herein reported provides a starting point for an improved understanding of bacterial biodiversity in the Southwestern Gulf of Mexico. It also represents a useful tool in public health as it may help identify infectious diseases associated with exposure to marine water and ingestion of fish or shellfish, and thus may be useful in predicting and preventing waterborne disease outbreaks. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Can Handheld Thermal Imaging Technology Improve Detection of Poachers in African Bushveldt?

    PubMed

    Hart, Adam G; Rolfe, Richard N; Dandy, Shantelle; Stubbs, Hannah; MacTavish, Dougal; MacTavish, Lynne; Goodenough, Anne E

    2015-01-01

    Illegal hunting (poaching) is a global threat to wildlife. Anti-poaching initiatives are making increasing use of technology, such as infrared thermography (IRT), to support traditional foot and vehicle patrols. To date, the effectiveness of IRT for poacher location has not been tested under field conditions, where thermal signatures are often complex. Here, we test the hypothesis that IRT will increase the distance over which a poacher hiding in African scrub bushveldt can be detected relative to a conventional flashlight. We also test whether any increase in effectiveness is related to the cost and complexity of the equipment by comparing comparatively expensive (22,000 USD) and relatively inexpensive (2000 USD) IRT devices. To test these hypotheses we employ a controlled, fully randomised, double-blind procedure to find a poacher in nocturnal field conditions in African bushveldt. Each of our 27 volunteer observers walked three times along a pathway using one detection technology on each pass in randomised order. They searched a prescribed search area of bushveldt within which the target was hiding. Hiding locations were pre-determined, randomised, and changed with each pass. Distances of first detection and positive detection were noted. All technologies could be used to detect the target. Average first detection distance for flashlight was 37.3 m, improving by 19.8 m to 57.1 m using LIRT and by a further 11.2m to 68.3m using HIRT. Although detection distances were significantly greater for both IRTs compared to flashlight, there was no significant difference between LIRT and HIRT. False detection rates were low and there was no significant association between technology and accuracy of detection. Although IRT technology should ideally be tested in the specific environment intended before significant investment is made, we conclude that IRT technology is promising for anti-poaching patrols and that for this purpose low cost IRT units are as effective as units

  1. Can Handheld Thermal Imaging Technology Improve Detection of Poachers in African Bushveldt?

    PubMed Central

    Dandy, Shantelle; Stubbs, Hannah; MacTavish, Dougal; MacTavish, Lynne

    2015-01-01

    Illegal hunting (poaching) is a global threat to wildlife. Anti-poaching initiatives are making increasing use of technology, such as infrared thermography (IRT), to support traditional foot and vehicle patrols. To date, the effectiveness of IRT for poacher location has not been tested under field conditions, where thermal signatures are often complex. Here, we test the hypothesis that IRT will increase the distance over which a poacher hiding in African scrub bushveldt can be detected relative to a conventional flashlight. We also test whether any increase in effectiveness is related to the cost and complexity of the equipment by comparing comparatively expensive (22000 USD) and relatively inexpensive (2000 USD) IRT devices. To test these hypotheses we employ a controlled, fully randomised, double-blind procedure to find a poacher in nocturnal field conditions in African bushveldt. Each of our 27 volunteer observers walked three times along a pathway using one detection technology on each pass in randomised order. They searched a prescribed search area of bushveldt within which the target was hiding. Hiding locations were pre-determined, randomised, and changed with each pass. Distances of first detection and positive detection were noted. All technologies could be used to detect the target. Average first detection distance for flashlight was 37.3m, improving by 19.8m to 57.1m using LIRT and by a further 11.2m to 68.3m using HIRT. Although detection distances were significantly greater for both IRTs compared to flashlight, there was no significant difference between LIRT and HIRT. False detection rates were low and there was no significant association between technology and accuracy of detection. Although IRT technology should ideally be tested in the specific environment intended before significant investment is made, we conclude that IRT technology is promising for anti-poaching patrols and that for this purpose low cost IRT units are as effective as units ten

  2. Rapid Method for Detection, Identification, and Susceptibility Testing of Enteric Pathogens

    PubMed Central

    Stager, Charles E.; Erikson, Eric; Davis, James R.

    1983-01-01

    Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35°C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion results

  3. Rapid method for detection, identification, and susceptibility testing of enteric pathogens.

    PubMed

    Stager, C E; Erikson, E; Davis, J R

    1983-01-01

    Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35 degrees C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion

  4. Microbial Diagnostic Microarrays for the Detection and Typing of Food- and Water-Borne (Bacterial) Pathogens

    PubMed Central

    Kostić, Tanja; Sessitsch, Angela

    2011-01-01

    Reliable and sensitive pathogen detection in clinical and environmental (including food and water) samples is of greatest importance for public health. Standard microbiological methods have several limitations and improved alternatives are needed. Most important requirements for reliable analysis include: (i) specificity; (ii) sensitivity; (iii) multiplexing potential; (iv) robustness; (v) speed; (vi) automation potential; and (vii) low cost. Microarray technology can, through its very nature, fulfill many of these requirements directly and the remaining challenges have been tackled. In this review, we attempt to compare performance characteristics of the microbial diagnostic microarrays developed for the detection and typing of food and water pathogens, and discuss limitations, points still to be addressed and issues specific for the analysis of food, water and environmental samples. PMID:27605332

  5. Rapid, portable, multiplexed detection of bacterial pathogens directly from clinical sample matrices

    DOE PAGES

    Phaneuf, Christopher R.; Mangadu, Betty Lou Bosano; Piccini, Matthew E.; ...

    2016-09-23

    Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. Furthermore, this platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated inmore » diarrheal and enteric diseases in less than 20 min.« less

  6. Detection and Characterization of Cancer Cells and Pathogenic Bacteria Using Aptamer-Based Nano-Conjugates

    PubMed Central

    Gedi, Vinayakumar; Kim, Young-Pil

    2014-01-01

    Detection and characterization of cells using aptamers and aptamer-conjugated nanoprobes has evolved a great deal over the past few decades. This evolution has been driven by the easy selection of aptamers via in vitro cell-SELEX, permitting sensitive discrimination between target and normal cells, which includes pathogenic prokaryotic and cancerous eukaryotic cells. Additionally, when the aptamer-based strategies are used in conjunction with nanomaterials, there is the potential for cell targeting and therapeutic effects with improved specificity and sensitivity. Here we review recent advances in aptamer-based nano-conjugates and their applications for detecting cancer cells and pathogenic bacteria. The multidisciplinary research utilized in this field will play an increasingly significant role in clinical medicine and drug discovery. PMID:25268922

  7. Modeling and simulation of DNA flow in a microfluidic-based pathogen detection system

    SciTech Connect

    Trebotich, D; Miller, G H

    2005-01-31

    We present simulation results from a new computational model of DNA flow in microfluidic devices. This work is important because computational models are needed to design miniaturized biomedical devices that are becoming the state-of-the-art in many significant applications including pathogen detection as well as continuous monitoring and drug delivery. Currently advanced algorithms in design tools are non-existent but necessary to understand the complex fluid and polymer dynamics involved in biological flow at small scales. Our model is based on a fully coupled fluid-particle numerical algorithm with both stochastic and deterministic components in a bead-rod polymer representation. We have applied this work to DNA extraction configurations in a microfluidic PCR chamber used in a pathogen detection system. We demonstrate our method on the test problem of flow of a single DNA molecule in a 2D packed array microchannel. We are also investigating mechanisms for molecular ''sticking'' using short range forces.

  8. The Dust at Altitude Recovery Technology (DART) System was Developed to Recover Plant, Human, and Animal Pathogens in Asian and African Dust Storms over North America

    NASA Astrophysics Data System (ADS)

    Schuerger, A. C.; Tench, B.; Nehr, A.; Emmons, T.; Valbuena, F.; Palaia, J.; Sugars, C.

    2014-12-01

    Dust emanates year-round from Africa and Asia and impacts air quality in North America. Asian dust plumes deliver up to 64 million tonnes of dust over the NW of the USA, and African dust storms deliver over 50 million tonnes of dust over Florida each year. Several recent studies have demonstrated that human and plant pathogens from Asian [1] African [2] aerosols can be transported to N. America in naturally occurring dust storms. What is unknown is whether these 'presumptive pathogens' impact human, plant, or animal health in the USA. In order to initiate a long-term monitoring program of pathogens in Asian and African dust plumes, we have developed a dust collection system called DART (Dust at Altitude Recovery Technology) (figure). The DART dust sampler can be mounted on a F104 Starfighter jet (figure) and a T6 Texan propeller driven airplane (not shown), and was test flown over FL in Dec. 2013 on the F104 and on the T6 in the summer of 2014. The DART system utilizes a high-volume pump to pass air through 6 separate filtration units where both aerosols and microbial cells are captured. The filtration systems exhibit flow rates from 25-142 L/min depending on the pore size and brand of filters used. Flow rates are directly correlated to increased air speed, and are inversely correlated to increased altitude. Filtration units can be turned on and off individually as required for specific science flight objectives. The DART dust sampler has performed nominally up to 7600 m, 0.92 Mach, and 3.5 +G's. During initial test flights in Dec. 2013, 5 of 8 genera of fungi recovered from the lower atmosphere over FL contained plant pathogens including species in the genera: Acremonium, Aspergillus, Cladosporium, Curvularia, and Fusarium. Numbers of recovered fungi, but not bacteria, increased significantly when 5 or 10 µm filters were used in the DART system compared to filter pore sizes ≤ 1.2 µm. Future sampling programs for both Asian and African dust events will be

  9. Flow-enhanced detection of biological pathogens using piezoelectric microcantilever arrays

    NASA Astrophysics Data System (ADS)

    McGovern, John-Paul

    The piezoelectric microcantilever sensor (PEMS) is an all-electrical resonant oscillator biosensor system capable of in-situ and label-free detection. With various insulation and antibody immobilization schemes, it is well-suited for sensitive, specific pathogen detection applications with limits of detection on the order of relevant lethal infectious dosages. Initial PEMS implementation demonstrated biodetection of just 36 total Bacillus anthracis (BA) spores in 0.8 ml of liquid. However, concerns of cross reactivity between the antibody and closely related species of the target pathogens casts doubts on the usefulness of antibody-based assays in terms of the specificity of detection. The goal of this thesis is to develop the PEMS as a method for in-situ, label-free, pathogen detection with better limits of detection than current antibody-based methods as well as high sensitivity and specificity, by exploring PEMS array detection and engineered fluidics specificity augmentation. Experimentation in an 8 mm wide channel revealed that optimal discriminatory detection of BA spores among close cousins (B. cereus (BC), thuringiensis (BT) and subtilis (BS)) was achieved at 14 ml/min. At this flow rate, the detection signals of BC, BT, and BS all fell to within the noise level of the sensor, while that of BA was still nearly optimal. Thus, it was deduced that the interaction forces of BC, BT, and BS were 100 pN. Implementation of array sensing systems enabled real-time, redundant biosensor assays and concurrent background determination by a reference PEMS. Consequentially, successful real-time detection of 10 BA spores/ml was achieved, and single Cryptosporidium parvum (CP) oocyst detection at 0.1 oocysts/ml was accomplished with step-wise resonance frequency shifts of 290 Hz and signal to noise ratios (SNR) greater than 5. In a 19 mm wide flow channel, optimal single oocyst detection efficiency was achieved at 2 ml/min. Optimal discrimination of CP from C. muris (CM

  10. Quantitative, multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array.

    PubMed

    Dunbar, Sherry A; Jacobson, James W

    2007-01-01

    We describe a suspension array hybridization assay for rapid detection and identification of Salmonella and other bacterial pathogens using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for (1) design of species-specific oligonucleotide capture probes and PCR amplification primers, (2) coupling oligonucleotide capture probes to carboxylated microspheres, (3) hybridization of coupled microspheres to oligonucleotide targets, (4) production of targets from DNA samples by PCR amplification, and (5) detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. The Luminex xMAP suspension array hybridization assay is rapid, requires few sample manipulations, and provides adequate sensitivity and specificity to detect and differentiate Salmonella and nine other test organisms through direct detection of species-specific DNA sequences.

  11. Rapid single-cell detection and identification of pathogens by using surface-enhanced Raman spectroscopy.

    PubMed

    Dina, N E; Zhou, H; Colniţă, A; Leopold, N; Szoke-Nagy, T; Coman, C; Haisch, C

    2017-05-21

    For the successful treatment of infections, real-time analysis and enhanced multiplex capacity, sensitivity and cost-effectiveness of the developed detection method are critical. In this work, surface-enhanced Raman scattering (SERS) was employed with the final aim of identification and discrimination of pathogenic bacteria, based on their detected SERS fingerprint at the single-cell level. Several genera of bacteria that are found in most of the isolated infections in bacteraemia were successfully identified in less than 5 minutes without the use of antibodies or other specific receptors. The key element of the SERS direct detection platform is the SERS substrate, which combines easy production at low costs with a high enhancement enabling single-cell detection. The innovative approach of detection required the in situ synthesis of silver nanoparticles (NPs), ensuring an intimate contact with the bacterial membrane. This protocol provided a good reproducibility of the single-cell SERS spectra and was successfully applied both on Gram-negative and Gram-positive microorganisms (E. coli, M. morganii, E. lactis, L. casei). Thus, a label-free SERS-based biosensor for pathogen detection was developed with low costs, minimal sample preparation, high-accuracy and a very short analysis time of less than 5 min, which is crucial for infection diagnosis.

  12. Evaluation of New bioMérieux Chromogenic CPS Media for Detection of Urinary Tract Pathogens.

    PubMed

    Rigaill, Josselin; Verhoeven, Paul O; Mahinc, Caroline; Jeraiby, Mohamed; Grattard, Florence; Fonsale, Nathalie; Pozzetto, Bruno; Carricajo, Anne

    2015-08-01

    Four chromogenic media were compared for their ability to detect urinary tract pathogens in 299 urine specimens, of which 175 were found positive, allowing the growth of 279 microorganisms. After 18 to 24 h of incubation, the CPS ID4, CPSE, CPSO (bioMérieux), and UriSelect4 (Bio-Rad) media showed sensitivities of 97.1%, 99.3%, 99.6%, and 99.6%, respectively.

  13. Engineering the stereochemistry of cephalosporin for specific detection of pathogenic carbapenemase-expressing bacteria.

    PubMed

    Shi, Haibin; Cheng, Yunfeng; Lee, Kyung Hyun; Luo, Robert F; Banaei, Niaz; Rao, Jianghong

    2014-07-28

    Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant β-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-β-lactamases in active bacterial pathogens.

  14. Microbial Biosensor for the Detection of Protease-Virulent Factors from Pathogens

    DTIC Science & Technology

    2017-04-28

    individuals. 15. SUBJECT TERMS synthetic biology , AOARD 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18. NUMBER OF PAGES      19   19a...University of Singapore Department: Department of Biochemistry, Yong Loo Lin School of Medicine, and NUS Synthetic Biology for Clinical and...biosensors for toxin-based pathogen detection. Employing synthetic biology approaches, our various constructs will endow military personnel with the

  15. Understanding Perceived Benefit of Early Cancer Detection: Community-Partnered Research with African American Women in South Los Angeles.

    PubMed

    Bazargan, Mohsen; Lucas-Wright, Anna; Jones, Loretta; Vargas, Roberto; Vadgama, Jaydutt V; Evers-Manly, Shirley; Maxwell, Annette E

    2015-09-01

    African American women have lower 5-year cancer survival rates than non-Latino White women. Differences in perceived benefits of early cancer detection among racial/ethnic groups may affect cancer-screening behaviors. This study assessed correlates of perceived benefits of early breast, cervical and colorectal cancer detection among 513 African American women. Using a community-partnered participatory research approach, we conducted a survey on cancer screening, risk behaviors, and related knowledge and attitudes among African American parishioners at 11 churches in South Los Angeles, a neighborhood that experiences one of the highest cancer mortality rates in California. African American women who participated in this study were more likely to believe that chances for survival are very good or good after early detection of breast cancer (74%) than after early detection of colorectal (51%) and cervical cancer (52%). Multivariate analyses show that perceived benefit of early cancer detection is associated with higher cancer knowledge and having discussed one's cancer risk with a doctor. Given that 5-year survival rates for early stage breast, cervical, and colorectal cancer range from 84% to 93%, our data suggest that a substantial proportion of African American women in South Los Angeles are not aware of the benefits of early detection, particularly of colorectal and cervical cancers. Programs that increase cancer knowledge and encourage a discussion of individual's cancer risk with a doctor may be able to increase perceived benefit of early detection, a construct that has been shown to be associated with cancer screening in some studies.

  16. A microfluidic platform with integrated arrays for immunologic assays for biological pathogen detection

    NASA Astrophysics Data System (ADS)

    Klemm, Richard; Becker, Holger; Hlawatsch, Nadine; Julich, Sandra; Miethe, Peter; Moche, Christian; Schattschneider, Sebastian; Tomaso, Herbert; Gärtner, Claudia

    2014-05-01

    The ability to integrate complete assays on a microfluidic chip helps to greatly simplify instrument requirements and allows the use of lab-on-a-chip technology in the field. A core application for such field-portable systems is the detection of pathogens in a CBRN scenario such as permanent monitoring of airborne pathogens, e.g. in subway stations or hospitals etc. An immunological assay was chosen as method for the pathogen identification. The conceptual approach was its realization as a lab-on-a-chip system, enabling an easy handling of the sample in an automated manner. The immunological detection takes place on an antibody array directly implemented in the microfluidic network. Different immobilization strategies will be presented showing the performance of the system. Central elements of the disposable microfluidic device like fluidic interface, turning valves, liquid introduction and waste storage, as well as the architecture of measurement and control fluidic network, will be introduced. Overall process times of about 30 minutes were achieved and assays for the detection of Francisella tularensis and Yersinia pestis are presented. An important feature of the integrated lab-on-a-chip approach is that all waste liquids remain on-chip and contamination risks can be avoided.

  17. Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections.

    PubMed

    Hong, Bang-Xing; Jiang, Li-Fang; Hu, Yu-Shan; Fang, Dan-Yun; Guo, Hui-Yu

    2004-09-01

    A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.

  18. Detection of enteric pathogens in Turkey flocks affected with severe enteritis, in Brazil.

    PubMed

    Moura-Alvarez, Joelma; Nuñez, Luis F N; Astolfi-Ferreira, Claudete S; Knöbl, Terezinha; Chacón, Jorge L; Moreno, Andrea M; Jones, Richard C; Ferreira, Antonio J Piantino

    2014-08-01

    Twenty-two flocks of turkeys affected by enteric problems, with ages between 10 and 104 days and located in the Southern region of Brazil, were surveyed for turkey by PCR for turkey astrovirus type 2 (TAstV-2), turkey coronavirus (TCoV), hemorrhagic enteritis virus (HEV), rotavirus, reovirus, Salmonella spp., and Lawsonia intracellularis (Li) infections. Eleven profiles of pathogen combination were observed. The most frequently encountered pathogen combinations were TCoV-Li, followed by TCoV-TAstV-2-Li, TCoV-TastV-2. Only TCoV was detected as the sole pathogen in three flocks. Eight and 19 flocks of the 22 were positive for TAstV-2 and TCoV, respectively. Six were positive for Salmonella spp. and L. intracellularis was detected in 12 turkey flocks. Reovirus and HEV were not detected in this survey. These results throw new light on the multiple etiology of enteritis in turkeys. The implications of these findings and their correlation with the clinical signs are comprehensively discussed, illustrating the complexity of the enteric diseases.

  19. Biosensors as innovative tools for the detection of food borne pathogens.

    PubMed

    Arora, Pooja; Sindhu, Annu; Dilbaghi, Neeraj; Chaudhury, Ashok

    2011-10-15

    The wholesomeness of food is the real proviso for healthy life. Food freed from microbial and chemical cross-contaminations adds on to its hygienic and nutritive value. Infectious diseases spreading every day through food have become a life-threatening problem for millions of people around the world. Food or food products are the potent transmitting agent of more than 250 known diseases. So far only in the United States, 76 million cases of food-borne illness, 32,500 cases of hospitalization and 5000 cases per annum of mortality are recognized. Health expert's estimate that the yearly cost of all the food borne diseases is approximately $5-6 billion. There is therefore, is an urgent need for the development of rapid, competent, and reliable methods for direct detection and identification of foodborne brown pathogens. In this overview, we have concentrated specifically on microbe-based biosensing methods such as optical, surface plasmon resonance (SPR), amperometric, potentiometric, whole-cell, electrochemical, impedimetric, piezoelectric for the rapid detection of food borne pathogens. Furthermore, we have focused our attention on the discussion of principal concepts, applications, and examples from analyte to the configuration of potential biosensors that have been achieved up until now to detect potential foodborne pathogens. The article presents foreseeable future trends in biosensor research activities for paving the way for fresh and healthy food proposal. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology

    PubMed Central

    Zhou, Linfu; Wu, Rui; Shi, Xiaodan; Feng, Dongyun; Feng, Guodong; Yang, Yining; Dai, Wen; Bian, Ting; Liu, Tingting; He, Ying; Shi, Ming; Zhao, Gang

    2016-01-01

    Early diagnosis and treatment are crucial for the outcome of central nervous system (CNS) infections. In this study, we developed a multiplex PCR-Luminex assay for the simultaneous detection of five major pathogens, including Mycobacterium tuberculosis, Cryptococcus neoformans, Streptococcus pneumoniae, and herpes simplex virus types 1 and 2, which frequently cause CNS infections. Through the hybridization reaction between multiplex PCR-amplified targets and oligonucleotide “anti-TAG” sequences, we found that the PCR-Luminex assay could detect as low as 101–102 copies of synthetic pathogen DNAs. Furthermore, 163 cerebrospinal fluid (CSF) specimens from patients with suspected CNS infections were used to evaluate the efficiency of this multiplex PCR-Luminex method. Compared with Ziehl-Neelsen stain, this assay showed a high diagnostic accuracy for tuberculosis meningitis (sensitivity, 90.7% and specificity, 99.1%). For cryptococcal meningitis, the sensitivity and specificity were 92% and 97.1%, respectively, compared with the May Grunwald Giemsa (MGG) stain. For herpes simplex virus types 1 and 2 encephalitis, the sensitivities were 80.8% and 100%, and the specificities were 94.2% and 99%, respectively, compared with Enzyme Linked Immunosorbent Assay (ELISA) assays. Taken together, this multiplex PCR-Luminex assay showed potential efficiency for the simultaneous detection of five pathogens and may be a promising supplement to conventional methods for diagnosing CNS infections. PMID:26861363

  1. Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens.

    PubMed

    Chaplen, Frank W R; Upson, Rosalyn H; Mcfadden, Philip N; Kolodziej, Wojtek

    2002-02-01

    Fish chromatophores from Betta splendens are used as the cytosensor element in the development of a portable microscale device capable of detecting certain environmental toxins and bacterial pathogens by monitoring changes in pigment granule distribution. The adaptation of chromatophores to a microscale environment has required the development of enabling technologies to produce miniaturized culture chambers, to integrate microfluidics for sample delivery, to miniaturize image capture, and to design new statistical methods for image analyses. Betta splendens chromatophores were selected as the cytosensor element because of their moderate size, their toleration of close contact, and most importantly, for their responses to a broad range of chemicals and pathogenic bacteria. A miniaturized culture chamber has been designed that supports chromatophore viability for as long as 3 months, and that can be easily transported without damage to the cells. New statistical methods for image analyses have been developed that increase sensitivity and also decrease the time required for detection of significant changes in pigment granule distribution. Betta chromatophores have been tested for their responses to selected pathogenic bacteria and chemical agents. We discuss in detail the aggregation of pigment granules seen when chromatophores are incubated with Bacillus cereus, a common cause of food poisoning. Also described are the more subtle responses of chromatophores to a class of environmental chemical toxins, polynuclear aromatic hydrocarbons. We show that the chromatophores are able to detect the presence of certain polynuclear aromatic hydrocarbons at concentrations lower than the Environment Protection Agency (EPA) 550.1 standards.

  2. Possible use of ail and foxA polymorphisms for detecting pathogenic Yersinia enterocolitica.

    PubMed

    Huang, Ying; Wang, Xin; Cui, Zhigang; Yang, Yuhuan; Xiao, Yuchun; Tang, Liuying; Kan, Biao; Xu, Jianguo; Jing, Huaiqi

    2010-08-07

    Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica. The genes ail and foxA in 271 pathogenic and 27 non-pathogenic Y. enterocolitica strains isolated from China and 10 reference strains were sequenced, aligned, compared to the ail and foxA sequences of Yersinia enterocolitica subsp. enterocolitica 8081 (Genbank: NC_008800), and analyzed for sequence polymorphism. The ail from the 282 strains showed 3 sequence patterns: 277 strains of serotypes O:3, O:9 and O:5, 27 with identical nucleic acid sequences formed pattern A1; 4 strains of serotype 1B/O:8 with identical nucleic acid sequences formed pattern A2; and one Chinese isolate 2/O:9 formed pattern A3. In the primary coding region of the foxA ORF (Genebank: X60447 nt 433-1866; nt 28 to 1,461 in the ORF), the sequences formed 3 groups and were further divided into 8 sequence patterns. The ail and foxA loci of pathogenic Y. enterocolitica have been analyzed. The ail sequence was highly conserved among the same serotype strains from different sources; and foxA was highly conserved among the pathogenic strains, although there was some sequence diversity. Fewer strains were used from outside China, which is a limitation of the study.

  3. Atypical sensors for direct and rapid neuronal detection of bacterial pathogens.

    PubMed

    Lim, Ji Yeon; Choi, Seung-In; Choi, Geunyeol; Hwang, Sun Wook

    2016-03-09

    Bacterial infection can threaten the normal biological functions of a host, often leading to a disease. Hosts have developed complex immune systems to cope with the danger. Preceding the elimination of pathogens, selective recognition of the non-self invaders is necessary. At the forefront of the body's defenses are the innate immune cells, which are equipped with particular sensor molecules that can detect common exterior patterns of invading pathogens and their secreting toxins as well as with phagocytic machinery. Inflammatory mediators and cytokines released from these innate immune cells and infected tissues can boost the inflammatory cascade and further recruit adaptive immune cells to maximize the elimination and resolution. The nervous system also seems to interact with this process, mostly known to be affected by the inflammatory mediators through the binding of neuronal receptors, consequently activating neural circuits that tune the local and systemic inflammatory states. Recent research has suggested new contact points: direct interactions of sensory neurons with pathogens. Latest findings demonstrated that the sensory neurons not only share pattern recognition mechanisms with innate immune cells, but also utilize endogenous and exogenous electrogenic components for bacterial pathogen detection, by which the electrical firing prompts faster information flow than what could be achieved when the immune system is solely involved. As a result, rapid pain generation and active accommodation of the immune status occur. Here we introduced the sensory neuron-specific detector molecules for directly responding to bacterial pathogens and their signaling mechanisms. We also discussed extended issues that need to be explored in the future.

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

    PubMed

    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.

  5. Evaluating Surveillance Strategies for the Early Detection of Low Pathogenicity Avian Influenza Infections

    PubMed Central

    Comin, Arianna; Stegeman, Arjan; Marangon, Stefano; Klinkenberg, Don

    2012-01-01

    In recent years, the early detection of low pathogenicity avian influenza (LPAI) viruses in poultry has become increasingly important, given their potential to mutate into highly pathogenic viruses. However, evaluations of LPAI surveillance have mainly focused on prevalence and not on the ability to act as an early warning system. We used a simulation model based on data from Italian LPAI epidemics in turkeys to evaluate different surveillance strategies in terms of their performance as early warning systems. The strategies differed in terms of sample size, sampling frequency, diagnostic tests, and whether or not active surveillance (i.e., routine laboratory testing of farms) was performed, and were also tested under different epidemiological scenarios. We compared surveillance strategies by simulating within-farm outbreaks. The output measures were the proportion of infected farms that are detected and the farm reproduction number (Rh). The first one provides an indication of the sensitivity of the surveillance system to detect within-farm infections, whereas Rh reflects the effectiveness of outbreak detection (i.e., if detection occurs soon enough to bring an epidemic under control). Increasing the sampling frequency was the most effective means of improving the timeliness of detection (i.e., it occurs earlier), whereas increasing the sample size increased the likelihood of detection. Surveillance was only effective in preventing an epidemic if actions were taken within two days of sampling. The strategies were not affected by the quality of the diagnostic test, although performing both serological and virological assays increased the sensitivity of active surveillance. Early detection of LPAI outbreaks in turkeys can be achieved by increasing the sampling frequency for active surveillance, though very frequent sampling may not be sustainable in the long term. We suggest that, when no LPAI virus is circulating yet and there is a low risk of virus introduction, a

  6. Development of saliva-based exposure assays for detecting exposure to waterborne pathogens

    EPA Pesticide Factsheets

    Identifying which pathogens we are exposed to can be challenging because many types of pathogens can be found in water and many pathogens have similar symptoms. EPA scientists have developed a simple way to measure human exposure to waterborne pathogens.

  7. Duplex Microfluidic SERS Detection of Pathogen Antigens with Nanoyeast Single-Chain Variable Fragments

    PubMed Central

    2015-01-01

    Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications. PMID:25192256

  8. Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.

    PubMed

    Wang, Yuling; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Schlücker, Sebastian; Trau, Matt

    2014-10-07

    Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

  9. Self-propelled, phage-based magnetoelastic biosentinels for detection of pathogens in liquid

    NASA Astrophysics Data System (ADS)

    Horikawa, Shin; Zhao, Ruiting; Chai, Yating; Wikle, Howard C.; Chin, Bryan A.

    2014-05-01

    This paper presents the concept of self-propelled magnetoelastic (ME) biosentinels that seek out and capture pathogenic bacteria in stagnant liquids. These biosentinels are composed of a free-standing, asymmetric-shaped ME resonator coated with a filamentous landscape phage that specifically binds with a pathogen of interest. When a time-varying magnetic pulse is applied, the ME biosentinels can be placed into mechanical resonance by magnetostriction. The resultant asymmetric vibration then generates a net force on the surroundings and hence generates autonomous motion in the liquid. As soon as the biosentinels find and bind with the target pathogen through the phage-based biomolecular recognition, a change in the biosentinel's resonant frequency occurs, and thereby the presence of the target pathogen can be detected. In order to actuate the ME biosentinels into mechanical resonance of a desired mode, modal analysis using the three-dimensional finite element method was performed. In addition, the design of a magnetic chamber that can control the orientation and/or translation of a biosentinel is discussed.

  10. Detection of African swine fever virus in infected pig tissues by immunocytochemistry and in sity hybridisation.

    PubMed

    Oura, C A; Powell, P P; Parkhouse, R M

    1998-06-01

    The techniques for determining cellular sites of establishment and persistence of African swine fever virus (ASFV) were established in susceptible domestic pigs and the resistant African reservoir hosts, the warthog and bushpig. Detection, both in vitro and in vivo, was achieved by in situ hybridisation and immunocytochemistry, focusing principally on specific probes for vp73, a major capsid protein. Hybridisation of radio-labelled probes for DNA and RNA was relatively insensitive and time consuming whereas hybridisation of non-radioactive DNA probes was quicker and more sensitive. Detection of vp73 protein by immunocytochemistry was as sensitive as non-radioactive DNA hybridisation, offering in addition improved speed, ease and morphology. Both non-radioactive DNA hybridisation and immunocytochemistry were therefore used to detect ASFV DNA and protein in a range of porcine cells infected in vitro with different ASFV isolates. Malta and Malawi isolates infected both alveolar and bone marrow macrophages, but infected negligible numbers of endothelial (< 1%) and kidney cells (IBRS2 cells). Attenuated Uganda isolate, however, infected a high percentage of endothelial cells and IBRS2 cells as well as alveolar and bone marrow macrophages. When used to investigate the cell tropism of ASFV in tissues from pigs infected with the highly virulent Malawi isolate of ASFV, both techniques identified virus principally in cells of the mononuclear phagocytic system. In the lung, double staining revealed that pulmonary intravascular macrophages, but not alveolar macrophages, were infected.

  11. Validation of ELISA for the detection of African horse sickness virus antigens and antibodies.

    PubMed

    Rubio, C; Cubillo, M A; Hooghuis, H; Sanchez-Vizcaino, J M; Diaz-Laviada, M; Plateau, E; Zientara, S; Crucière, C; Hamblin, C

    1998-01-01

    The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y Produccion Animal (LSPA) in Algete, Spain was charged with the responsibility of co-ordinating and supplying samples of viruses and antisera to the participating laboratories in Spain, France and the United Kingdom. The panels comprised 76 antigen samples for assay by indirect sandwich ELISAs and 53 serum samples for antibody detection by either indirect or competitive ELISAs. Results generated by ELISA for each laboratory were analysed in LSPA in terms of their relative sensitivities and specificities. There was a good agreement between the ELISAs used for either antigen or antibody detection. The participating groups agreed that any field sample giving a doubtful result would always be retested by ELISA and an alternative assay.

  12. Detection of novel sequences related to african Swine Fever virus in human serum and sewage.

    PubMed

    Loh, Joy; Zhao, Guoyan; Presti, Rachel M; Holtz, Lori R; Finkbeiner, Stacy R; Droit, Lindsay; Villasana, Zoilmar; Todd, Collin; Pipas, James M; Calgua, Byron; Girones, Rosina; Wang, David; Virgin, Herbert W

    2009-12-01

    The family Asfarviridae contains only a single virus species, African swine fever virus (ASFV). ASFV is a viral agent with significant economic impact due to its devastating effects on populations of domesticated pigs during outbreaks but has not been reported to infect humans. We report here the discovery of novel viral sequences in human serum and sewage which are clearly related to the asfarvirus family but highly divergent from ASFV. Detection of these sequences suggests that greater genetic diversity may exist among asfarviruses than previously thought and raises the possibility that human infection by asfarviruses may occur.

  13. Detection of human bacterial pathogens in ticks collected from Louisiana black bears (Ursus americanus luteolus).

    PubMed

    Leydet, Brian F; Liang, Fang-Ting

    2013-04-01

    There are 4 major human-biting tick species in the northeastern United States, which include: Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, and Ixodes scapularis. The black bear is a large mammal that has been shown to be parasitized by all the aforementioned ticks. We investigated the bacterial infections in ticks collected from Louisiana black bears (Ursus americanus subspecies luteolus). Eighty-six ticks were collected from 17 black bears in Louisiana from June 2010 to March 2011. All 4 common human-biting tick species were represented. Each tick was subjected to polymerase chain reaction (PCR) targeting select bacterial pathogens and symbionts. Bacterial DNA was detected in 62% of ticks (n=53). Rickettsia parkeri, the causative agent of an emerging spotted fever group rickettsiosis, was identified in 66% of A. maculatum, 28% of D. variabilis, and 11% of I. scapularis. The Lyme disease bacterium, Borrelia burgdorferi, was detected in 2 I. scapularis, while one A. americanum was positive for Borrelia bissettii, a putative human pathogen. The rickettsial endosymbionts Candidatus Rickettsia andeanae, rickettsial endosymbiont of I. scapularis, and Rickettsia amblyommii were detected in their common tick hosts at 21%, 39%, and 60%, respectively. All ticks were PCR-negative for Anaplasma phagocytophilum, Ehrlichia spp., and Babesia microti. This is the first reported detection of R. parkeri in vector ticks in Louisiana; we also report the novel association of R. parkeri with I. scapularis. Detection of both R. parkeri and B. burgdorferi in their respective vectors in Louisiana demands further investigation to determine potential for human exposure to these pathogens. Copyright © 2013 Elsevier GmbH. All rights reserved.

  14. Electrochemical genosensor for specific detection of the food-borne pathogen, Vibrio cholerae.

    PubMed

    Low, Kim-Fatt; Chuenrangsikul, Kritsanaporn; Rijiravanich, Patsamon; Surareungchai, Werasak; Chan, Yean-Yean

    2012-04-01

    A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.

  15. Bacteria Murmur: Application of an Acoustic Biosensor for Plant Pathogen Detection

    PubMed Central

    Dimopoulou, Anastasia; Glynos, Paraskevas; Gizeli, Electra

    2015-01-01

    A multi-targeting protocol for the detection of three of the most important bacterial phytopathogens, based on their scientific and economic importance, was developed using an acoustic biosensor (the Quartz Crystal Microbalance) for DNA detection. Acoustic detection was based on a novel approach where DNA amplicons were monitored and discriminated based on their length rather than mass. Experiments were performed during real time monitoring of analyte binding and in a direct manner, i.e. without the use of labels for enhancing signal transduction. The proposed protocol improves time processing by circumventing gel electrophoresis and can be incorporated as a routine detection method in a diagnostic lab or an automated lab-on-a-chip system for plant pathogen diagnostics. PMID:26177507

  16. Bacteria Murmur: Application of an Acoustic Biosensor for Plant Pathogen Detection.

    PubMed

    Papadakis, George; Skandalis, Nicholas; Dimopoulou, Anastasia; Glynos, Paraskevas; Gizeli, Electra

    2015-01-01

    A multi-targeting protocol for the detection of three of the most important bacterial phytopathogens, based on their scientific and economic importance, was developed using an acoustic biosensor (the Quartz Crystal Microbalance) for DNA detection. Acoustic detection was based on a novel approach where DNA amplicons were monitored and discriminated based on their length rather than mass. Experiments were performed during real time monitoring of analyte binding and in a direct manner, i.e. without the use of labels for enhancing signal transduction. The proposed protocol improves time processing by circumventing gel electrophoresis and can be incorporated as a routine detection method in a diagnostic lab or an automated lab-on-a-chip system for plant pathogen diagnostics.

  17. Molecular detection of pathogens in ticks infesting cattle in Nampula province, Mozambique.

    PubMed

    Matsimbe, Ana Marcília; Magaia, Vlademiro; Sanches, Gustavo Seron; Neves, Luís; Noormahomed, Emília; Antunes, Sandra; Domingos, Ana

    2017-08-30

    Ticks are ectoparasites that can act as vectors of a large number of pathogens in wild and domestic animals, pets, and occasionally humans. The global threat of emerging or re-emerging tick-borne diseases supports the need for research focused in the zoonotic transmission, especially in countries like Mozambique where rural populations are in close contact with domestic animals. The present study aims to: (1) identify tick species infesting cattle from Monapo and Nacala Porto, districts of Nampula province, Mozambique; and (2) investigate the presence of pathogens in the collected ticks. A total of 646 ticks were collected from cattle and morphologically identified as Amblyomma variegatum, Rhipicephalus microplus, and R. evertsi evertsi. For convenience, 72 A. variegatum and 15 R. microplus from Monapo, and 30 A. variegatum from Nacala Porto were screened for the presence of the selected pathogens: Rickettsia spp. (A. variegatum), and Babesia/Theileria spp. and Anaplasma/Ehrlichia spp. (R. microplus). Rickettsia africae was detected in four of the 72 A. variegatum collected in Monapo (5.6%). Additionally, one R. microplus tick (6.7%) was positive for Theileria velifera, one positive for Colpodella spp., one positive for Candidatus Midichloria mitochondrii, and another one positive for Anaplasma ovis. Using the present approach, no microorganisms were detected in tick samples from Nacala Porto. These findings expand our knowledge about the repertoire of tick-borne microorganisms in ticks in Nampula province, Mozambique.

  18. [Detection of biofilm formation by selected pathogens relevant to the food industry].

    PubMed

    Šilhová-Hrušková, L; Moťková, P; Šilha, D; Vytřasová, J

    2015-09-01

    Detection of biofilm formation by microbial pathogens relevant to the food industry and comparison of biofilm formation under different conditions of culture. The following microorganisms were selected for the study: Staphylococcus aureus, Listeria innocua, Listeria ivanovii, Cronobacter sakazakii, Cronobacter muytjensii, Arcobacter butzleri, Arcobacter cryaerophilus, Campylobacter jejuni, and Campylobacter coli. To detect biofilm formation the microtiter plate assay, as described by Christensen and culture on stainless steel coupons were used. The biofilm forming capacity was confirmed in all microorganisms tested, both on the microtiter plates and stainless steel coupons. Biofilm formation was influenced by the culture medium, material used, and culture duration as well as by the test microorganism. It was found that different species and strains of the same genus differ in biofilm formation. Differences were also found between the collection strains and isolates from the environment. Some bacteria tended to form biofilm more readily on the surface of the polyethylene microtiter plates and less readily on stainless steel coupons while others appeared to have an opposite tendency. Some pathogens were able to increase the planktonic cell density in the initial suspension even by three orders of magnitude within 72 hours while producing plenty of biofilm. The study of biofilm formation by high risk pathogens is of utmost importance, not only to the food industry. From the obtained results, it is evident that bacterial biofilms form rapidly (within 24 hours in the present study). Due to their architecture, these biofilms are difficult to eradicate, and therefore, it is crucial to prevent biofilm formation.

  19. Agarose-based microfluidic device for point-of-care concentration and detection of pathogen.

    PubMed

    Li, Yiwei; Yan, Xinghua; Feng, Xiaojun; Wang, Jie; Du, Wei; Wang, Yachao; Chen, Peng; Xiong, Liang; Liu, Bi-Feng

    2014-11-04

    Preconcentration of pathogens from patient samples represents a great challenge in point-of-care (POC) diagnostics. Here, a low-cost, rapid, and portable agarose-based microfluidic device was developed to concentrate biological fluid from micro- to picoliter volume. The microfluidic concentrator consisted of a glass slide simply covered by an agarose layer with a binary tree-shaped microchannel, in which pathogens could be concentrated at the end of the microchannel due to the capillary effect and the strong water permeability of the agarose gel. The fluorescent Escherichia coli strain OP50 was used to demonstrate the capacity of the agarose-based device. Results showed that 90% recovery efficiency could be achieved with a million-fold volume reduction from 400 μL to 400 pL. For concentration of 1 × 10(3) cells mL(-1) bacteria, approximately ten million-fold enrichment in cell density was realized with volume reduction from 100 μL to 1.6 pL. Urine and blood plasma samples were further tested to validate the developed method. In conjugation with fluorescence immunoassay, we successfully applied the method to the concentration and detection of infectious Staphylococcus aureus in clinics. The agarose-based microfluidic concentrator provided an efficient approach for POC detection of pathogens.

  20. Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions.

    PubMed

    Wu, Qingzhong; Prager, Katherine C; Goldstein, Tracey; Alt, David P; Galloway, Renee L; Zuerner, Richard L; Lloyd-Smith, James O; Schwacke, Lori

    2014-08-11

    Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

  1. New Trends in Impedimetric Biosensors for the Detection of Foodborne Pathogenic Bacteria

    PubMed Central

    Wang, Yixian; Ye, Zunzhong; Ying, Yibin

    2012-01-01

    The development of a rapid, sensitive, specific method for the foodborne pathogenic bacteria detection is of great importance to ensure food safety and security. In recent years impedimetric biosensors which integrate biological recognition technology and impedance have gained widespread application in the field of bacteria detection. This paper presents an overview on the progress and application of impedimetric biosensors for detection of foodborne pathogenic bacteria, particularly the new trends in the past few years, including the new specific bio-recognition elements such as bacteriophage and lectin, the use of nanomaterials and microfluidics techniques. The applications of these new materials or techniques have provided unprecedented opportunities for the development of high-performance impedance bacteria biosensors. The significant developments of impedimetric biosensors for bacteria detection in the last five years have been reviewed according to the classification of with or without specific bio-recognition element. In addition, some microfluidics systems, which were used in the construction of impedimetric biosensors to improve analytical performance, are introduced in this review. PMID:22737018

  2. Ultrarapid detection of pathogenic bacteria using a 3D immunomagnetic flow assay.

    PubMed

    Lee, Wonjae; Kwon, Donghoon; Chung, Boram; Jung, Gyoo Yeol; Au, Anthony; Folch, Albert; Jeon, Sangmin

    2014-07-01

    We developed a novel 3D immunomagnetic flow assay for the rapid detection of pathogenic bacteria in a large-volume food sample. Antibody-functionalized magnetic nanoparticle clusters (AbMNCs) were magnetically immobilized on the surfaces of a 3D-printed cylindrical microchannel. The injection of a Salmonella-spiked sample solution into the microchannel produced instant binding between the AbMNCs and the Salmonella bacteria due to their efficient collisions. Nearly perfect capture of the AbMNCs and AbMNCs-Salmonella complexes was achieved under a high flow rate by stacking permanent magnets with spacers inside the cylindrical separator to maximize the magnetic force. The concentration of the bacteria in solution was determined using ATP luminescence measurements. The detection limit was better than 10 cfu/mL, and the overall assay time, including the binding, rinsing, and detection steps for a 10 mL sample took less than 3 min. To our knowledge, the 3D immunomagnetic flow assay described here provides the fastest high-sensitivity, high-capacity method for the detection of pathogenic bacteria.

  3. Aptamer-based hydrogel barcodes for the capture and detection of multiple types of pathogenic bacteria.

    PubMed

    Xu, Yueshuang; Wang, Huan; Luan, Chengxin; Liu, Yuxiao; Chen, Baoan; Zhao, Yuanjin

    2017-09-20

    Rapid and sensitive diagnosing hematological infections based on the separation and detection of pathogenic bacteria in the patient's blood is a significant challenge. To address this, we herein present a new barcodes technology that can simultaneously capture and detect multiple types of pathogenic bacteria from a complex sample. The barcodes are poly (ethylene glycol) (PEG) hydrogel inverse opal particles with characteristic reflection peak codes that remain stable during bacteria capture on their surfaces. As the spherical surface of the particles has ordered porous nanostructure, the barcodes can provide not only more surface area for probe immobilization and reaction, but also a nanopatterned platform for highly efficient bioreactions. In addition, the PEG hydrogel scaffold could decrease the non-specificity adsorption by its anti-adhesive effect, and the decorated aptamer probes in the scaffolds could increase the sensitivity, reliability, and specificity of the bacteria capture and detection. Moreover, the tagged magnetic nanoparticles in the PEG scaffold could impart the barcodes with controllable movement under magnetic fields, which can be used to significantly increase the reaction speed and simplify the processing of the bioassays. Based on the describe barcodes, it was demonstrated that the bacteria could be captured and identified even at low bacterial concentrations (100 CFU mL(-1)) within 2.5h, which is effectively shortened in comparison with the "gold standard" in clinic. These features make the barcodes ideal for capturing and detecting multiple bacteria from clinical samples for hematological infection diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Molecular detection of pathogenic leptospiral protein encoding gene (lipL32) in environmental aquatic biofilms.

    PubMed

    Vinod Kumar, K; Lall, C; Vimal Raj, R; Vedhagiri, K; Vijayachari, P

    2016-04-01

    Leptospirosis is a zoonotic disease often encountered during post-monsoon season due to exposure with contaminated water. Leptospires have long been regarded as solitary organisms that persist in soil and aquatic environments. Here, the presence of leptospires in the aquatic biofilm exposed in the paddy field, sewers and stagnant rain water was demonstrated. Biofilm samples from paddy field water, submerged paddy leaves, sewers and stagnant rain waters from urban and rural areas were collected. Total genomic DNA was extracted and pathogenic leptospiral specific gene amplification was carried out to determine the spatial distribution of the bacteria. The degree of pathogenic Leptospira in biofilms from paddy field surface water, submerged leaf, were 33·3% and 27·2% respectively, whereas in rural and urban area, the sampling sites such as stagnant rain water, domestic sewer and collective sewers showed 11·1%, 13% and 16·6% with leptospires respectively. Higher proportion of pathogenic Leptospira in aquatic ecosystems, such as paddy field, could be one of the main factors for the occurrence of disease, more among the agricultural workers. This study would help to identify various survival strategies of leptospires in the environment and thus disease transmission. Little is known regarding the mechanisms by which pathogenic leptospires persist in aqueous environment, outside the mammalian host. In this view this is the first report of the distribution of Leptospira in environmental biofilm such as sewers and paddy leaf surfaces. This ability of pathogenic Leptospira to survive in aquatic ecosystems especially in biofilms could be one of the main factors which facilitate its survival in the environment, and thus disease transmission among the risk groups, such as sewage and agriculture worker. This study will encourage researchers in the field to consider biofilm as an important factor, when detecting leptospires in environment. © 2015 The Society for Applied

  5. Nucleic Acid-Based Detection and Identification of Bacterial and Fungal Plant Pathogens - Final Report

    SciTech Connect

    Kingsley, Mark T

    2001-03-13

    The threat to American interests from terrorists is not limited to attacks against humans. Terrorists might seek to inflict damage to the U.S. economy by attacking our agricultural sector. Infection of commodity crops by bacterial or fungal crop pathogens could adversely impact U.S. agriculture, either directly from damage to crops or indirectly from damage to our ability to export crops suspected of contamination. Recognizing a terrorist attack against U.S. agriculture, to be able to prosecute the terrorists, is among the responsibilities of the members of Hazardous Material Response Unit (HMRU) of the Federal Bureau of Investigation (FBI). Nucleic acid analysis of plant pathogen strains by the use of polymerase chain reaction (PCR) amplification techniques is a powerful method for determining the exact identity of pathogens, as well as their possible region of origin. This type of analysis, however, requires that PCR assays be developed specific to each particular pathogen strain, an d analysis protocols developed that are specific to the particular instrument used for detection. The objectives of the work described here were threefold: (1) to assess the potential terrorist threat to U.S. agricultural crops, (2) to determine whether suitable assays exist to monitor that threat, and (3) where assays are needed for priority plant pathogen threats, to modify or develop those assays for use by specialists at the HMRU. The assessment of potential threat to U.S. commodity crops and the availability of assays for those threats were described in detail in the Technical Requirements Document (9) and will be summarized in this report. This report addresses development of specific assays identified in the Technical Requirements Document, and offers recommendations for future development to ensure that HMRU specialists will be prepared with the PCR assays they need to protect against the threat of economic terrorism.

  6. Nucleic Acid-Based Detection and Identification of Bacterial and Fungal Plant Pathogens - Final Report

    SciTech Connect

    Kingsley, Mark T.

    2001-03-13

    The threat to American interests from terrorists is not limited to attacks against humans. Terrorists might seek to inflict damage to the U.S. economy by attacking our agricultural sector. Infection of commodity crops by bacterial or fungal crop pathogens could adversely impact U.S. agriculture, either directly from damage to crops or indirectly from damage to our ability to export crops suspected of contamination. Recognizing a terrorist attack against U.S. agriculture, to be able to prosecute the terrorists, is among the responsibilities of the members of Hazardous Material Response Unit (HMRU) of the Federal Bureau of Investigation (FBI). Nucleic acid analysis of plant pathogen strains by the use of polymerase chain reaction (PCR) amplification techniques is a powerful method for determining the exact identity of pathogens, as well as their possible region of origin. This type of analysis, however, requires that PCR assays be developed specific to each particular pathogen strain, and analysis protocols developed that are specific to the particular instrument used for detection. The objectives of the work described here were threefold: 1) to assess the potential terrorist threat to U.S. agricultural crops, 2) to determine whether suitable assays exist to monitor that threat, and 3) where assays are needed for priority plant pathogen threats, to modify or develop those assays for use by specialists at the HMRU. The assessment of potential threat to U.S. commodity crops and the availability of assays for those threats were described in detail in the Technical Requirements Document (9) and will be summarized in this report. This report addresses development of specific assays identified in the Technical Requirements Document, and offers recommendations for future development to ensure that HMRU specialists will be prepared with the PCR assays they need to protect against the threat of economic terrorism.

  7. Coliform detection in cheese is associated with specific cheese characteristics, but no association was found with pathogen detection.

    PubMed

    Trmčić, A; Chauhan, K; Kent, D J; Ralyea, R D; Martin, N H; Boor, K J; Wiedmann, M

    2016-08-01

    Coliform detection in finished products, including cheese, has traditionally been used to indicate whether a given product has been manufactured under unsanitary conditions. As our understanding of the diversity of coliforms has improved, it is necessary to assess whether coliforms are a good indicator organism and whether coliform detection in cheese is associated with the presence of pathogens. The objective of this study was (1) to evaluate cheese available on the market for presence of coliforms and key pathogens, and (2) to characterize the coliforms present to assess their likely sources and public health relevance. A total of 273 cheese samples were tested for presence of coliforms and for Salmonella, Staphylococcus aureus, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and other Listeria species. Among all tested cheese samples, 27% (75/273) tested positive for coliforms in concentrations >10cfu/g. Pasteurization, pH, water activity, milk type, and rind type were factors significantly associated with detection of coliforms in cheese; for example, a higher coliform prevalence was detected in raw milk cheeses (42% with >10cfu/g) compared with pasteurized milk cheese (21%). For cheese samples contaminated with coliforms, only water activity was significantly associated with coliform concentration. Coliforms isolated from cheese samples were classified into 13 different genera, including the environmental coliform genera Hafnia, Raoultella, and Serratia, which represent the 3 genera most frequently isolated across all cheeses. Escherichia, Hafnia, and Enterobacter were significantly more common among raw milk cheeses. Based on sequencing of the housekeeping gene clpX, most Escherichia isolates were confirmed as members of fecal commensal clades of E. coli. All cheese samples tested negative for Salmonella, Staph. aureus, and Shiga toxin-producing E. coli. Listeria spp. were found in 12 cheese samples, including 5 samples positive for L

  8. Widespread detection of highly pathogenic H5 influenza viruses in wild birds from the Pacific Flyway of the United States

    USDA-ARS?s Scientific Manuscript database

    A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza ...

  9. Comparison of four multiplex PCR assays for the detection of viral pathogens in respiratory specimens.

    PubMed

    Anderson, Trevor P; Werno, Anja M; Barratt, Kevin; Mahagamasekera, Patalee; Murdoch, David R; Jennings, Lance C

    2013-08-01

    Multiplex PCR has become the test of choice for the detection of multiple respiratory viruses in clinical specimens. However, there are few direct comparisons of different PCR assays. This study compares 4 different multiplex PCR assays for the recovery of common respiratory viruses. We tested 213 respiratory specimens using four different multiplex PCR assays: the xTAG respiratory viral panel fast (Abbott Molecular Laboratories), Fast-track Respiratory Pathogen assay (Fast-track Diagnostics), Easyplex respiratory pathogen 12 kit (Ausdiagnostics), and an in-house multiplex real-time PCR assay. The performance of the four assays was very similar, with 93-100% agreement for all comparisons. Other issues, such as through-put, technical requirements and cost, are likely to be as important for making a decision about which of these assays to use given their comparative performance.

  10. Detection of virulence factors and molecular typing of pathogenic Leptospira from capybara (Hydrochaeris hydrochaeris).

    PubMed

    Jorge, Sérgio; Monte, Leonardo G; Coimbra, Marco Antonio; Albano, Ana Paula; Hartwig, Daiane D; Lucas, Caroline; Seixas, Fabiana K; Dellagostin, Odir A; Hartleben, Cláudia P

    2012-10-01

    Leptospirosis is a globally prevalent zoonosis caused by pathogenic Leptospira spp.; several serologic variants have reservoirs in synanthropic rodents. The capybara is the largest living rodent in the world, and it has a wide geographical distribution in Central and South America. This rodent is a significant source of Leptospira since the agent is shed via urine into the environment and is a potential public health threat. In this study, we isolated and identified by molecular techniques a pathogenic Leptospira from capybara in southern Brazil. The isolated strain was characterized by partial rpoB gene sequencing and variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, to confirm the expression of virulence factors, the bacterial immunoglobulin-like proteins A and B expression was detected by indirect immunofluorescence using leptospiral specific monoclonal antibodies. This report identifies capybaras as an important source of infection and provides insight into the epidemiology of leptospirosis.

  11. Detection of pathogenic viruses in sewage provided early warnings of hepatitis A virus and norovirus outbreaks.

    PubMed

    Hellmér, Maria; Paxéus, Nicklas; Magnius, Lars; Enache, Lucica; Arnholm, Birgitta; Johansson, Annette; Bergström, Tomas; Norder, Heléne

    2014-11-01

    Most persons infected with enterically transmitted viruses shed large amounts of virus in feces for days or weeks, both before and after onset of symptoms. Therefore, viruses causing gastroenteritis may be detected in wastewater, even if only a few persons are infected. In this study, the presence of eight pathogenic viruses (norovirus, astrovirus, rotavirus, adenovirus, Aichi virus, parechovirus, hepatitis A virus [HAV], and hepatitis E virus) was investigated in sewage to explore whether their identification could be used as an early warning of outbreaks. Samples of the untreated sewage were collected in proportion to flow at Ryaverket, Gothenburg, Sweden. Daily samples collected during every second week between January and May 2013 were pooled and analyzed for detection of viruses by concentration through adsorption to milk proteins and PCR. The largest amount of noroviruses was detected in sewage 2 to 3 weeks before most patients were diagnosed with this infection in Gothenburg. The other viruses were detected at lower levels. HAV was detected between weeks 5 and 13, and partial sequencing of the structural VP1protein identified three different strains. Two strains were involved in an ongoing outbreak in Scandinavia and were also identified in samples from patients with acute hepatitis A in Gothenburg during spring of 2013. The third strain was unique and was not detected in any patient sample. The method used may thus be a tool to detect incipient outbreaks of these viruses and provide early warning before the causative pathogens have been recognized in health care. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. African penguins (Spheniscus demersus) can detect dimethyl sulphide, a prey-related odour.

    PubMed

    Cunningham, Gregory B; Strauss, Venessa; Ryan, Peter G

    2008-10-01

    Although it is well established that certain procellariiform seabirds use odour cues to find prey, it is not clear whether penguins use olfactory cues to forage. It is commonly assumed that penguins lack a sense of smell, yet they are closely related to procellariiforms and forage on similar types of prey in similar areas to many procellariiforms. Such regions are characterized by having high levels of dimethyl sulphide (DMS) a scented compound that many marine animals use to locate foraging grounds. If penguins can smell, DMS may be a biologically relevant scented compound that they may be sensitive to in nature. To test this hypothesis, we investigated whether adult African penguins (Spheniscus demersus) could detect DMS using two separate experiments. We tested wild penguins on Robben Island, South Africa, by deploying mumolar DMS solutions in the colonies, and found that birds slowed down their walking speeds. We also tested captive penguins in a Y-maze. In both cases, our data convincingly demonstrate that African penguins have a functioning sense of smell and are attracted to DMS. The implication of this work is that the detection of changes in the odour landscape (DMS) may assist penguins in identifying productive areas of the ocean for foraging. At-sea studies are needed to investigate this issue further.

  13. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    SciTech Connect

    Cary,; Bruce, R; Stubben, Christopher J

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  14. Multifunctional Nanotechnology-Enabled Sensors for Rapid Capture and Detection of Pathogens.

    PubMed

    Mustafa, Fatima; Hassan, Rabeay Y A; Andreescu, Silvana

    2017-09-15

    Nanomaterial-based sensing approaches that incorporate different types of nanoparticles (NPs) and nanostructures in conjunction with natural or synthetic receptors as molecular recognition elements provide opportunities for the design of sensitive and selective assays for rapid detection of contaminants. This review summarizes recent advancements over the past ten years in the development of nanotechnology-enabled sensors and systems for capture and detection of pathogens. The most common types of nanostructures and NPs, their modification with receptor molecules and integration to produce viable sensing systems with biorecognition, amplification and signal readout are discussed. Examples of all-in-one systems that combine multifunctional properties for capture, separation, inactivation and detection are also provided. Current trends in the development of low-cost instrumentation for rapid assessment of food contamination are discussed as well as challenges for practical implementation and directions for future research.

  15. High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy.

    PubMed

    Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

    2011-03-10

    Historical records suggest that multiple burial sites from the 14th-16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

  16. High Throughput, Multiplexed Pathogen Detection Authenticates Plague Waves in Medieval Venice, Italy

    PubMed Central

    Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

    2011-01-01

    Background Historical records suggest that multiple burial sites from the 14th–16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. Methodology/Principal Findings High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. Conclusions These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century. PMID:21423736

  17. Multifunctional Nanotechnology-Enabled Sensors for Rapid Capture and Detection of Pathogens

    PubMed Central

    Mustafa, Fatima; Andreescu, Silvana

    2017-01-01

    Nanomaterial-based sensing approaches that incorporate different types of nanoparticles (NPs) and nanostructures in conjunction with natural or synthetic receptors as molecular recognition elements provide opportunities for the design of sensitive and selective assays for rapid detection of contaminants. This review summarizes recent advancements over the past ten years in the development of nanotechnology-enabled sensors and systems for capture and detection of pathogens. The most common types of nanostructures and NPs, their modification with receptor molecules and integration to produce viable sensing systems with biorecognition, amplification and signal readout are discussed. Examples of all-in-one systems that combine multifunctional properties for capture, separation, inactivation and detection are also provided. Current trends in the development of low-cost instrumentation for rapid assessment of food contamination are discussed as well as challenges for practical implementation and directions for future research. PMID:28914769

  18. Salvage microbiology: opportunities and challenges in the detection of bacterial pathogens following initiation of antimicrobial treatment

    PubMed Central

    Farrell, John J.; Hujer, Andrea M.; Sampath, Rangarajan; Bonomo, Robert A.

    2015-01-01

    Broad-range 16S ribosomal RNA gene PCR coupled with Sanger sequencing was originally employed by soil scientists and was subsequently adapted for clinical applications. PCR coupled with electrospray ionization mass spectrometry has also progressed from initial applications in the detection of organisms from environmental samples into the clinical realm and has demonstrated promise in detection of pathogens in clinical specimens obtained from patients with suspected infection but negative cultures. We review studies of multiplex PCR, 16S ribosomal RNA gene PCR and sequencing and PCR coupled with electrospray ionization mass spectrometry for detection of bacteria in specimens that were obtained from patients during or after administration of antibiotic treatment, and examine the role of each for assisting in antimicrobial treatment and stewardship efforts. Following an exploration of the available data in this field we discuss the opportunities that the preliminary investigations reveal, as well as the challenges faced with implementation of these strategies in clinical practice. PMID:25523281

  19. Detection of Quiescent Infections with Multiple Elephant Endotheliotropic Herpesviruses (EEHVs), Including EEHV2, EEHV3, EEHV6, and EEHV7, within Lymphoid Lung Nodules or Lung and Spleen Tissue Samples from Five Asymptomatic Adult African Elephants

    PubMed Central

    Zong, Jian-Chao; Heaggans, Sarah Y.; Long, Simon Y.; Latimer, Erin M.; Nofs, Sally A.; Bronson, Ellen; Casares, Miguel; Fouraker, Michael D.; Pearson, Virginia R.; Richman, Laura K.

    2015-01-01

    ABSTRACT More than 80 cases of lethal hemorrhagic disease associated with elephant endotheliotropic herpesviruses (EEHVs) have been identified in young Asian elephants worldwide. Diagnostic PCR tests detected six types of EEHV in blood of elephants with acute disease, although EEHV1A is the predominant pathogenic type. Previously, the presence of herpesvirus virions within benign lung and skin nodules from healthy African elephants led to suggestions that African elephants may be the source of EEHV disease in Asian elephants. Here, we used direct PCR-based DNA sequencing to detect EEHV genomes in necropsy tissue from five healthy adult African elephants. Two large lung nodules collected from culled wild South African elephants contained high levels of either EEHV3 alone or both EEHV2 and EEHV3. Similarly, a euthanized U.S. elephant proved to harbor multiple EEHV types distributed nonuniformly across four small lung nodules, including high levels of EEHV6, lower levels of EEHV3 and EEHV2, and a new GC-rich branch type, EEHV7. Several of the same EEHV types were also detected in random lung and spleen samples from two other elephants. Sanger PCR DNA sequence data comprising 100 kb were obtained from a total of 15 different strains identified, with (except for a few hypervariable genes) the EEHV2, EEHV3, and EEHV6 strains all being closely related to known genotypes from cases of acute disease, whereas the seven loci (4.0 kb) obtained from EEHV7 averaged 18% divergence from their nearest relative, EEHV3. Overall, we conclude that these four EEHV species, but probably not EEHV1, occur commonly as quiescent infections in African elephants. IMPORTANCE Acute hemorrhagic disease characterized by high-level viremia due to infection by members of the Proboscivirus genus threatens the future breeding success of endangered Asian elephants worldwide. Although the genomes of six EEHV types from acute cases have been partially or fully characterized, lethal disease predominantly

  20. Detection of African swine fever virus DNA in blood samples stored on FTA cards from asymptomatic pigs in Mbeya region, Tanzania.

    PubMed

    Braae, U C; Johansen, M V; Ngowi, H A; Rasmussen, T B; Nielsen, J; Uttenthal, Å

    2015-02-01

    The aim of the study was to assess whether blood samples collected onto FTA(®) cards could be used in combination with real-time PCR for the detection of African swine fever virus (ASFV) DNA in samples from resource-poor settings under the assumption that asymptomatically (sub-clinically) infected pigs may be present. Blood samples were collected from clinically healthy pigs from Mbeya Region, Tanzania. The blood samples were stored on FTA(®) cards and analysed by real-time PCR assays in duplicate; three pigs had high levels of viral DNA (Ct values of 27-29), and three pigs had a low level of viral DNA (Ct 36-45). Four pigs were positive in one of the duplicate samples only, but clear products of the expected size were obtained when the reactions were analysed by gel electrophoresis. For comparison, blood samples from pigs experimentally infected with either a pathogenic (OURT T88/1) or a non-pathogenic (OURT T88/3) isolate of ASFV were collected, stored on FTA(®) cards and analysed in the same way. The blood from pigs infected with the OURT T88/1 isolate showed high levels of viral DNA (Ct 22-33), whereas infection with non-pathogenic OURT T88/3 isolate resulted in only low levels of viral DNA (Ct 39) in samples collected at 10-14 days after inoculation.

  1. [Real-time PCR kits for the detection of the African Swine Fever virus].

    PubMed

    Latyshev, O E; Eliseeva, O V; Grebennikova, T V; Verkhovskiĭ, O A; Tsibezov, V V; Chernykh, O Iu; Dzhailidi, G A; Aliper, T I

    2014-01-01

    The results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the DNA of the African Swine Fever in the pathological material, as well as in the culture fluid, are presented. A high sensitivity and specificity for detection of the DNA in the organs and tissues of animals was shown to be useful for detection in the European Union referentiality reagent kits for DNA detection by real time PCR of ASFV. More rapid and effective method of DNA extraction using columns mini spin Quick gDNA(TM) MiniPrep was suggested and compared to the method of DNA isolation on the inorganic sorbent. High correlation of the results of the DNA detection of ASFV by real-time PCR and antigen detection results ASFV by competitive ELISA obtained with the ELISA SEROTEST/INGEZIM COMRAC PPA was demonstrated. The kit can be used in the veterinary services for effective monitoring of ASFV to contain, eliminate and prevent further spread of the disease.

  2. Integrated electrochemical microsystems for genetic detection of pathogens at the point of care.

    PubMed

    Hsieh, Kuangwen; Ferguson, B Scott; Eisenstein, Michael; Plaxco, Kevin W; Soh, H Tom

    2015-04-21

    The capacity to achieve rapid, sensitive, specific, quantitative, and multiplexed genetic detection of pathogens via a robust, portable, point-of-care platform could transform many diagnostic applications. And while contemporary technologies have yet to effectively achieve this goal, the advent of microfluidics provides a potentially viable approach to this end by enabling the integration of sophisticated multistep biochemical assays (e.g., sample preparation, genetic amplification, and quantitative detection) in a monolithic, portable device from relatively small biological samples. Integrated electrochemical sensors offer a particularly promising solution to genetic detection because they do not require optical instrumentation and are readily compatible with both integrated circuit and microfluidic technologies. Nevertheless, the development of generalizable microfluidic electrochemical platforms that integrate sample preparation and amplification as well as quantitative and multiplexed detection remains a challenging and unsolved technical problem. Recognizing this unmet need, we have developed a series of microfluidic electrochemical DNA sensors that have progressively evolved to encompass each of these critical functionalities. For DNA detection, our platforms employ label-free, single-step, and sequence-specific electrochemical DNA (E-DNA) sensors, in which an electrode-bound, redox-reporter-modified DNA "probe" generates a current change after undergoing a hybridization-induced conformational change. After successfully integrating E-DNA sensors into a microfluidic chip format, we subsequently incorporated on-chip genetic amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to enable genetic detection at clinically relevant target concentrations. To maximize the potential point-of-care utility of our platforms, we have further integrated sample preparation via immunomagnetic separation, which

  3. Assessment of an extraction protocol to detect the major mastitis-causing pathogens in bovine milk.

    PubMed

    Cressier, B; Bissonnette, N

    2011-05-01

    Despite all efforts to control its spread, mastitis remains the most costly disease for dairy farmers worldwide. One key component of better control of this disease is identification of the causative bacterial agent during udder infections in cows. Mastitis is complex, however, given the diversity of pathogens that must be identified. Development of a rapid and efficient bacterial species identification tool is thus necessary. This study was conducted to demonstrate the feasibility of bacterial DNA extraction for the automated molecular detection of major mastitis-causing pathogens directly in milk samples to complement traditional microbiological identification. Extraction and detection procedures were designed and optimized to achieve detection in a respectable time frame, at a reasonable cost, and with a high throughput capacity. The following species were identified: Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Klebsiella spp. (including Klebsiella oxytoca and Klebsiella pneumoniae). The detection procedure includes specific genomic DNA amplification by multiplex PCR for each species, separation by capillary electrophoresis, and laser-assisted automated detection. The specificity of the primers was assessed with a panel of bacteria representing mastitis-negative control species. The extraction protocol comprised multiple steps, starting with centrifugation for fat removal, followed by heating in the presence of a cation exchange resin to trap divalent ions. The analytical sensitivity was 100 cfu/mL for milk samples spiked with Staph. aureus, Strep. dysgalactiae, and E. coli, with a tendency for K. pneumoniae. The detection limit was 500 cfu/mL for Strep. uberis and Strep. agalactiae. The overall diagnostic sensitivity (95.4%) and specificity (97.3%) were determined in a double-blind randomized assay by processing 172 clinical milk samples with microbiological characterization as the

  4. Light-emission-based biosensor for detection of food pathogens: a review

    NASA Astrophysics Data System (ADS)

    Singh, Ashutosh; Singh, Rakesh K.; Bhunia, Arun K.

    2001-03-01

    Rapid detection, identification and enumeration of pathogenic microorganisms is highly important to the food industry to address the food safety concerns. Biosensors are devices that promise to achieve these objectives, these analytical devices can accurately and selectively estimate the levels of foodborne pathogens. The most widely applied biosensors are based on the optical properties like absorption, fluorescence, reflection, refraction, dispersion etc. This review is done to provide an overview of optic based biosensor and their application in the area of food safety. Working principles of Surface plasmon resonance , resonant mirror and fiber optic based biosensor are described in the article. Resonant mirror based biosensor has been used for the estimation of S. aureus, it was proven selective for the strain being tested (Cowan-1). The sensitivity of the assay was increased by using colloidal-gold conjugates in sandwich assay format. An integrated fiber optic based biosensor is used for detection of Salmonella typhimurium reducing the detection time to 30 min. Fiber optic based biosensors offer advantage of compactness, flexibility , resistance to electrical noise and small probe size.

  5. Leptospira wolffii, a potential new pathogenic Leptospira species detected in human, sheep and dog.

    PubMed

    Zakeri, Sedigheh; Khorami, Nargess; Ganji, Zahra F; Sepahian, Neda; Malmasi, Abdol-Ali; Gouya, Mohammad Mehdi; Djadid, Navid D

    2010-03-01

    Leptospirosis is the most common zoonotic disease, which is transmitted to humans through contaminated water or direct exposure to the urine of infected animals. In this study, the presence and prevalence of Leptospira species in the infected samples of human (n=369) and sheep (n=75) sera and also dogs' urine (n=150), collected from four provinces of Iran, were investigated by using nested-PCR/RFLP assay followed by sequencing analysis. Nested-PCR assay detected that 98/369 (26.5%) human, 13/75 (17.33%) of sheep's sera and 33/150 (22%) dogs' urine samples were positive for Leptospira DNA. RFLP assay detected that all positive cases had either pathogenic or intermediate Leptospira species. By sequence analysis, Leptospira interrogans was the most prevalent species among the examined samples of human (53/82, 64.6%) and sheep (11/13, 84.6%). However, in dog samples, Leptospira wolffii (27/29, 93.1%) was detected for the first time and was the dominant species. The presence of L. wolffii with 100% identity in clinical human samples and animals suspected with Leptospira may provide evidence for circulation of L. wolffii and its role in transmission cycle within human and animal hosts. In addition, this species can be potentially pathogenic to human and probably animal hosts. A large epidemiology survey would be needed to define the presence and the prevalence of this species in global endemic regions.

  6. Magnetostrictive particle based biosensors for in situ and real-time detection of pathogens in water.

    PubMed

    Zhang, Kewei; Fu, Liling; Zhang, Lin; Cheng, Z-Y; Huang, Tung-Shi

    2014-11-01

    Biosensors for in situ detection of pathogenic bacteria in liquid are developed using magnetostrictive particles (MSP) as the sensor platform. The sensing elements used are phage E2 against Salmonella typhimurium, monoclonal antibody against Listeria monocytogenes, polyclonal antibody against Escherichia coli, and polyclonal antibody against Staphylococcus aureus, respectively. These biosensors were characterized in cultures with different populations ranging from 5 × 10(1) to 5 × 10(8)  cfu/mL. It is found that the MSP-based biosensors work well in water and have a rapid response with a response time in minutes, which makes the MSP-based sensors suitable for in situ and real-time detection of pathogenic bacteria in liquid. The experimental results show that all MSP-phage and MSP-antibody biosensors in size of 1.0 mm × 0.3 mm × 15 µm exhibit a detection limit better than 100 cfu/mL. Based on the Hill plot, it is concluded that each bacterial cell is bound onto the sensor surface through about four-to-five sites. When the cultures with low population (<10(6)  cfu/mL) are tested, both MSP-phage and MSP-antibody sensors exhibit the similar response. However, the phage-MSP sensors exhibit a higher capability in the capture of target bacterial cell. © 2014 Wiley Periodicals, Inc.

  7. A Pathogenic Mosaic TP53 Mutation in Two Germ Layers Detected by Next Generation Sequencing

    PubMed Central

    Williams, Richard D.; Side, Lucy; Hubank, Mike; West, Rebecca; Pearson, Katie; Sebire, Neil; Tarpey, Patrick; Futreal, Andrew; Brooks, Tony; Stratton, Michael R.; Anderson, John

    2014-01-01

    Background Li-Fraumeni syndrome is caused by germline TP53 mutations and is clinically characterized by a predisposition to a range of cancers, most commonly sarcoma, brain tumours and leukemia. Pathogenic mosaic TP53 mutations have only rarely been described. Methods and Findings We describe a 2 years old child presenting with three separate cancers over a 6 month period; two soft tissue mesenchymal tumors and an aggressive metastatic neuroblastoma. As conventional testing of blood DNA by Sanger sequencing for mutations in TP53, ALK, and SDH was negative, whole exome sequencing of the blood DNA of the patient and both parents was performed to screen more widely for cancer predisposing mutations. In the patient's but not the parents' DNA we found a c.743 G>A, p.Arg248Gln (CCDS11118.1) TP53 mutation in 3–20% of sequencing reads, a level that would not generally be detectable by Sanger sequencing. Homozygosity for this mutation was detected in all tumor samples analyzed, and germline mosaicism was demonstrated by analysis of the child's newborn blood spot DNA. The occurrence of separate tumors derived from different germ layers suggests that this de novo mutation occurred early in embryogenesis, prior to gastrulation. Conclusion The case demonstrates pathogenic mosaicim, detected by next generation deep sequencing, that arose in the early stages of embryogenesis. PMID:24810334

  8. The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

    PubMed

    Wang, Lih-Chiann; Kuo, Ya-Ting; Chueh, Ling-Ling; Huang, Dean; Lin, Jiunn-Horng

    2017-05-01

    Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control.

  9. Laser diagnostic technology for early detection of pathogen infestation in orange fruits

    NASA Astrophysics Data System (ADS)

    Giubileo, Gianfranco; Lai, Antonella; Piccinelli, Delinda; Puiu, Adriana

    2010-11-01

    Due to an increased expectation of food products that respect high quality and safety standards, there is a need for the growth of accurate, fast, objective and non-destructive technologies for quality determination of food and agricultural products. For this purpose, a diagnostic system based on laser photoacoustic spectroscopy (LPAS) was developed at ENEA Frascati Molecular Spectroscopy Laboratory (Italy). In the design of the photoacoustic detector, particular emphasis was placed in attaining a high sensitivity in detecting ethylene (ET) down to sub-parts per billion level (minimum detectable concentration 0.2 ppb). This was required due to the necessity to monitor and follow up ET production at a single fruit scale. ET is normally synthesised in very low amounts by healthy citrus fruits; however stress conditions such as pathogen attack may induce a substantial increase in the synthesised ET. In the present paper, the comparison between the ET emitted by healthy oranges ( Citrus sinensis L. Osbeck) cv Navel and by Phytophthora citrophthora infested Navel orange fruits are reported. The obtained results show a well evident increase in ET emission from the infested fruit with respect to the healthy one, even 24 h after the inoculation with the pathogen; at that time the tissue necrosis was not yet visible, and the fruit was also not yet damaged. The possibility to perform a real time non-destructive detection of ET traces makes the LPAS a powerful tool for monitoring the healthy state of the citrus fruits.

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

    SciTech Connect

    Rahman, Salma

    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. Comparative evaluation of two commercial chromogenic media for detection and presumptive identification of urinary tract pathogens.

    PubMed

    Scarparo, C; Piccoli, P; Ricordi, P; Scagnelli, M

    2002-04-01

    The performance of two commercial chromogenic media for the isolation and presumptive identification of urinary tract pathogens, the CPS ID2 (bioMérieux, France) and the CHROMagar Orientation (BBL Becton Dickinson, USA), was evaluated and compared with that of cystine-lactose-electrolyte-deficient agar and tryptic soy agar with 5% sheep blood. The detection, determination of bacterial counts, and presumptive identification of bacteria causing urinary tract infections were evaluated in 3,000 urine specimens. The two chromogenic media showed excellent correlation with the standard media for the detection and the bacterial count of urinary pathogens. The Escherichia coli strains produced the expected colour on the CHROMagar Orientation and the CPS ID2 media in 99% and 90% of the cases, respectively. The Klebsiella-Enterobacter-Citrobacter and the Proteus-Morganella-Providencia groups were easily identified on both chromogenic media, but further biochemical tests were needed to differentiate them to a species level. Both media enabled the differentiation, with varying degrees of difficulty, of Pseudomonas spp. strains from members of the family Enterobacteriaceae. All isolates of Enterococcus spp. were correctly identified and were easily distinguished from the Streptococcus agalactiae isolates. Staphylococcus saprophyticus isolates were easy to identify only on the CHROMagar Orientation medium. No substantial difference was observed when comparing the results of the susceptibility tests, which were performed according to the standardized disk diffusion method as described by the National Committee for Clinical Laboratory Standards, for colonies recovered from the blood agar versus those recovered from the chromogenic media. In conclusion, the CPS ID2 and CHROMagar Orientation media enabled excellent detection, count determination, and presumptive identification of urinary pathogens, both in pure and mixed cultures, and reliable and accurate antimicrobial

  12. Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks.

    PubMed

    Latrofa, Maria Stefania; Dantas-Torres, Filipe; Giannelli, Alessio; Otranto, Domenico

    2014-10-01

    The Rhipicephalus sanguineus group includes some of the most widespread dog ticks, whose identification currently represents a difficult task due to the morphological similarities of these ixodid species. Recently, following the morphological and molecular characterization of tick specimens collected from dogs in all continents, Rhipicephalus sanguineus sensu lato (s.l.), Rhipicephalus turanicus and three different operational taxonomic units (namely Rhipicephalus sp. I-III) have been identified. Therefore, a comprehensive molecular study has been herein carried out to detect selected canine vector-borne pathogens (i.e., Anaplasma platys, Cercopithifilaria spp., Ehrlichia canis and Hepatozoon canis) in ticks belonging to the R. sanguineus group. A total of 204 tick specimens from dogs were examined and identified as R. sanguineus s.l. (n=81), R. turanicus (n=17), Rhipicephalus sp. I (n=66), Rhipicephalus sp. II (n=37), and Rhipicephalus sp. III (n=3). PCR assays were performed to detect mitochondrial and ribosomal target genes of Cercopithifilaria spp., A. platys, E. canis and H. canis. Out of 204 specimens examined, 2.5%, 7.4% and 21.6% scored positive to A. platys, H. canis and Cercopithifilaria spp., respectively. In addition, co-infections with two pathogens (i.e., Cercopithifilaria bainae and A. platys or H. canis) were detected in four tick specimens. This study suggests a relationship between ticks belonging to the R. sanguineus group and the geographical distribution of A. platys, H. canis and Cercopithifilaria spp. However, the role of each representative tick species within the R. sanguineus group in the transmission of these canine pathogens needs to be further investigated.

  13. PCR detection of African horse sickness virus serogroup based on genome segment three sequence analysis.

    PubMed

    Aradaib, Imadeldin E

    2009-07-01

    A nested reverse transcriptase (RT) polymerase chain reaction (RT-PCR), for rapid detection of African horse sickness virus (AHSV) double-stranded ribonucleic acid (dsRNA) in cell culture and tissue samples, was developed and evaluated. Using an outer pair of primers (P1 and P2), selected from genome segment three of AHSV serotype 6 (AHSV-6), the RT-PCR-based assay resulted in amplification of a 890 base pair (bp) primary PCR product. RNAs from the nine vaccine strains of AHSV, and a number of AHSV field isolates including the Central African isolates of AHSV-9 and AHSV-6, propagated in cell cultures, were detected by this assay. A second pair of nested primers (P3 and P4) was used to produce a 240-bp PCR product. The RT-PCR described below detected as little as 0.1 fg of AHSV RNA, which is equivalent to six viral particles. The nested amplification confirmed the integrity of the primary PCR product and increased the sensitivity of the PCR assay by at least 1000-fold. Application of this RT-PCR assay to clinical samples resulted in direct detection of AHSV dsRNA from blood and a variety of tissue samples collected from equines infected experimentally and naturally. The specificity studies indicated that the primary or the nested PCR products were not amplified from, closely related orbiviruses including, bluetongue virus (BTV) prototypes serotypes 1, 2, 4, 10, 16 and 17; epizootic hemorrhagic disease of deer virus (EHDV) prototypes serotypes 1 and 2; EHDV-318, Sudanese isolates of palyam serogroup of orbiviruses; total nucleic acid extracts from uninfected Vero cells; or unfractionated blood from horses and donkeys that were AHSV-seronegative and virus isolation negative. The RT-PCR provides a valuable tool for study of the epidemiology of AHSV and can be recommended for rapid diagnosis during an outbreak of the disease among susceptible equines.

  14. Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting Fluorescent Probe, CDy11.

    PubMed

    Kim, Jun-Young; Sahu, Srikanta; Yau, Yin-Hoe; Wang, Xu; Shochat, Susana Geifman; Nielsen, Per Halkjær; Dueholm, Morten Simonsen; Otzen, Daniel E; Lee, Jungyeol; Delos Santos, May Margarette Salido; Yam, Joey Kuok Hoong; Kang, Nam-Young; Park, Sung-Jin; Kwon, Hawyoung; Seviour, Thomas; Yang, Liang; Givskov, Michael; Chang, Young-Tae

    2016-01-13

    Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-associated infections relies heavily on culturing methods, which fail to detect nonculturable bacteria. Identification of novel fluorescent probes for biofilm imaging will greatly facilitate diagnosis of pathogenic bacterial infection. Herein, we report a novel fluorescent probe, CDy11 (compound of designation yellow 11), which targets amyloid in the Pseudomonas aeruginosa biofilm matrix through a diversity oriented fluorescent library approach (DOFLA). CDy11 was further demonstrated for in vivo imaging of P. aeruginosa in implant and corneal infection mice models.

  15. Detection of pathogenic micro-organisms on children's hands and toys during play.

    PubMed

    Martínez-Bastidas, T; Castro-del Campo, N; Mena, K D; Castro-del Campo, N; León-Félix, J; Gerba, C P; Chaidez, C

    2014-06-01

    This study aimed to determine if the children's leisure activities impact the presence of pathogens on their hands and toys. To assess the microbiological hazard in playground areas, a pilot study that included 12 children was conducted. We then conducted an intervention study; children's hands and toys were washed before playing. Faecal coliforms, pathogenic bacteria and Giardia lamblia were quantified by membrane filtration, selective media and flotation techniques, respectively; rotavirus, hepatitis A and rhinovirus by RT-PCR. Pilot study results revealed faecal contamination on children's hands and toys after playing on sidewalks and in public parks. Pathogenic bacteria, hepatitis A and G. lamblia on children's hands were also found. In the intervention study, Staphylococcus aureus and Klebsiella pneumoniae were found on children's hands at concentrations up to 2·5 × 10(4) and 1 × 10(4) CFU hands(-1), respectively. E. coli and Kl. pneumoniae were detected on toys (2·4 × 10(3) and 2·7 × 10(4) CFU toy(-1), respectively). Salmonella spp, Serratia spp and G. lamblia cysts were also present on toys. Children's play activities influence microbial presence on hands and toys; the transfer seems to occur in both ways. Control strategy needs to be implemented to protect children from infectious diseases. © 2014 The Society for Applied Microbiology.

  16. Pathogens of Food Animals: Sources, Characteristics, Human Risk, and Methods of Detection.

    PubMed

    Logue, Catherine M; Barbieri, Nicolle L; Nielsen, Daniel W

    Pathogens associated with food production (livestock) animals come in many forms causing a multitude of disease for humans. For the purpose of this review, these infectious agents can be divided into three broad categories: those that are associated with bacterial disease, those that are associated with viruses, and those that are parasitic in nature. The goal of this chapter is to provide the reader with an overview of the most common pathogens that cause disease in humans through exposure via the food chain and the consequence of this exposure as well as risk and detection methods. We have also included a collection of unusual pathogens that although rare have still caused disease, and their recognition is warranted in light of emerging and reemerging diseases. These provide the reader an understanding of where the next big outbreak could occur. The influence of the global economy, the movement of people, and food makes understanding production animal-associated disease paramount to being able to address new diseases as they arise. © 2017 Elsevier Inc. All rights reserved.

  17. A microfluidic-based hybrid SPR/molecular imaging biosensor for the multiplexed detection of foodborne pathogens

    NASA Astrophysics Data System (ADS)

    Zordan, Michael D.; Grafton, Meggie M. G.; Acharya, Ghanashyam; Reece, Lisa M.; Aronson, Arthur I.; Park, Kinam; Leary, James F.

    2009-02-01

    It is important to screen our food supply for pathogen contaminations. Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time-costly growth in cultures. There is need for portable, real-time, multiplex pathogen detection technology that can predict the safety of food where it is produced or distributed. Surface plasmon resonance (SPR) imaging is a sensitive, label-free method that can detect the binding of an analyte to a surface due to changes in refractive index that occur upon binding. It can be used for label-free detection of the presence of potential pathogens. Simultaneous fluorescence molecular imaging on the other side of the biochip can be used to ascertain pathogen status or functional state which may affect its potential danger to humans or animals. We are designing and testing hybrid microfluidic biochips to detect multiple pathogens using a combination of SPRI and fluorescence imaging. The device consists of an array of gold spots, each functionalized with a peptide targeting a specific pathogen. This peptide biosensor array is enclosed by a PDMS microfluidic flow chamber that delivers a magnetically concentrated sample to be tested. An SPR image is taken from the bottom of the biochip. Image analysis is used to quantify the amount of pathogen (both live and dead) bound to each spot. Since PDMS is very transmissive to visible light, an epi-fluorescence image is taken from the top of the biochip. Fluorescence imaging determines the live:dead ratio of each pathogen using an inexpensive SYTO 9(R)-Propidium Iodide assay. The volume of sample that the biochip can analyze is small, so possible pathogens are pre-concentrated using immunomagnetic separation. Functionalized magnetic particles are bound to pathogens present in the sample, and a magnet is used to separate them from the bulk fluid.

  18. Quantitative Molecular Detection of 19 Major Pathogens in the Interdental Biofilm of Periodontally Healthy Young Adults

    PubMed Central

    Carrouel, Florence; Viennot, Stéphane; Santamaria, Julie; Veber, Philippe; Bourgeois, Denis

    2016-01-01

    pathogen in adult periodontal disease, represents 8.08% of the 19 bacteria analyzed. P. gingivalis was detected in 19% of healthy subjects and represents 0.02% of the interdental biofilm. T. forsythensis and T. denticola (0.02 and 0.04% of the interdental biofilm) were detected in 93 and 49% of healthy subjects, respectively. The effective presence of periodontal pathogens is a strong indicator of the need to develop new methods for disrupting interdental biofilm in daily oral hygiene. PMID:27313576

  19. Surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety.

    PubMed

    Bergwerff, Aldert A; van Knapen, Frans

    2006-01-01

    This review describes the exploitation of exclusively optical surface plasmon resonance (SPR) biosensors for the direct and indirect detection of pathogenic microorganisms in food chains and the environment. Direct detection is, in most cases, facilitated by the use of defined monoclonal or polyclonal antibodies raised against (a part of) the target pathogenic microorganisms. The antibodies were immobilized to a solid phase of the sensor to capture the microbe from the sample. Alternatively, antibodies were used in an inhibition-like assay involving incubation with the target organism prior to analysis of nonbound antibodies. The free immunoglobins were screened on a sensor surface coated with either purified antigens or with Fc or Fab binding antibodies. Discussed examples of these approaches are the determination of Escherichia coli O1 57:H7, Salmonella spp., and Listeria monocytogenes. Another direct detection strategy involved SPR analysis of polymerase chain reaction products of Shiga toxin-2 genes reporting the presence of E. coli O157:H7 in human stool. Metabolic products have been exploited as biomarkers for the presence of a microbial agent, such as enterotoxin B and a virulence factor for the occurrence of Staphylococcus aureus and Streptococcus suis, respectively. Indirect detection, on the other hand, is performed by analysis of a humoral immune response of the infected animal or human. By immobilization of specific antigenic structures, infections with Herpes simplex and human immunodeficiency viruses, Salmonella and Treponema pallidum bacteria, and Schistosoma spp. parasites were revealed using human, avian, and porcine sera and avian eggs. Bound antibodies were easily isotyped using an SPR biosensor to reveal the infection history of the individual. Discussed studies show the recent recognition of the suitability of this type of instrument for (rapid) detection of health-threatening microbes to food and environmental microbial safety.

  20. Multiplex 16S rRNA‐derived geno‐biochip for detection of 16 bacterial pathogens from contaminated foods

    PubMed Central

    Shin, Hwa Hui; Hwang, Byeong Hee

    2016-01-01

    Abstract Foodborne diseases caused by various pathogenic bacteria occur worldwide. To prevent foodborne diseases and minimize their impacts, it is important to inspect contaminated foods and specifically detect many types of pathogenic bacteria. Several DNA oligonucleotide biochips based on 16S rRNA have been investigated to detect bacteria; however, a mode of detection that can be used to detect diverse pathogenic strains and to examine the safety of food matrixes is still needed. In the present work, a 16S rRNA gene‐derived geno‐biochip detection system was developed after screening DNA oligonucleotide specific capture probes, and it was validated for multiple detection of 16 pathogenic strains that frequently occur with a signature pattern. rRNAs were also used as detection targets directly obtained from cell lysates without any purification and amplification steps in the bacterial cells separated from 8 food matrixes by simple pretreatments. Thus, the developed 16S rRNA‐derived geno‐biochip can be successfully used for the rapid and multiple detection of the 16 pathogenic bacteria frequently isolated from contaminated foods that are important for food safety. PMID:27492058

  1. Detecting signals of chronic shedding to explain pathogen persistence: Leptospira interrogans in California sea lions.

    PubMed

    Buhnerkempe, Michael G; Prager, Katherine C; Strelioff, Christopher C; Greig, Denise J; Laake, Jeff L; Melin, Sharon R; DeLong, Robert L; Gulland, Frances M D; Lloyd-Smith, James O

    2017-05-01

    Identifying mechanisms driving pathogen persistence is a vital component of wildlife disease ecology and control. Asymptomatic, chronically infected individuals are an oft-cited potential reservoir of infection, but demonstrations of the importance of chronic shedding to pathogen persistence at the population-level remain scarce. Studying chronic shedding using commonly collected disease data is hampered by numerous challenges, including short-term surveillance that focuses on single epidemics and acutely ill individuals, the subtle dynamical influence of chronic shedding relative to more obvious epidemic drivers, and poor ability to differentiate between the effects of population prevalence of chronic shedding vs. intensity and duration of chronic shedding in individuals. We use chronic shedding of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus) as a case study to illustrate how these challenges can be addressed. Using leptospirosis-induced strands as a measure of disease incidence, we fit models with and without chronic shedding, and with different seasonal drivers, to determine the time-scale over which chronic shedding is detectable and the interactions between chronic shedding and seasonal drivers needed to explain persistence and outbreak patterns. Chronic shedding can enable persistence of L. interrogans within the sea lion population. However, the importance of chronic shedding was only apparent when surveillance data included at least two outbreaks and the intervening inter-epidemic trough during which fadeout of transmission was most likely. Seasonal transmission, as opposed to seasonal recruitment of susceptibles, was the dominant driver of seasonality in this system, and both seasonal factors had limited impact on long-term pathogen persistence. We show that the temporal extent of surveillance data can have a dramatic impact on inferences about population processes, where the failure to identify both short- and

  2. Hyperspectral imaging using a color camera and its application for pathogen detection

    NASA Astrophysics Data System (ADS)

    Yoon, Seung-Chul; Shin, Tae-Sung; Heitschmidt, Gerald W.; Lawrence, Kurt C.; Park, Bosoon; Gamble, Gary

    2015-02-01

    This paper reports the results of a feasibility study for the development of a hyperspectral image recovery (reconstruction) technique using a RGB color camera and regression analysis in order to detect and classify colonies of foodborne pathogens. The target bacterial pathogens were the six representative non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) grown in Petri dishes of Rainbow agar. The purpose of the feasibility study was to evaluate whether a DSLR camera (Nikon D700) could be used to predict hyperspectral images in the wavelength range from 400 to 1,000 nm and even to predict the types of pathogens using a hyperspectral STEC classification algorithm that was previously developed. Unlike many other studies using color charts with known and noise-free spectra for training reconstruction models, this work used hyperspectral and color images, separately measured by a hyperspectral imaging spectrometer and the DSLR color camera. The color images were calibrated (i.e. normalized) to relative reflectance, subsampled and spatially registered to match with counterpart pixels in hyperspectral images that were also calibrated to relative reflectance. Polynomial multivariate least-squares regression (PMLR) was previously developed with simulated color images. In this study, partial least squares regression (PLSR) was also evaluated as a spectral recovery technique to minimize multicollinearity and overfitting. The two spectral recovery models (PMLR and PLSR) and their parameters were evaluated by cross-validation. The QR decomposition was used to find a numerically more stable solution of the regression equation. The preliminary results showed that PLSR was more effective especially with higher order polynomial regressions than PMLR. The best classification accuracy measured with an independent test set was about 90%. The results suggest the potential of cost-effective color imaging using hyperspectral image

  3. Early detection of communication delays with the PEDS tools in at-risk South African infants

    PubMed Central

    Swanepoel, De Wet; Hanekom, Linique; Lemmer, Tasha; Schoeman, Karla; Glascoe, Frances Page; Vinck, Bart

    2016-01-01

    Background Prevalence of communication delays or disorders is increasing, possibly because of various environmental risk factors. Selection and implementation of effective screening tools are important to detect at-risk infants as early as possible. This study aimed to evaluate the accuracy of the Parents’ Evaluation of Developmental Status (PEDS), PEDS-Developmental Milestones and PEDS tools to detect communication delays in infants (6–12 months) in a South African primary healthcare context. Method A comparative study design evaluated the accuracy of the PEDS tools to detect communication delays, using an internationally accepted diagnostic assessment tool, the Rossetti Infant-Toddler Language Scale (RITLS). A convenience sample of 201 infants was selected at primary healthcare clinics. Results Expressive and receptive language sensitivity scores were low across all three screens (ranging between 14% and 44%). The PEDS tools had high sensitivity (71%) and specificity (73%) ratings for the receptive and expressive language and socio-emotional domain in combination. Conclusion In the sample population, the PEDS tools did not accurately detect receptive and expressive language delays; however, communication delays in general were identified. Future research determining accuracy of the PEDS, PEDS-Developmental Milestones and PEDS tools for children aged 2–5 years in detecting communication delays should be prioritised. PMID:28730050

  4. Detection of bacterial aggregation in cell suspensions treated with pathogenic bacteria

    USDA-ARS?s Scientific Manuscript database

    The early interaction between plant cells and pathogenic bacteria were studied using tobacco cell suspensions treated with pathogenic and nonpathogenic Pseudomonas species. Previous studies of this system have documented that interactions with pathogens that cause a hypersensitive response on whole...

  5. Development of an updated PCR assay for detection of African swine fever virus.

    PubMed

    Luo, Yuzi; Atim, Stella A; Shao, Lina; Ayebazibwe, Chrisostom; Sun, Yuan; Liu, Yan; Ji, Shengwei; Meng, Xing-Yu; Li, Su; Li, Yongfeng; Masembe, Charles; Ståhl, Karl; Widén, Frederik; Liu, Lihong; Qiu, Hua-Ji

    2017-01-01

    Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

  6. Pathogenic Chytrid Fungus Batrachochytrium dendrobatidis, but Not B. salamandrivorans, Detected on Eastern Hellbenders

    PubMed Central

    Bales, Emma K.; Hyman, Oliver J.; Loudon, Andrew H.; Harris, Reid N.; Lipps, Gregory; Chapman, Eric; Roblee, Kenneth; Kleopfer, John D.; Terrell, Kimberly A.

    2015-01-01

    Recent worldwide declines and extinctions of amphibian populations have been attributed to chytridiomycosis, a disease caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Until recently, Bd was thought to be the only Batrachochytrium species that infects amphibians; however a newly described species, Batrachochytrium salamandrivorans (Bs), is linked to die-offs in European fire salamanders (Salamandra salamandra). Little is known about the distribution, host range, or origin of Bs. In this study, we surveyed populations of an aquatic salamander that is declining in the United States, the eastern hellbender (Cryptobranchus alleganiensis alleganiensis), for the presence of Bs and Bd. Skin swabs were collected from a total of 91 individuals in New York, Pennsylvania, Ohio, and Virginia, and tested for both pathogens using duplex qPCR. Bs was not detected in any samples, suggesting it was not present in these hellbender populations (0% prevalence, 95% confidence intervals of 0.0–0.04). Bd was found on 22 hellbenders (24% prevalence, 95% confidence intervals of 0.16 ≤ 0.24 ≤ 0.34), representing all four states. All positive samples had low loads of Bd zoospores (12.7 ± 4.9 S.E.M. genome equivalents) compared to other Bd susceptible species. More research is needed to determine the impact of Batrachochytrium infection on hellbender fitness and population viability. In particular, understanding how hellbenders limit Bd infection intensity in an aquatic environment may yield important insights for amphibian conservation. This study is among the first to evaluate the distribution of Bs in the United States, and is consistent with another, which failed to detect Bs in the U.S. Knowledge about the distribution, host-range, and origin of Bs may help control the spread of this pathogen, especially to regions of high salamander diversity, such as the eastern United States. PMID:25695636

  7. Characterization and molecular methods for detection of a novel spiroplasma pathogenic to Penaeus vannamei.

    PubMed

    Nunan, Linda M; Pantoja, Carlos R; Salazar, Marcela; Aranguren, Fernando; Lightner, Donald V

    2004-12-13

    Traditionally, Spiroplasma spp. have only been isolated from the surfaces of flowers and other plant parts, from the guts and hemolymph of various insects, and from vascular plant fluids (phloem sap) and insects that feed on these fluids. In this article, we report the first pathogenic spiroplasma to be discovered in shrimp and the results of its characterization through histological evaluation, in situ hybridization assays, transmission electron microscopy, 16S rRNA sequence homology, and injection infectivity studies. In addition, molecular methods are described that were developed for the detection of this microorganism, which was determined to be the causative disease agent in Colombian farm-raised Penaeus vannamei suffering from high mortalities. Using standard histological methods and in situ hybridization assays, it was confirmed that P. vannamei was infected with this pathogenic spiroplasma. Histological analysis revealed systemic inflammatory reactions in affected organs/tissues. In an attempt to identify the bacteria, frozen infected P. vannamei samples, from the initial epizootic, were used to sequence the 16S rRNA gene and develop molecular detection methods. The 16S rRNA gene was amplified by PCR and then sequenced. The sequence data were analyzed using the GenBank BLAST search and the results revealed a 98% homology with Spiroplasma citri, a pathogen of citrus trees. The 16S rRNA sequence data were evaluated for development of unique PCR primers to the putative spiroplasma. Using PCR primers developed for the spiralin gene of Spiroplasma spp., a digoxigenin-labeled probe was developed and tested. This probe was species-specific, with no positive reactions or cross-reactivity occurring with other bacterial samples tested in this format.

  8. Pathogenic chytrid fungus Batrachochytrium dendrobatidis, but not B. salamandrivorans, detected on eastern hellbenders.

    PubMed

    Bales, Emma K; Hyman, Oliver J; Loudon, Andrew H; Harris, Reid N; Lipps, Gregory; Chapman, Eric; Roblee, Kenneth; Kleopfer, John D; Terrell, Kimberly A

    2015-01-01

    Recent worldwide declines and extinctions of amphibian populations have been attributed to chytridiomycosis, a disease caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Until recently, Bd was thought to be the only Batrachochytrium species that infects amphibians; however a newly described species, Batrachochytrium salamandrivorans (Bs), is linked to die-offs in European fire salamanders (Salamandra salamandra). Little is known about the distribution, host range, or origin of Bs. In this study, we surveyed populations of an aquatic salamander that is declining in the United States, the eastern hellbender (Cryptobranchus alleganiensis alleganiensis), for the presence of Bs and Bd. Skin swabs were collected from a total of 91 individuals in New York, Pennsylvania, Ohio, and Virginia, and tested for both pathogens using duplex qPCR. Bs was not detected in any samples, suggesting it was not present in these hellbender populations (0% prevalence, 95% confidence intervals of 0.0-0.04). Bd was found on 22 hellbenders (24% prevalence, 95% confidence intervals of 0.16 ≤ 0.24 ≤ 0.34), representing all four states. All positive samples had low loads of Bd zoospores (12.7 ± 4.9 S.E.M. genome equivalents) compared to other Bd susceptible species. More research is needed to determine the impact of Batrachochytrium infection on hellbender fitness and population viability. In particular, understanding how hellbenders limit Bd infection intensity in an aquatic environment may yield important insights for amphibian conservation. This study is among the first to evaluate the distribution of Bs in the United States, and is consistent with another, which failed to detect Bs in the U.S. Knowledge about the distribution, host-range, and origin of Bs may help control the spread of this pathogen, especially to regions of high salamander diversity, such as the eastern United States.

  9. Performance of a multiplexed serological microarray for the detection of antibodies against central nervous system pathogens.

    PubMed

    Jääskeläinen, Anne J; Viitala, Sari M; Kurkela, Satu; Hepojoki, Satu; Sillanpää, Heidi; Kallio-Kokko, Hannimari; Bergström, Tomas; Suni, Jukka; Närvänen, Ale; Vapalahti, Olli; Vaheri, Antti

    2014-05-01

    Central nervous system (CNS) infections have multiple potential causative agents for which simultaneous pathogen screening can provide a useful tool. This study evaluated a multiplexed microarray for the simultaneous detection of antibodies against CNS pathogens. The performance of selected microarray antigens for the detection of IgG antibodies against herpes simplex virus 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), adenovirus, Mycoplasma pneumoniae and Borrelia burgdorferi sensu lato, was evaluated using serum sample panels tested with reference assays used in a routine diagnostic laboratory. The microarray sensitivity for HSV-1, HSV-2, VZV, adenovirus and M. pneumonia ranged from 77% to 100%, and the specificity ranged from 74% to 97%. Very variable sensitivities and specificities were found for borrelial antigens of three different VlsE protein IR(6) peptide variants (IR6p1, IR6p2, IR6p4) and three recombinant decorin binding proteins A (DbpA; DbpAIa, DbpA91, DbpAG40). For single antigens, good specificity was shown for antigens of IR6p4 and DbpAIa (96%), while DbpA91, IR6p1 and IR6p2 were moderately specific (88-92%). The analytical sensitivity of the microarray was dependent on the borrelial IgG concentration of the specimen. The overall performance and technical features of the platform showed that the platform supports both recombinant proteins, whole viruses and peptides as antigens. This study showed diagnostic potential for all six CNS pathogens, including Borrelia burgdorferi sensu lato, using glutaraldehyde based microarray, and further highlighted the importance of careful antigen selection and the requirement for the use of multiple borrelial antigens in order to increase specificity without a major lack of sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays

    PubMed Central

    Jin, Lian-Qun; Li, Jun-Wen; Wang, Sheng-Qi; Chao, Fu-Huan; Wang, Xin-Wei; Yuan, Zheng-Quan

    2005-01-01

    AIM: To detect the common intestinal pathogenic bacteria quickly and accurately. METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays. RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified. CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost. PMID:16437687

  11. Rapid detection of pathogenic leptospires by lyophilized reagent-based polymerase chain reaction.

    PubMed

    Lee, S V; Tai, E S; Mutalib, A R; Khairani-Bejo, S; Bahaman, A R

    2011-12-01

    A simple and reliable tool for the early diagnosis of leptospirosis is urgently needed. We report the development of a lyophilized reagent-based polymerase chain reaction (PCR) assay targeting lipL32 gene, which is present only in pathogenic leptospires. To determine the effectiveness of the newly developed assay in the early diagnosis of leptospirosis, the sensitivity and specificity was evaluated. In simulated clinical samples, the assay was able to detect 10² and 10³ leptospires/ml in spiked urine and blood samples, respectively. In experimentally infected animals, leptospiral DNA could be detected in blood and lung samples as early as Day 1 post infection. This assay was also shown to be stable and remained sensitive for up to five months at ambient temperature. Hence, this lyophilized reagent-based PCR assay with high specificity, sensitivity and stability would provide a simple, rapid and reliable method in diagnosing acute leptospirosis, especially in the field of veterinary medicine.

  12. A facile and sensitive detection of pathogenic bacteria using magnetic nanoparticles and optical nanocrystal probes.

    PubMed

    Joo, Jinmyoung; Yim, Changyong; Kwon, Donghoon; Lee, Jaejin; Shin, Hwa Hui; Cha, Hyung Joon; Jeon, Sangmin

    2012-08-21

    We report a facile and sensitive analytical method for the detection of pathogenic bacteria. Salmonella bacteria in milk were captured by antibody-conjugated magnetic nanoparticles (MNPs) and separated from analyte samples by applying an external magnetic field. The MNP-Salmonella complexes were re-dispersed in a buffer solution then exposed to antibody-immobilized TiO(2) nanocrystals (TNs), which absorb UV light. After magnetically separating the MNP-Salmonella-TN complexes from solution, the UV-Vis absorption spectrum of the unbound TN solution was obtained. Because the light absorption intensity was reversely proportional to the Salmonella concentration, the assay exhibited high sensitivity toward low concentrations of Salmonella bacteria. The detection limit of Salmonella in milk was found to be more than 100 cfu mL(-1).

  13. Detection of human pathogenic Ehrlichia muris-like agent in Peromyscus leucopus.

    PubMed

    Castillo, Caroline G; Eremeeva, Marina E; Paskewitz, Susan M; Sloan, Lynne M; Lee, Xia; Irwin, William E; Tonsberg, Stefan; Pritt, Bobbi S

    2015-03-01

    An Ehrlichia muris-like (EML) bacterium was recently detected in humans and Ixodes scapularis ticks in Minnesota and Wisconsin. The reservoir for this agent is unknown. To investigate the occurrence of the EML agent, groEL PCR testing and sequencing was performed on blood from small mammals and white-tailed deer that were collected in areas where human and tick infections were previously demonstrated. DNA of the EML agent was detected in two Peromyscus leucopus of 146 small mammals (1.4%); while 181 O. virginianus tested negative. This report provides the first evidence that DNA from the EML agent is found in P. leucopus, the same animal that is a reservoir for Anaplasma phagocytophilum in this region. The role of white-tailed deer remains inconclusive. Further sampling is warranted to understand the spatial and temporal distribution, transmission and maintenance of this pathogen. Copyright © 2014 Elsevier GmbH. All rights reserved.

  14. A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

    PubMed

    Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

    2015-05-21

    Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.

  15. Molecular detection of tick-borne pathogens in Ixodes ricinus from Moldova collected in 1960.

    PubMed

    Movila, Alexandru; Toderas, Ion; Uspenskaia, Inga; Conovalov, Jurii

    2013-06-01

    This study is the first report about the prevalence of tick-borne pathogens, as well as their (co-)infection rates, in the museum-archived I. ricinus female ticks collected in Moldova in 1960. A total of 16.7% (21/126) ticks was mono-infected. Borrelia burgdorferi sensu stricto was revealed as the most abundant species (4.8%) followed by B. garinii (1.6%), B. afzelii (0.8%), B. valaisiana (0.8%), and B. lusitaniae (0.8%). DNA of Rickettsia helvetica (2.4%), R. monacensis (2.4%), Anaplasma phagocytophilum (2.4%), 'Candidatus Neoehrlichia mikurensis' (0.8%), and Babesia microti (0.8%) were also detected, indicating the occurrence of these emerging tick-borne microorganisms in Moldova since 1960 at least. In this study, we detected a co-infection (0.8%; 1/126 tested ticks) between B. microti and R. helvetica. Additional investigations are warranted to further characterize a historical snapshot of the distribution of tick-borne pathogens in Europe.

  16. Effect of temperature of incubation on performance of media in the detection of enteric pathogens.

    PubMed

    Taylor, W I; Schelhart, D

    1973-06-01

    The effect of incubation temperatures on the efficacies of both plating media and transport or enrichment broths was determined by the analysis of 391 diarrheal stools for salmonellae and shigellae. Each analysis resulted in 90 observations. Stool specimens were homogenized in saline and used to inoculate eosin methylene blue (EMB), Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) agar plates, Amies and Cary-Blair (CB) transport media, and gram-negative (GN) enrichment broth. All media were incubated at 25, 30, and 35 C for 24 and 48 h. In order of efficacy, GN and saline were significantly better than Amies and CB, which were still better than direct streaking for both salmonellae and shigellae. Forty-eight hours was a significant improvement over 24 h only at 25 C on direct streaking for both pathogens. Salmonella detection was also improved at 30 over 25 C on direct streaking. In direct plating, XLD was better than both SS and EMB for both pathogens. After broths, for salmonellae, XLD > SS > EMB, and for shigellae, XLD > EMB > SS, with all differences significant. SS agar was significantly improved for detection of shigellae with 48-h broth inocula versus 24-h broth inocula. The differences thus observed at the various temperatures tested proved to be less important than the media used. The efficient media, GN broth, saline-stool, and XLD were shown to be affected very little by either temperature or time variance of the magnitude tested.

  17. Beta-Hemolytic Bacteria Selectively Trigger Liposome Lysis, Enabling Rapid and Accurate Pathogen Detection.

    PubMed

    Sum, Rongji; Swaminathan, Muthukaruppan; Rastogi, Sahil Kumar; Piloto, Obdulio; Cheong, Ian

    2017-09-20

    For more than a century, blood agar plates have been the only test for beta-hemolysis. Although blood agar cultures are highly predictive for bacterial pathogens, they are too slow to yield actionable information. Here, we show that beta-hemolytic pathogens are able to lyse and release fluorophores encapsulated in sterically stabilized liposomes whereas alpha and gamma-hemolytic bacteria have no effect. By analyzing fluorescence kinetics, beta-hemolytic colonies cultured on agar could be distinguished in real time with 100% accuracy within 6 h. Additionally, end point analysis based on fluorescence intensity and machine-extracted textural features could discriminate between beta-hemolytic and cocultured control colonies with 99% accuracy. In broth cultures, beta-hemolytic bacteria were detectable in under an hour while control bacteria remained negative even the next day. This strategy, called beta-hemolysis triggered-release assay (BETA) has the potential to enable the same-day detection of beta-hemolysis with single-cell sensitivity and high accuracy.

  18. A quantum-dot-based fluoroassay for detection of food-borne pathogens.

    PubMed

    Mohamadi, Elaheh; Moghaddasi, Mohammadali; Farahbakhsh, Afshin; Kazemi, Abbass

    2017-09-01

    Evaluation of the distribution capability of food-borne pathogens existing in food products by taking the advantage of quantum dots (QDs) for their photoluminescence properties was carried out. Bacteria namely Escherichia coli (E. coli) labelled with CdSe-QDs were examined both on an Agar nutrient and ground fish substrates in order to observe their growth rate in different environments in the Lab. A sample with an appropriate concentration ratio 10(7)CFU/mL of bacteria/CdSe-QDs was empirically selected from the samples which were grown on the Agar containing plates. The selected sample was also tested on a ground fish substrate as a real food sample. The bacterial growth was observed under the irradiation of UV light and the growth patterns were investigated for 3 successive days. The growth patterns indicated that E. coli can stay alive and can be distributed on food products so that the growth can be easily monitored. This approach makes bacterial growth on food products detectable so that it can be used as a bacteria-QD assay for an easy detection of food borne pathogens grown on a food sample. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR.

    PubMed

    Lamarche, Josyanne; Potvin, Amélie; Pelletier, Gervais; Stewart, Don; Feau, Nicolas; Alayon, Dario I O; Dale, Angela L; Coelho, Aaron; Uzunovic, Adnan; Bilodeau, Guillaume J; Brière, Stephan C; Hamelin, Richard C; Tanguay, Philippe

    2015-01-01

    Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.

  20. Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

    PubMed Central

    Lamarche, Josyanne; Potvin, Amélie; Pelletier, Gervais; Stewart, Don; Feau, Nicolas; Alayon, Dario I. O.; Dale, Angela L.; Coelho, Aaron; Uzunovic, Adnan; Bilodeau, Guillaume J.; Brière, Stephan C.; Hamelin, Richard C.; Tanguay, Philippe

    2015-01-01

    Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada. PMID:26274489

  1. Detection of Tick-Borne Pathogens in the Korean Water Deer (Hydropotes inermis argyropus) from Jeonbuk Province, Korea.

    PubMed

    Seong, Giyong; Han, Yu-Jung; Oh, Sung-Suck; Chae, Joon-Seok; Yu, Do-Hyeon; Park, Jinho; Park, Bae-Keun; Yoo, Jae-Gyu; Choi, Kyoung-Seong

    2015-10-01

    The objective of this study was to investigate the prevalence of tick-borne pathogens in the Korean water deer (Hydropotes inermis argyropus). Pathogens were identified using PCR which included Anaplasma, Ehrlichia, Rickettsia, and Theileria. Rickettsia was not detected, whereas Anaplasma, Ehrlichia, and Theileria infections were detected in 4, 2, and 8 animals, respectively. The most prevalent pathogen was Theileria. Of the 8 Theileria-positive animals, 2 were mixed-infected with 3 pathogens (Anaplasma, Ehrlichia, and Theileria) and another 2 animals showed mixed-infection with 2 pathogens (Anaplasma and Theileria). Sequencing analysis was used to verify the PCR results. The pathogens found in this study were identified as Anaplasma phagocytophilum, Ehrlichia canis, and Theileria sp. To the best of our knowledge, this is the first report identifying these 3 pathogens in the Korean water deer. Our results suggest that the Korean water deer may serve as a major reservoir for these tick-borne pathogens, leading to spread of tick-borne diseases to domestic animals, livestock, and humans. Further studies are needed to investigate their roles in this respect.

  2. Molecular and serological detection of tick-borne pathogens in donkeys (Equus asinus) in Italy.

    PubMed

    Veronesi, Fabrizia; Morganti, Giulia; Ravagnan, Silvia; Laus, Fulvio; Spaterna, Andrea; Diaferia, Manuela; Moretti, Annabella; Fioretti, Daniela Piergili; Capelli, Gioia

    2014-10-10

    Donkeys, owing to the frequent outdoor activity, are exposed to a high risk of infection with tick-borne pathogens. This work aimed to detect exposure to Theileria equi, Babesia caballi, Anaplasma phagocytophilum and Borrelia burgdorferi s.l. of donkeys reared in Central Italy. For this purpose 122 adult donkeys were selected within 11 herds and submitted to blood collection. IgG antibodies to T. equi, B. caballi, A. phagocytophilum and B. burgdorferi s.l. were detected by IFAT. Conventional PCRs targeting the genes MSP2 and the flagellin were used for the detection of A. phagocytophilum and B. burgdorferi s.l. respectively and a Real Time PCR Sybr Green was used to detect Babesia/Theileria spp…. The species identity was determined by amplicons sequencing. Forty eight (39.3%) and 58 (47.5%) animals tested positive for T. equi and B. caballi antibodies, respectively; nine animals (7.4%) were found positive for antibodies against A. phagocytophilum whereas negative results were obtained for B. burgdorferi s.l. Twenty-six (21.3%) animals showed antibodies for both T. equi and B. caballi. Twenty-three (18.8%) donkeys were positive to Babesia/Theileria spp. PCR assay. Out of 21 sequenced amplicons, 20 were identified as T. equi, belonging to three main groups designated A, B and D and one as B. caballi group A. Neither A. phagocytophilum nor B. burgdorferi PCR results were positive. The study showed a high exposure of donkeys to tick-borne pathogens and provides information on the genetic identity of the T. equi strains circulating in Central Italy.

  3. Method for detection of a few pathogenic bacteria and determination of live versus dead cells

    NASA Astrophysics Data System (ADS)

    Horikawa, Shin; Chen, I.-Hsuan; Du, Songtao; Liu, Yuzhe; Wikle, Howard C.; Suh, Sang-Jin; Barbaree, James M.; Chin, Bryan A.

    2016-05-01

    This paper presents a method for detection of a few pathogenic bacteria and determination of live versus dead cells. The method combines wireless phage-coated magnetoelastic (ME) biosensors and a surface-scanning dectector, enabling real-time monitoring of the growth of specific bacteria in a nutrient broth. The ME biosensor used in this investigation is composed of a strip-shaped ME resonator upon which an engineered bacteriophage is coated to capture a pathogen of interest. E2 phage with high binding affinity for Salmonella Typhimurium was used as a model study. The specificity of E2 phage has been reported to be 1 in 105 background bacteria. The phage-coated ME biosensors were first exposed to a low-concentration Salmonella suspension to capture roughly 300 cells on the sensor surface. When the growth of Salmonella in the broth occurs, the mass of the biosensor increases, which results in a decrease in the biosensor's resonant frequency. Monitoring of this mass- induced resonant frequency change allows for real-time detection of the presence of Salmonella. Detection of a few bacteria is also possible by growing them to a sufficient number. The surface-scanning detector was used to measure resonant frequency changes of 25 biosensors sequentially in an automated manner as a function of time. This methodology offers direct, real-time detection, quantification, and viability determination of specific bacteria. The rate of the sensor's resonant frequency change was found to be largely dependent on the number of initially bound cells and the efficiency of cell growth.

  4. Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis.

    PubMed

    Bletz, M C; Rebollar, E A; Harris, R N

    2015-02-10

    Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens.

  5. Molecular detection of bacterial and parasitic pathogens in hard ticks from Portugal.

    PubMed

    Maia, Carla; Ferreira, Andreia; Nunes, Mónica; Vieira, Maria Luísa; Campino, Lenea; Cardoso, Luís

    2014-06-01

    Ticks are important vector arthropods of human and animal pathogens. As information about agents of disease circulating in vectors in Portugal is limited, the aim of the present study was to detect bacteria and parasites with veterinary and zoonotic importance in ticks collected from dogs, cats, and field vegetation. A total of 925 ticks, comprising 888 (96.0%) adults, 8 (0.9%) nymphs, and 29 (3.1%) larvae, were collected in 4 geographic areas (districts) of Portugal. Among those, 620 (67.0%) were removed from naturally infested dogs, 42 (4.5%) from cats, and 263 (28.4%) were questing ticks obtained from field vegetation. Rhipicephalus sanguineus was the predominant tick species, and the only one collected from dogs and vegetation, while all Ixodes ricinus specimens (n=6) were recovered from cats. Rickettsia massiliae and Rickettsia conorii were identified in 35 ticks collected from cats and dogs and in 3 ticks collected from dogs. Among ticks collected from cats or dogs, 4 Rh. sanguineus specimens were detected with Hepatozoon felis, 3 with Anaplasma platys, 2 with Hepatozoon canis, one with Anaplasma phagocytophilum, one with Babesia vogeli, one with Borrelia burgdorferi sensu lato and one with Cercopithifilaria spp. Rickettsia helvetica was detected in one I. ricinus tick collected from a cat. To the best of our knowledge, this was the first time that Cercopithifilaria spp., Ba. vogeli, H. canis, and H. felis have been detected in ticks from Portugal. The wide range of tick-borne pathogens identified, some of zoonotic concern, suggests a risk for the emergence of tick-borne diseases in domestic animals and humans in Portugal. Further studies on these and other tick-borne agents should be performed to better understand their epidemiological and clinical importance, and to support the implementation of effective control measures.

  6. Evaluation of Use of a New Chromogenic Agar in Detection of Urinary Tract Pathogens

    PubMed Central

    Samra, Z.; Heifetz, M.; Talmor, J.; Bain, E.; Bahar, J.

    1998-01-01

    CHROMagar Orientation, a new chromogenic medium, was evaluated for the detection and differentiation of gram-positive and gram-negative pathogenic microorganisms in 900 urine samples from hospitalized patients. Performance characteristics of the medium were evaluated in comparison to those of 5% sheep blood and MacConkey agars by direct inoculation of the urine samples on the three media. Four gram-negative and two gram-positive strains as well as one yeast control strain from the American Type Culture Collection were used to ensure quality control. CHROMagar Orientation succeeded in detecting all the urine pathogens that were detected by the reference media, including gram-negative bacilli, staphylococci, streptococci, and yeasts. Colony color and morphology on CHROMagar Orientation accurately differentiated Escherichia coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, and Acinetobacter spp. Owing to the similarity in the pigmentation produced by Klebsiella, Enterobacter, and Citrobacter isolates, the medium failed to distinguish among them; however, these isolates were easily recognized as coliforms because of their metallic blue coloration. Staphylococci were clearly perceptible: S. aureus and S. epidermidis grow in regular-size colonies that range from opaque white to yellowish, and S. saprophyticus produces opaque pink colonies. All streptococcus strains, including those from groups B and C, were detected. They grow as undifferentiated flat dry diffused colonies, and additional tests were required for identification. Enterococci were easily discriminated by their strong turquoise pigmentation and their typical growth on the agar’s surface. Yeast grow in typical creamy wet convex colonies. The accuracy of antibiotic susceptibility determinations according to standard methods was also tested by picking isolates directly from CHROMagar Orientation. The results showed excellent correlation with those obtained with microorganisms picked from

  7. Evaluation of use of a new chromogenic agar in detection of urinary tract pathogens.

    PubMed

    Samra, Z; Heifetz, M; Talmor, J; Bain, E; Bahar, J

    1998-04-01

    CHROMagar Orientation, a new chromogenic medium, was evaluated for the detection and differentiation of gram-positive and gram-negative pathogenic microorganisms in 900 urine samples from hospitalized patients. Performance characteristics of the medium were evaluated in comparison to those of 5% sheep blood and MacConkey agars by direct inoculation of the urine samples on the three media. Four gram-negative and two gram-positive strains as well as one yeast control strain from the American Type Culture Collection were used to ensure quality control. CHROMagar Orientation succeeded in detecting all the urine pathogens that were detected by the reference media, including gram-negative bacilli, staphylococci, streptococci, and yeasts. Colony color and morphology on CHROMagar Orientation accurately differentiated Escherichia coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, and Acinetobacter spp. Owing to the similarity in the pigmentation produced by Klebsiella, Enterobacter, and Citrobacter isolates, the medium failed to distinguish among them; however, these isolates were easily recognized as coliforms because of their metallic blue coloration. Staphylococci were clearly perceptible: S. aureus and S. epidermidis grow in regular-size colonies that range from opaque white to yellowish, and S. saprophyticus produces opaque pink colonies. All streptococcus strains, including those from groups B and C, were detected. They grow as undifferentiated flat dry diffused colonies, and additional tests were required for identification. Enterococci were easily discriminated by their strong turquoise pigmentation and their typical growth on the agar's surface. Yeast grow in typical creamy wet convex colonies. The accuracy of antibiotic susceptibility determinations according to standard methods was also tested by picking isolates directly from CHROMagar Orientation. The results showed excellent correlation with those obtained with microorganisms picked from

  8. Detection of fish pathogens by loop-mediated isothermal amplification (LAMP) technique.

    PubMed

    Soliman, Hatem; Saleh, Mona; El-Matbouli, Mansour

    2015-01-01

    Rapid detection of fish pathogens is mandatory for applying the crucial preventive and control measures to reduce fish losses and, consequently, minimize the economic impact of diseases on the fish farm owners. The currently used molecular diagnostic tools of fish infectious agents, such as PCR and RT-PCR, are sensitive and specific but still have some drawbacks. These tools are usually time consuming and laborious, need skilled persons, and require sophisticated devices to be performed. Therefore, next-generation tools for rapid diagnosis of fish infectious diseases were developed to conquer these shortages. One of these novel tools is the loop-mediated isothermal amplification (LAMP) technique. LAMP is considered a more advantageous tool than PCR because it needs only a heating block or a thermostatically controlled water bath as a source of constant temperature. It is considered to be more specific than the PCR assay as it uses 4-6 primers, which may diminish the occurrence of false-positive results. The time required for the amplification process by LAMP is ranging from 30 min to 1 h comparing to 3-5 h in the case of PCR. The visual detection methods coupled with the LAMP assay eliminates the post-run processing for detection of the amplification products. Its sensitivity is either comparable with the PCR or better than it. A variety of LAMP assays were developed for simple and rapid detection of a diversity of fish pathogens. Herein, we describe how to perform a LAMP assay and troubleshoot any potential problem arising during the process.

  9. Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira.

    PubMed

    Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A

    2014-05-08

    Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning.

  10. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction.

    PubMed Central

    Burg, J L; Grover, C M; Pouletty, P; Boothroyd, J C

    1989-01-01

    We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses. Images PMID:2768467

  11. Differential detection of pathogenic Yersinia spp. by fluorescence in situ hybridization.

    PubMed

    Rohde, Alexander; Hammerl, Jens Andre; Appel, Bernd; Dieckmann, Ralf; Al Dahouk, Sascha

    2017-04-01

    Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis are pathogens of major medical importance, which are responsible for a considerable number of infections every year. The detection of these species still relies on cultural methods, which are slow, labour intensive and often hampered by the presence of high amounts of accompanying flora. In this study, fluorescence in situ hybridization (FISH) was used to develop a fast, sensitive and reliable alternative to detect viable bacteria in food. For this purpose, highly specific probes targeting the 16S and 23S ribosomal RNA were employed to differentially detect each of the three species. In order to enable the differentiation of single nucleotide polymorphisms (SNPs), suitable competitor oligonucleotides and locked nucleic acids (LNAs) were used. Starved cells still showed a strong signal and a direct viable count (DVC) approach combined with FISH optimized live/dead discrimination. Sensitivity of the FISH test was high and even a single cell per gram of spiked minced pork meat could be detected within a day, demonstrating the applicability to identify foodborne hazards at an early stage. In conclusion, the established FISH tests proved to be promising tools to compensate existing drawbacks of the conventional cultural detection of these important zoonotic agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Development of a Recombinase Polymerase Amplification Assay for the Detection of Pathogenic Leptospira

    PubMed Central

    Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A.

    2014-01-01

    Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning. PMID:24814943

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Evaluation of a real-time PCR test for the detection and discrimination of theileria species in the African buffalo (Syncerus caffer).

    PubMed

    Chaisi, Mamohale E; Janssens, Michiel E; Vermeiren, Lieve; Oosthuizen, Marinda C; Collins, Nicola E; Geysen, Dirk

    2013-01-01

    A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.

  15. Field-deployable colorimetric biosensor system for the rapid detection of pathogenic organisms

    NASA Astrophysics Data System (ADS)

    Duy, Janice

    The rapid identification of pathogenic organisms is necessary for recognizing and managing human and environmental health risks. Numerous detection schemes are available, but most are difficult to employ in non-laboratory settings due to their need for bulky, specialized equipment, multiple reagents, or highly trained personnel. To address this problem, a rapid, field-compatible biosensor system based on the colorimetric detection of nucleic acid hybrids was developed. Peptide nucleic acid (PNA) probes were used to capture ribosomal RNA sequences from environmental samples. Non-target nucleic acids, including single-base mismatches flanked by adenines and uracils, were removed with a micrococcal nuclease digestion step. Matched PNA-RNA hybrids remained intact and were indicated by the cyanine dye DiSC2(5). PNA-containing duplexes function as templates for the aggregation of DiSC2(5), visualized as a change in solution color from blue to purple. This transition can be measured as an increase in the solution absorbance at 540 nm (dye aggregate) at the expense of the dye monomer peak at 650 nm. These concomitant spectral changes were used to calculate a "hybridization signal" using the ratio A aggregate/Amonomer ≈ A540/A650. Testing with pathogenic environmental samples was accomplished using two model organisms: the harmful algal bloom-causing dinoflagellate Alexandrium species, and the potato wart disease-causing fungus Synchytrium endobioticum. In both cases, the colorimetric approach was able to distinguish the targets with sensitivities rivaling those of established techniques, but with the advantages of decreased hands-on time and cost. Assay fieldability was tested with a portable colorimeter designed to quantify the dye-indicated hybridization signal and assembled from commercially available components. Side-by-side testing revealed no difference in the sensing performance of the colorimeter compared to a laboratory spectrophotometer (Pearson's r=0

  16. Detection of Termites and Other Insects Consumed by African Great Apes using Molecular Fecal Analysis

    PubMed Central

    Hamad, Ibrahim; Delaporte, Eric; Raoult, Didier; Bittar, Fadi

    2014-01-01

    The consumption of insects by apes has previously been reported based on direct observations and/or trail signs in feces. However, DNA-based diet analyses may have the potential to reveal trophic links for these wild species. Herein, we analyzed the insect-diet diversity of 9 feces obtained from three species of African great apes, gorilla (Gorilla gorilla gorilla), chimpanzee (Pan troglodytes) and bonobo (Pan paniscus), using two mitochondrial amplifications for arthropods. A total of 1056 clones were sequenced for Cyt-b and COI gene libraries, which contained 50 and 56 operational taxonomic units (OTUs), respectively. BLAST research revealed that the OTUs belonged to 32 families from 5 orders (Diptera, Isoptera, Lepidoptera, Coleoptera, and Orthoptera). While ants were not detected by this method, the consumption of flies, beetles, moths, mosquitoes and termites was evident in these samples. Our findings indicate that molecular techniques can be used to analyze insect food items in wild animals. PMID:24675424

  17. Detection of termites and other insects consumed by African great apes using molecular fecal analysis.

    PubMed

    Hamad, Ibrahim; Delaporte, Eric; Raoult, Didier; Bittar, Fadi

    2014-03-27

    The consumption of insects by apes has previously been reported based on direct observations and/or trail signs in feces. However, DNA-based diet analyses may have the potential to reveal trophic links for these wild species. Herein, we analyzed the insect-diet diversity of 9 feces obtained from three species of African great apes, gorilla (Gorilla gorilla gorilla), chimpanzee (Pan troglodytes) and bonobo (Pan paniscus), using two mitochondrial amplifications for arthropods. A total of 1056 clones were sequenced for Cyt-b and COI gene libraries, which contained 50 and 56 operational taxonomic units (OTUs), respectively. BLAST research revealed that the OTUs belonged to 32 families from 5 orders (Diptera, Isoptera, Lepidoptera, Coleoptera, and Orthoptera). While ants were not detected by this method, the consumption of flies, beetles, moths, mosquitoes and termites was evident in these samples. Our findings indicate that molecular techniques can be used to analyze insect food items in wild animals.

  18. Concurrent Detection of Human Norovirus and Bacterial Pathogens in Water Samples from an Agricultural Region in Central California Coast.

    PubMed

    Tian, Peng; Yang, David; Shan, Lei; Wang, Dapeng; Li, Qianqian; Gorski, Lisa; Lee, Bertram G; Quiñones, Beatriz; Cooley, Michael B

    2017-01-01

    Bacterial pathogens and human norovirus (HuNoV) are major cause for acute gastroenteritis caused by contaminated food and water. Public waterways can become contaminated from a variety of sources and flood after heavy rain events, leading to pathogen contamination of produce fields. We initiated a survey of several public watersheds in a major leafy green produce production region of the Central California Coast to determine the prevalence of HuNoV as well as bacterial pathogens. Moore swabs were used to collect environmental samples bi-monthly at over 30 sampling sites in the region. High prevalence of HuNoV and bacterial pathogens were detected in environmental water samples in the region. The overall detection rates of HuNoV, O157 Shiga toxin-producing Escherichia coli (STEC), non-O157 STEC, Salmonella, and Listeria were 25.58, 7.91, 9.42, 59.65, and 44.30%, respectively. The detection rates of Salmonella and L. monocytogenes were significantly higher in the spring. Fall and spring had elevated detection rates of O157 STEC. The overall detection rates of non-O157 STEC in the fall were lower than the other seasons but not significant. The overall detection rates of HuNoV were highest in fall, followed by spring and winter, with summer being lowest and significantly lower than other seasons. This study presented the first study of evaluating the correlation between the detection rate of HuNoV and the detection rates of four bacterial pathogens from environmental water. Overall, there was no significant difference in HuNoV detection rates between samples testing positive or negative for the four bacterial pathogens tested. Pathogens in animal-impacted and human-impacted areas were investigated. There were significant higher detection rates in animal-impacted areas than that of human-impacted areas for bacterial pathogens. However, there was no difference in HuNoV detection rates between these two areas. The overall detection levels of generic E. coli and detection

  19. Monitoring invasive pathogens in plant nurseries for early-detection and to minimise the probability of escape.

    PubMed

    Alonso Chavez, Vasthi; Parnell, Stephen; VAN DEN Bosch, Frank

    2016-10-21

    The global increase in the movement of plant products in recent years has triggered an increase in the number of introduced plant pathogens. Plant nurseries importing material from abroad may play an important role in the introduction and spread of diseases such as ash dieback and sudden oak death which are thought to have been introduced through trade. The economic, environmental and social costs associated with the spread of invasive pathogens become considerably larger as the incidence of the pathogen increases. To control the movement of pathogens across the plant trade network it is crucial to develop monitoring programmes at key points of the network such as plant nurseries. By detecting the introduction of invasive pathogens at low incidence, the control and eradication of an epidemic is more likely to be successful. Equally, knowing the likelihood of having sold infected plants once a disease has been detected in a nursery can help designing tracing plans to control the onward spread of the disease. Here, we develop an epidemiological model to detect and track the movement of an invasive plant pathogen into and from a plant nursery. Using statistical methods, we predict the epidemic incidence given that a detection of the pathogen has occurred for the first time, considering that the epidemic has an asymptomatic period between infection and symptom development. Equally, we calculate the probability of having sold at least one infected plant during the period previous to the first disease detection. This analysis can aid stakeholder decisions to determine, when the pathogen is first discovered in a nursery, the need of tracking the disease to other points in the plant trade network in order to control the epidemic. We apply our method to high profile recent introductions including ash dieback and sudden oak death in the UK and citrus canker and Huanglongbing disease in Florida. These results provide new insight for the design of monitoring strategies at key

  20. Microbial Quality, Safety, and Pathogen Detection by Using Quantitative PCR of Raw Salad Vegetables Sold in Dhanbad City, India.

    PubMed

    Mritunjay, Sujeet K; Kumar, Vipin

    2017-01-01

    Consumption of ready-to-eat fresh vegetables has increased worldwide, with a consequent increase in outbreaks caused by foodborne pathogens. In the Indian subcontinent, raw fresh vegetables are usually consumed without washing or other decontamination procedures, thereby leading to new food safety threats. In this study, the microbiological quality and pathogenic profile of raw salad vegetables was evaluated through standard protocols. In total, 480 samples (60 each of eight different salad vegetables) of cucumber, tomato, carrot, coriander, cabbage, beetroot, radish, and spinach were collected from different locations in Dhanbad, a city famous for its coal fields and often called the "Coal Capital of