Science.gov

Sample records for rapid detection method

  1. Radiometric method for the rapid detection of Leptospira organisms

    SciTech Connect

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

    1986-02-01

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

  2. Rapid Methods for High-Throughput Detection of Sulfoxides▿

    PubMed Central

    Shainsky, Janna; Derry, Netta-Lee; Leichtmann-Bardoogo, Yael; Wood, Thomas K.; Fishman, Ayelet

    2009-01-01

    Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment. PMID:19465532

  3. Rapid surface enhanced Raman scattering detection method for chloramphenicol residues.

    PubMed

    Ji, Wei; Yao, Weirong

    2015-06-01

    Chloramphenicol (CAP) is a widely used amide alcohol antibiotics, which has been banned from using in food producing animals in many countries. In this study, surface enhanced Raman scattering (SERS) coupled with gold colloidal nanoparticles was used for the rapid analysis of CAP. Density functional theory (DFT) calculations were conducted with Gaussian 03 at the B3LYP level using the 3-21G(d) and 6-31G(d) basis sets to analyze the assignment of vibrations. Affirmatively, the theoretical Raman spectrum of CAP was in complete agreement with the experimental spectrum. They both exhibited three strong peaks characteristic of CAP at 1104 cm(-1), 1344 cm(-1), 1596 cm(-1), which were used for rapid qualitative analysis of CAP residues in food samples. The use of SERS as a method for the measurements of CAP was explored by comparing use of different solvents, gold colloidal nanoparticles concentration and absorption time. The method of the detection limit was determined as 0.1 μg/mL using optimum conditions. The Raman peak at 1344 cm(-1) was used as the index for quantitative analysis of CAP in food samples, with a linear correlation of R(2)=0.9802. Quantitative analysis of CAP residues in foods revealed that the SERS technique with gold colloidal nanoparticles was sensitive and of a good stability and linear correlation, and suited for rapid analysis of CAP residue in a variety of food samples. PMID:25754387

  4. Rapid surface enhanced Raman scattering detection method for chloramphenicol residues

    NASA Astrophysics Data System (ADS)

    Ji, Wei; Yao, Weirong

    2015-06-01

    Chloramphenicol (CAP) is a widely used amide alcohol antibiotics, which has been banned from using in food producing animals in many countries. In this study, surface enhanced Raman scattering (SERS) coupled with gold colloidal nanoparticles was used for the rapid analysis of CAP. Density functional theory (DFT) calculations were conducted with Gaussian 03 at the B3LYP level using the 3-21G(d) and 6-31G(d) basis sets to analyze the assignment of vibrations. Affirmatively, the theoretical Raman spectrum of CAP was in complete agreement with the experimental spectrum. They both exhibited three strong peaks characteristic of CAP at 1104 cm-1, 1344 cm-1, 1596 cm-1, which were used for rapid qualitative analysis of CAP residues in food samples. The use of SERS as a method for the measurements of CAP was explored by comparing use of different solvents, gold colloidal nanoparticles concentration and absorption time. The method of the detection limit was determined as 0.1 μg/mL using optimum conditions. The Raman peak at 1344 cm-1 was used as the index for quantitative analysis of CAP in food samples, with a linear correlation of R2 = 0.9802. Quantitative analysis of CAP residues in foods revealed that the SERS technique with gold colloidal nanoparticles was sensitive and of a good stability and linear correlation, and suited for rapid analysis of CAP residue in a variety of food samples.

  5. Rapid Screening Method for Detection of Bacteria in Platelet Concentrates

    PubMed Central

    Ribault, S.; Harper, K.; Grave, L.; Lafontaine, C.; Nannini, P.; Raimondo, A.; Faure, I. Besson

    2004-01-01

    Public awareness has long focused on the risks of the transmission of viral agents through blood product transfusion. This risk, however, pales in comparison to the less publicized danger associated with the transfusion of blood products contaminated with bacteria, in particular, platelet concentrates. Up to 1,000 cases of clinical sepsis after the transfusion of platelet concentrates are reported annually in the United States. The condition is characterized by acute reaction symptoms and the rapid onset of septicemia and carries a 20 to 40% mortality rate. The urgent need for a method for the routine screening of platelet concentrates to improve patient safety has long been recognized. We describe the development of a rapid and highly sensitive method for screening for bacteria in platelet concentrates for transfusion. No culture period is required; and the entire procedure, from the time of sampling to the time that the final result is obtained, takes less than 90 min. The method involves three basic stages: the selective removal of platelets by filtration following activation with a monoclonal antibody, DNA-specific fluorescent labeling of bacteria, and concentration of the bacteria on a membrane surface for enumeration by solid-phase cytometry. The method offers a universal means of detection of live, nondividing, or dead gram-negative and gram-positive bacteria in complex cellular blood products. The sensitivity is higher than those of the culture-based methods available at present, with a detection limit of 10 to 102 CFU/ml, depending upon the bacterial strain. PMID:15131147

  6. Rapid screening method for detection of bacteria in platelet concentrates.

    PubMed

    Ribault, S; Harper, K; Grave, L; Lafontaine, C; Nannini, P; Raimondo, A; Faure, I Besson

    2004-05-01

    Public awareness has long focused on the risks of the transmission of viral agents through blood product transfusion. This risk, however, pales in comparison to the less publicized danger associated with the transfusion of blood products contaminated with bacteria, in particular, platelet concentrates. Up to 1,000 cases of clinical sepsis after the transfusion of platelet concentrates are reported annually in the United States. The condition is characterized by acute reaction symptoms and the rapid onset of septicemia and carries a 20 to 40% mortality rate. The urgent need for a method for the routine screening of platelet concentrates to improve patient safety has long been recognized. We describe the development of a rapid and highly sensitive method for screening for bacteria in platelet concentrates for transfusion. No culture period is required; and the entire procedure, from the time of sampling to the time that the final result is obtained, takes less than 90 min. The method involves three basic stages: the selective removal of platelets by filtration following activation with a monoclonal antibody, DNA-specific fluorescent labeling of bacteria, and concentration of the bacteria on a membrane surface for enumeration by solid-phase cytometry. The method offers a universal means of detection of live, nondividing, or dead gram-negative and gram-positive bacteria in complex cellular blood products. The sensitivity is higher than those of the culture-based methods available at present, with a detection limit of 10 to 10(2) CFU/ml, depending upon the bacterial strain. PMID:15131147

  7. A rapid method to improve protein detection by indirect ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measureable substrate. Numerous incarnations of th...

  8. Rapid Methods for the Detection of General Fecal Indicators

    EPA Science Inventory

    Specified that EPA should develop: appropriate and effective indicators for improving detection in a timely manner of pathogens in coastal waters appropriate, accurate, expeditious and cost-effective methods for the timely detection of pathogens in coastal waters

  9. Three rapid methods compared with a conventional method for detection of urease production in anaerobic bacteria.

    PubMed Central

    Mills, C K; Grimes, B Y; Gherna, R L

    1987-01-01

    Three rapid methods (spot test, disk, and tube) for detecting urease production in anaerobic bacteria yielded results faster than the conventional method. The results were more consistent with the disk and tube methods than with the spot test. Blood agar plate growth gave more consistent results than growth from chopped-meat slants. PMID:3320087

  10. Development of a rapid detection method for Yellow Dwarf Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley and Cereal yellow dwarf viruses (B/CYDVs), constitute the most economically important group of oat viruses. A multiplex reverse transcription polymerase chain reaction method was developed for the simultaneous detection and discrimination of five B/CYDVs viruses. The protocol uses specific pr...

  11. Methods for the rapid detection of biological and chemical weapons

    SciTech Connect

    Castro, A.; Hemberger, P.H.; Swanson, B.I.

    1997-08-01

    This is the final report of a one-year, Laboratory-Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). This work undertook the development of technology for the detection of chemical and biological agents. The project consisted of three tasks: (1) modifying a transportable mass spectrometer for the detection of chemical gents; (2) demonstrating the detection of a specific bacterial DNA sequence using a fluorescence-based single- copy gene detector; and (3) upgrading a surface acoustic wave measurement station.

  12. 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

  13. 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. PMID:25628612

  14. A Rapid and Simple Bioassay Method for Herbicide Detection

    PubMed Central

    Li, Xiu-Qing; Ng, Alan; King, Russell; Durnford, Dion G.

    2008-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, has been used in bioassay detection of a variety of toxic compounds such as pesticides and toxic metals, but mainly using liquid culture systems. In this study, an algal lawn—agar system for semi-quantitative bioassay of herbicidal activities has been developed. Sixteen different herbicides belonging to 11 different categories were applied to paper disks and placed on green alga lawns in Petri dishes. Presence of herbicide activities was indicated by clearing zones around the paper disks on the lawn 2–3 days after application. The different groups of herbicides induced clearing zones of variable size that depended on the amount, mode of action, and chemical properties of the herbicides applied to the paper disks. This simple, paper-disk-algal system may be used to detect the presence of herbicides in water samples and act as a quick and inexpensive semi-quantitative screening for assessing herbicide contamination. PMID:19578512

  15. Electrical impedance measurements: rapid method for detecting and monitoring microorganisms.

    PubMed Central

    Cady, P; Dufour, S W; Shaw, J; Kraeger, S J

    1978-01-01

    A conceptually simple and east-to-use technique is described that uses continuous impedance measurements for automated monitoring of microbial growth and metabolism. The method has been applied to a wide range of microorganisms. Optical clarity is not required. The sensitivity and reproducibility of the method are demonstrated. The mechanism whereby microbial growth alters the impedance of the medium is discussed, as well as potential applications of the method to clinical microbiology. Images PMID:348718

  16. Commercially Available Rapid Methods for Detection of Selected Food-borne Pathogens.

    PubMed

    Valderrama, Wladir B; Dudley, Edward G; Doores, Stephanie; Cutter, Catherine N

    2016-07-01

    Generally, the enumeration and isolation of food-borne pathogens is performed using culture-dependent methods. These methods are sensitive, inexpensive, and provide both qualitative and quantitative assessment of the microorganisms present in a sample, but these are time-consuming. For this reason, researchers are developing new techniques that allow detection of food pathogens in shorter period of time. This review identifies commercially available methods for rapid detection and quantification of Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and Shiga toxin-producing Escherichia coli in food samples. Three categories are discussed: immunologically based methods, nucleic acid-based assays, and biosensors. This review describes the basic mechanism and capabilities of each method, discusses the difficulties of choosing the most convenient method, and provides an overview of the future challenges for the technology for rapid detection of microorganisms. PMID:25749054

  17. Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

    PubMed

    Melendez, Johan H; Santaus, Tonya M; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A; Geddes, Chris D

    2016-10-01

    Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

  18. Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

    PubMed

    Tsai, Shinn Shyong; Chang, Yeng Ling; Huang, Yen Li; Liu, Hung Jen; Ke, Guan Ming; Chiou, Chwei Jang; Hsieh, Yao Ching; Chang, Tsung Chou; Cheng, Li Ting; Chuang, Kuo Pin

    2014-05-01

    There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity. PMID:24193953

  19. Apparatus and method for rapid separation and detection of hydrocarbon fractions in a fluid stream

    SciTech Connect

    Sluder, Charles S.; Storey, John M.; Lewis, Sr., Samuel A.

    2013-01-22

    An apparatus and method for rapid fractionation of hydrocarbon phases in a sample fluid stream are disclosed. Examples of the disclosed apparatus and method include an assembly of elements in fluid communication with one another including one or more valves and at least one sorbent chamber for removing certain classifications of hydrocarbons and detecting the remaining fractions using a detector. The respective ratios of hydrocarbons are determined by comparison with a non separated fluid stream.

  20. A Rapid Method for Viral Particle Detection in Viral-Induced Gastroenteritis: A TEM Study

    NASA Astrophysics Data System (ADS)

    Hicks, M. John; Barrish, James P.; Hayes, Elizabeth S.; Leer, Laurie C.; Estes, Mary K.; Cubitt, W. D.

    1995-10-01

    Infectious gastroenteritis is a common cause of hospitalization in the pediatric population. The most frequent cause of gastroenteritis is viral in origin. The purpose of this study was to compare a rapid modified negative-staining TEM method with the conventional pseudoreplica technique in detection of viral particles in fecal samples from children with viral gastroenteritis. The modified negative-staining method resulted in a significantly higher (2.5 ± 0.5, p = 0.02) viral rating score than that for the conventional pseudoreplica technique (1.7 ± 0.4). In addition, the preparation time for the negative-staining method was approximately one fifth that for the conventional pseudoreplica technique. Rapid diagnosis of viral gastroenteritis may be made by ultrastructural detection of viral particles in fecal samples using the negative staining technique.

  1. Application of a rapid screening method to detect irradiated meat in Brazil

    NASA Astrophysics Data System (ADS)

    Villavicencio, A. L. C. H. A. L. C. H.; Mancini-Filho, J. J.; Delincée, H.

    2000-03-01

    Based on the enormous potential for food irradiation in Brazil, and to ensure free consumer choice, there is a need to find a convenient and rapid method for detection of irradiated food. Since treatment with ionising radiation causes DNA fragmentation, the analysis of DNA damage might be promising. In this paper, the DNA Comet Assay was used to identify exotic meat (boar, jacaré and capybara), irradiated with 60Co gamma rays. The applied radiation doses were 0, 1.5, 3.0 and 4.5 kGy. Analysis of the DNA migration enabled a rapid identification of the radiation treatment.

  2. An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models

    PubMed Central

    Fu, Kaifei; Li, Jun; Wang, Yuxiao; Liu, Jianfei; Yan, He; Shi, Lei; Zhou, Lijun

    2016-01-01

    Vibrio alginolyticus is one of the most common pathogenic marine Vibrio species, and has been found to cause serious seafood-poisoning or fatal extra-intestinal infections in humans, such as necrotizing soft-tissue infections, bacteremia, septic shock, and multiple organ failures. Delayed accurate diagnosis and treatment of most Vibrio infections usually result to high mortality rates. The objective of this study was to establish a rapid diagnostic method to detect and identify the presence of V. alginolyticus in different samples, so as to facilitate timely treatment. The widely employed conventional methods for detection of V. alginolyticus include biochemical identification and a variety of PCR methods. The former is of low specificity and time-consuming (2–3 days), while the latter has improved accuracy and processing time. Despite such advancements, these methods are still complicated, time-consuming, expensive, require expertise and advanced laboratory systems, and are not optimal for field use. With the goal of providing a simple and efficient way to detect V. alginolyticus, we established a rapid diagnostic method based on loop-mediated Isothermal amplification (LAMP) technology that is feasible to use in both experimental and field environments. Three primer pairs targeting the toxR gene of V. alginolyticus were designed, and amplification was carried out in an ESE tube scanner and Real-Time PCR device. We successfully identified 93 V. alginolyticus strains from a total of 105 different bacterial isolates and confirmed their identity by 16s rDNA sequencing. We also applied this method on infected mouse blood and contaminated scallop samples, and accurate results were both easily and rapidly (20–60 min) obtained. Therefore, the RT-LAMP assay we developed can be conveniently used to detect the presence of V. alginolyticus in different samples. Furthermore, this method will also fulfill the gap for real-time screening of V. alginolyticus infections

  3. A rapid and enhanced DNA detection method for crop cultivar discrimination.

    PubMed

    Monden, Yuki; Takasaki, Kazuto; Futo, Satoshi; Niwa, Kousuke; Kawase, Mitsuo; Akitake, Hiroto; Tahara, Makoto

    2014-09-20

    In many crops species, the development of a rapid and precise cultivar discrimination system has been required for plant breeding and patent protection of plant cultivars and agricultural products. Here, we successfully evaluated strawberry cultivars via a novel method, namely, the single tag hybridization (STH) chromatographic printed array strip (PAS) using the PCR products of eight genomic regions. In a previous study, we showed that genotyping of eight genomic regions derived from FaRE1 retrotransposon insertion site enabled to discriminate 32 strawberry cultivars precisely, however, this method required agarose/acrylamide gel electrophoresis, thus has the difficulty for practical application. In contrast, novel DNA detection method in this study has some great advantages over standard DNA detection methods, including agarose/acrylamide gel electrophoresis, because it produces signals for DNA detection with dramatically higher sensitivity in a shorter time without any preparation or staining of a gel. Moreover, this method enables the visualization of multiplex signals simultaneously in a single reaction using several independent amplification products. We expect that this novel method will become a rapid and convenient cultivar screening assay for practical purposes, and will be widely applied to various situations, including laboratory research, and on-site inspection of plant cultivars and agricultural products. PMID:24954682

  4. Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples ▿

    PubMed Central

    Létant, Sonia E.; Murphy, Gloria A.; Alfaro, Teneile M.; Avila, Julie R.; Kane, Staci R.; Raber, Ellen; Bunt, Thomas M.; Shah, Sanjiv R.

    2011-01-01

    In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples. PMID:21764960

  5. Apparatus and method for rapid detection of explosives residue from the deflagration signature thereof

    DOEpatents

    Funsten, H.O.; McComas, D.J.

    1999-06-15

    Apparatus and method are disclosed for rapid detection of explosives residue from the deflagration signature thereof. A property inherent to most explosives is their stickiness, resulting in a strong tendency of explosive particulate to contaminate the environment of a bulk explosive. An apparatus for collection of residue particulate, burning the collected particulate, and measurement of the ultraviolet emission produced thereby, is described. The present invention can be utilized for real-time screening of personnel, cars, packages, suspected devices, etc., and provides an inexpensive, portable, and noninvasive means for detecting explosives. 4 figs.

  6. Apparatus and method for rapid detection of explosives residue from the deflagration signature thereof

    DOEpatents

    Funsten, Herbert O.; McComas, David J.

    1997-01-01

    Apparatus and method for rapid detection of explosives residue from the deflagration signature thereof. A property inherent to most explosives is their stickiness, resulting in a strong tendency of explosive particulate to contaminate the environment of a bulk explosive. An apparatus for collection of residue particulate, burning the collected particulate, and measurement of the optical emission produced thereby is described. The present invention can be utilized for real-time screening of personnel, cars, packages, suspected devices, etc., and provides an inexpensive, portable, and noninvasive means for detecting explosives.

  7. Apparatus and method for rapid detection of explosives residue from the deflagration signature thereof

    DOEpatents

    Funsten, Herbert O.; McComas, David J.

    1999-01-01

    Apparatus and method for rapid detection of explosives residue from the deflagration signature thereof. A property inherent to most explosives is their stickiness, resulting in a strong tendency of explosive particulate to contaminate the environment of a bulk explosive. An apparatus for collection of residue particulate, burning the collected particulate, and measurement of the ultraviolet emission produced thereby, is described. The present invention can be utilized for real-time screening of personnel, cars, packages, suspected devices, etc., and provides an inexpensive, portable, and noninvasive means for detecting explosives.

  8. Rapid Molecular Detection Methods for Arboviruses of Livestock of Importance to Northern Europe

    PubMed Central

    Johnson, Nicholas; Voller, Katja; Phipps, L. Paul; Mansfield, Karen; Fooks, Anthony R.

    2012-01-01

    Arthropod-borne viruses (arboviruses) have been responsible for some of the most explosive epidemics of emerging infectious diseases over the past decade. Their impact on both human and livestock populations has been dramatic. The early detection either through surveillance or diagnosis of virus will be a critical feature in responding and resolving the emergence of such epidemics in the future. Although some of the most important emerging arboviruses are human pathogens, this paper aims to highlight those diseases that primarily affect livestock, although many are zoonotic and some occasionally cause human mortality. This paper also highlights the molecular detection methods specific to each virus and identifies those emerging diseases for which a rapid detection methods are not yet developed. PMID:22219660

  9. 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

  10. Rapid method for glutathione quantitation using high-performance liquid chromatography with coulometric electrochemical detection.

    PubMed

    Bayram, Banu; Rimbach, Gerald; Frank, Jan; Esatbeyoglu, Tuba

    2014-01-15

    A rapid, sensitive, and direct method (without derivatization) was developed for the detection of reduced glutathione (GSH) in cultured hepatocytes (HepG2 cells) using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The method was validated according to the guidelines of the U.S. Food and Drug Administration in terms of linearity, lower limit of quantitation (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, and stabilities of GSH standards and quality control samples. The total analysis time was 5 min, and the retention time of GSH was 1.78 min. Separation was carried out isocratically using 50 mM sodium phosphate (pH 3.0) as a mobile phase with a fused-core column. The detector response was linear between 0.01 and 80 μmol/L, and the regression coefficient (R(2)) was >0.99. The LOD for GSH was 15 fmol, and the intra- and interday recoveries ranged between 100.7 and 104.6%. This method also enabled the rapid detection (in 4 min) of other compounds involved in GSH metabolism such as uric acid, ascorbic acid, and glutathione disulfite. The optimized and validated HPLC-ECD method was successfully applied for the determination of GSH levels in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor, and α-lipoic acid (α-LA), an inducer of GSH synthesis. As expected, the amount of GSH concentration-dependently decreased with BSO and increased with α-LA treatments in HepG2 cells. This method could also be useful for the quantitation of GSH, uric acid, ascorbic acid, and glutathione disulfide in other biological matrices such as tissue homogenates and blood. PMID:24328299

  11. Rapid, simple and efficient method for detection of viral genomes on raspberries.

    PubMed

    Perrin, A; Loutreul, J; Boudaud, N; Bertrand, I; Gantzer, C

    2015-11-01

    In recent years, foodborne viruses, especially human noroviruses (NoV) and hepatitis A virus (HAV), have been increasingly reported as the causes of foodborne disease outbreaks. Soft red fruits, especially raspberries, have a high incidence among the types of food concerned. Due to low infectious doses and low concentrations of enteric viruses in food samples, it is necessary to have an efficient and rapid detection method to implement prevention measures. A standard method for virus detection and quantification in food, including raspberries (XP CEN ISO/TS 15216-1 and -2, 2013) is currently available. This method proposes a consensus detection approach by RT-real time PCR (RT-qPCR) but also a virus extraction procedure based on the elution-concentration principle. In this study, an alternative method of extraction in which RNAs are directly extracted from food matrices (based on direct RNA extraction) has been optimized. First, each step was improved to make it a highly rapid, specific and simple method. Second, the standard virus concentration method was compared with the optimized direct RNA extraction one. Human enteric viral surrogates, Murine Norovirus (MNV) and F-specific RNA bacteriophage GA, were selected according to their adhesion properties and resistance to pH close to our main targets (NoV and HAV). Raspberries were artificially contaminated using two different techniques (immersion and spotting) in order to define a recovery rate and the amounts of virus recovered. Results showed that the direct RNA extraction method revealed significantly higher viral extraction efficiency (46.2%) than the elution-concentration method (20.3%), with similar proportions of inhibitors for both. In the same way with inoculation by spotting, the best recovery rate of GA phage (39.7% against 0.7%) and MNV (42.8% against 0.5%) was observed by direct RNA extraction. For the lowest concentrations of phage and virus in the immersion bath, only the direct RNA extraction method

  12. Novel method for rapid identification of Nocardia species by detection of preformed enzymes.

    PubMed Central

    Biehle, J R; Cavalieri, S J; Felland, T; Zimmer, B L

    1996-01-01

    The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The beta-naphthylamide-labeled substrate L-pyrrolidonyl-beta-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-alpha-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp. PMID:8748283

  13. Rapid methods to detect organic mercury and total selenium in biological samples

    PubMed Central

    2011-01-01

    Background Organic mercury (Hg) is a global pollutant of concern and selenium is believed to afford protection against mercury risk though few approaches exist to rapidly assess both chemicals in biological samples. Here, micro-scale and rapid methods to detect organic mercury (< 1.5 ml total sample volume, < 1.5 hour) and total selenium (Se; < 3.0 ml total volume, < 3 hour) from a range of biological samples (10-50 mg) are described. Results For organic Hg, samples are digested using Tris-HCl buffer (with sequential additions of protease, NaOH, cysteine, CuSO4, acidic NaBr) followed by extraction with toluene and Na2S2O3. The final product is analyzed via commercially available direct/total mercury analyzers. For Se, a fluorometric assay has been developed for microplate readers that involves digestion (HNO3-HClO4 and HCl), conjugation (2,3-diaminonaphthalene), and cyclohexane extraction. Recovery of organic Hg (86-107%) and Se (85-121%) were determined through use of Standard Reference Materials and lemon shark kidney tissues. Conclusions The approaches outlined provide an easy, rapid, reproducible, and cost-effective platform for monitoring organic Hg and total Se in biological samples. Owing to the importance of organic Hg and Se in the pathophysiology of Hg, integration of such methods into established research monitoring efforts (that largely focus on screening total Hg only) will help increase understanding of Hg's true risks. PMID:21232132

  14. Rapid staining method to detect and identify downy mildew (Peronospora belbahrii) in basil1

    PubMed Central

    Koroch, Adolfina R.; Villani, Thomas S.; Pyne, Robert M.; Simon, James E.

    2013-01-01

    • Premise of the study: Demand for fresh-market sweet basil continues to increase, but in 2009 a new pathogen emerged, threatening commercial field/greenhouse production and leading to high crop losses. This study describes a simple and effective staining method for rapid microscopic detection of basil downy mildew (Peronospora belbahrii) from leaves of basil (Ocimum basilicum). • Methods and Results: Fresh leaf sections infected with P. belbahrii were placed on a microscope slide, cleared with Visikol™, and stained with iodine solution followed by one drop of 70% sulfuric acid. Cell walls of the pathogen were stained with a distinct coloration, providing a high-contrast image between the pathogen and plant. • Conclusions: This new staining method can be used successfully to identify downy mildew in basil, which then can significantly reduce its spread if identified early, coupled with mitigation strategies. This technique can facilitate the control of the disease, without expensive and specialized equipment. PMID:25202569

  15. Simple, specific, sensitive and rapid loop-mediated method for detecting Yersinia enterocolitica.

    PubMed

    Xu, Yun-Ming; Liu, Xi-Lin; Ma, Jing; Li, Yan-Song; Hu, Pan; Zou, De-Ying; Guo, Xing; Chen, Xiao-Feng; Tang, Feng; Liu, Nan-Nan; Wei, Li-Bin; Zhou, Yu; Liu, Zeng-Shan; Ren, Hong-Lin; Lu, Shi-Ying

    2014-05-01

    Yersinia enterocolitica (YE) is a main pathogenic bacterium causing diarrhea and yersiniosis occurs in both developed and developing countries with high incidence. YE in contaminated food is able to survive for a long duration even under cold storage, thereby enhancing the risk of food infection. In this study, a new loop-mediated isothermal amplification (LAMP) method showing the characteristics of simplicity, rapidity, high specificity and sensitivity was established by targeting outL of pathogenic YE. Two inner-primers and outer-primers were designed and LAMP reaction was optimized for Mg2+, betaine, dNTPs and inner primers concentrations, reaction temperature and time. Sensitivity and specificity of the LAMP assay was evaluated using YE genomic DNA and those of 44 different bacteria strains, respectively. Validation of LAMP detection method was by employing meat samples spiked with varying CFU of YE. The optimized LAMP assay was specific, capable of detecting 97 fg of genomic DNA (equivalent to 37 genome copies) of YE (100-fold more sensitive than PCR) and 80 CFU/ml of YE-spiked meat samples based on ethidium bromide stained amplicon bands on agarose gel-electrophoresis and on GelRed fluorescence of the LAMP reaction solution, respectively. This rapid, sensitive and specific LAMP technique should enable application in field inspection of Y. enterocolitica in food. PMID:24974652

  16. Comparison of concentration methods for rapid detection of hookworm ova in wastewater matrices using quantitative PCR.

    PubMed

    Gyawali, P; Ahmed, W; Jagals, P; Sidhu, J P S; Toze, S

    2015-12-01

    Hookworm infection contributes around 700 million infections worldwide especially in developing nations due to increased use of wastewater for crop production. The effective recovery of hookworm ova from wastewater matrices is difficult due to their low concentrations and heterogeneous distribution. In this study, we compared the recovery rates of (i) four rapid hookworm ova concentration methods from municipal wastewater, and (ii) two concentration methods from sludge samples. Ancylostoma caninum ova were used as surrogate for human hookworm (Ancylostoma duodenale and Necator americanus). Known concentration of A. caninum hookworm ova were seeded into wastewater (treated and raw) and sludge samples collected from two wastewater treatment plants (WWTPs) in Brisbane and Perth, Australia. The A. caninum ova were concentrated from treated and raw wastewater samples using centrifugation (Method A), hollow fiber ultrafiltration (HFUF) (Method B), filtration (Method C) and flotation (Method D) methods. For sludge samples, flotation (Method E) and direct DNA extraction (Method F) methods were used. Among the four methods tested, filtration (Method C) method was able to recover higher concentrations of A. caninum ova consistently from treated wastewater (39-50%) and raw wastewater (7.1-12%) samples collected from both WWTPs. The remaining methods (Methods A, B and D) yielded variable recovery rate ranging from 0.2 to 40% for treated and raw wastewater samples. The recovery rates for sludge samples were poor (0.02-4.7), although, Method F (direct DNA extraction) provided 1-2 orders of magnitude higher recovery rate than Method E (flotation). Based on our results it can be concluded that the recovery rates of hookworm ova from wastewater matrices, especially sludge samples, can be poor and highly variable. Therefore, choice of concentration method is vital for the sensitive detection of hookworm ova in wastewater matrices. PMID:26358269

  17. A low complexity rapid molecular method for detection of Clostridium difficile in stool.

    PubMed

    McElgunn, Cathal J; Pereira, Clint R; Parham, Nicholas J; Smythe, James E; Wigglesworth, Michael J; Smielewska, Anna; Parmar, Surendra A; Gandelman, Olga A; Brown, Nicholas M; Tisi, Laurence C; Curran, Martin D

    2014-01-01

    Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB(+), n = 111; tcdB(-), n= 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB(+), n = 47; tcdB(-), n= 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB(+), n = 25; tcdB(-), n= 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings. PMID:24416173

  18. A Low Complexity Rapid Molecular Method for Detection of Clostridium difficile in Stool

    PubMed Central

    McElgunn, Cathal J.; Pereira, Clint R.; Parham, Nicholas J.; Smythe, James E.; Wigglesworth, Michael J.; Smielewska, Anna; Parmar, Surendra A.; Gandelman, Olga A.; Brown, Nicholas M.; Tisi, Laurence C.; Curran, Martin D.

    2014-01-01

    Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB+, n =  111; tcdB−, n  = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB+, n =  47; tcdB−, n  = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB+, n =  25; tcdB−, n  = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings. PMID:24416173

  19. Rapid and highly sensitive method for influenza A (H1N1) virus detection.

    PubMed

    Su, Li-Chen; Chang, Chung-Ming; Tseng, Ya-Ling; Chang, Ying-Feng; Li, Ying-Chang; Chang, Yu-Sun; Chou, Chien

    2012-05-01

    In this study, we applied the developed paired surface plasma waves biosensor (PSPWB) in a dual-channel biosensor for rapid and sensitive detection of swine-origin influenza A (H1N1) virus (S-OIV). In conjunction with the amplitude ratio of the signal and the reference channel, the stability of the PSPWB system is significantly improved experimentally. The theoretical limit of detection (LOD) of the dual-channel PSPWB for S-OIV is 30 PFU/mL (PFU, plaque-forming unit), which was calculated from the fitting curve of the surface plasmon resonance signal with a S-OIV clinical isolate concentration in phosphate-buffered saline (PBS) over a range of 18-1.8 × 10(6) PFU/mL. The LOD is 2 orders of magnitude more sensitive than the commercial rapid influenza diagnostic test at worst and an order of magnitude less sensitive than real-time quantitative polymerase chain reaction (PCR) whose LOD for S-OIV in PBS was determined to be 3.5 PFU/mL in this experiment. Furthermore, under in vivo conditions, this experiment demonstrates that the assay successfully measured S-OIV at a concentration of 1.8 × 10(2) PFU/mL in mimic solution, which contained PBS-diluted normal human nasal mucosa. Most importantly, the assay time took less than 20 min. From the results, the dual-channel PSPWB potentially offers great opportunity in developing an alternative PCR-free diagnostic method for rapid, sensitive, and accurate detection of viral pathogens with epidemiological relevance in clinical samples by using an appropriate pathogen-specific antibody. PMID:22401570

  20. Development of a rapid cyprinid herpesvirus 2 detection method by loop-mediated isothermal amplification.

    PubMed

    Liang, L-G; Xie, J; Luo, D

    2014-10-01

    Cyprinid herpesvirus 2 (CyHV2) is a pathogen that causes severe disease and high mortality in goldfish and Prussian carp. We developed a six primer loop-mediated isothermal amplification (LAMP) assay targeting the intercapsomeric triplex protein gene. CyHV-2 DNA was 10-fold serially diluted (10(8)-10(0) copies μl(-1)) and was used as the template to determine primer sensitivity. LAMP assays were performed with DNA templates from other pathogens to determine specificity. The LAMP assay had an unequivocal detection limit of 10 copies μl(-1), which was 100 times lower than that of the polymerase chain reaction. Other pathogen strains were not amplified by the LAMP primers, indicating good specificity. SYBR Green I was added to visually detect the amplification products. Assay applicability was evaluated in 120 samples of Carassius auratus gibelio, and a positive rate of 92·5% was obtained. In conclusion, a conventional LAMP assay has high convenience, rapidity, sensitivity and specificity for detecting CyHV-2 in infected aquatic organisms. Significance and impact of the study: Herpesviral haematopoietic necrosis, caused by cyprinid herpesvirus 2 (CyHV-2), is a severe disease of goldfish and Prussian carp associated with high mortality. We developed a loop-mediated isothermal amplification (LAMP) assay to detect CyHV-2 at relatively low plasmid DNA copy levels. The results show that the LAMP assay has a number of advantages (simple, sensitive, rapid and specific) over the conventional polymerase chain reaction and can be applied in the laboratory and field. Particularly, the method is highly applicable to facilitate surveillance and early diagnosis of CyHV-2. PMID:24935791

  1. Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia.

    PubMed

    Tan, Liping; Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Zheng, Guochao; Hu, Wei; Song, Meiran; Wang, Zhen; Jiang, Biao; Li, Guoqing

    2015-11-01

    Giardia lamblia is a zoonotic flagellate protozoan in the intestine of human and many mammals including dogs. To assess a threat of dog-derived G. lamblia to humans, the common dog-derived G. lamblia assemblages A, C, and D were genotyped by high-resolution melting (HRM) technology. According to β-giardin gene sequence, the qPCR-HRM primers BG5 and BG7 were designed. A series of experiments on the stability, sensitivity, and accuracy of the HRM method were also tested. Results showed that the primers BG5 and BG7 could distinguish among three assemblages A, C, and D, which Tm value differences were about 1 °C to each other. The melting curves of intra-assay reproducibility were almost coincided, and those of inter-assay reproducibility were much the same shape. The lowest detection concentration was about 5 × 10(-6)-ng/μL sample. The genotyping results from 21 G. lamblia samples by the HRM method were in complete accordance with sequencing results. It is concluded that the HRM genotyping method is rapid, stable, specific, highly sensitive, and suitable for clinical detection and molecular epidemiological survey of dog-derived G. lamblia. PMID:26212101

  2. A Rapid and Specific Method for the Detection of Indole in Complex Biological Samples

    PubMed Central

    Chappell, Cynthia; Gonzales, Christopher; Okhuysen, Pablo

    2015-01-01

    Indole, a bacterial product of tryptophan degradation, has a variety of important applications in the pharmaceutical industry and is a biomarker in biological and clinical specimens. Yet, specific assays to quantitate indole are complex and require expensive equipment and a high level of training. Thus, indole in biological samples is often estimated using the simple and rapid Kovács assay, which nonspecifically detects a variety of commonly occurring indole analogs. We demonstrate here a sensitive, specific, and rapid method for measuring indole in complex biological samples using a specific reaction between unsubstituted indole and hydroxylamine. We compared the hydroxylamine-based indole assay (HIA) to the Kovács assay and confirmed that the two assays are capable of detecting microgram amounts of indole. However, the HIA is specific to indole and does not detect other naturally occurring indole analogs. We further demonstrated the utility of the HIA in measuring indole levels in clinically relevant biological materials, such as fecal samples and bacterial cultures. Mean and median fecal indole concentrations from 53 healthy adults were 2.59 mM and 2.73 mM, respectively, but varied widely (0.30 mM to 6.64 mM) among individuals. We also determined that enterotoxigenic Escherichia coli strain H10407 produces 3.3 ± 0.22 mM indole during a 24-h period in the presence of 5 mM tryptophan. The sensitive and specific HIA should be of value in a variety of settings, such as the evaluation of various clinical samples and the study of indole-producing bacterial species in the gut microbiota. PMID:26386049

  3. Improved method for rapid detection of phthalates in bottled water by gas chromatography-mass spectrometry.

    PubMed

    Otero, Paz; Saha, Sushanta Kumar; Moane, Siobhan; Barron, John; Clancy, Gerard; Murray, Patrick

    2015-08-01

    An improved gas chromatography-mass spectrometry (GC-MS) method for simple, rapid and precise quantification of phthalates in drinking water is presented. This method was validated for bis (2-n-butoxyethyl) phthalate (DBEP), bis (2-n-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP), di-butyl phthalate (DBP), diethyl phthalate (DEP), dihexyl phthalate (DHP), dimethyl phthalate (DMP), di-n-octyl phthalate (DNOP) and dinonyl phthalate (DINP). Linearity of 0.9984>r(2)>0.9975 in the range of 0.075-4.8μg/mL for the selected phthalates was obtained. Accuracy values were in the range of 93-114% and RSD% for the analysis of 1.2μg/mL of each phthalate was below 2.3% (n=9). This new method design has significantly improved the detection in terms of rapidity, specificity, repeatability and accuracy compared to available methods. The procedure has been applied to the analyses of three different brands of commercially available bottled mineral water and the corresponding plastic bottles. Phthalates were extracted with dichloromethane and re-constituted in cyclohexane prior to GC-MS analysis. When the validated GC-MS method was applied to the quantification of the selected phthalates in the samples, only DBP (up to 0.0675±0.0018μg/mL) and DEHP (up to 1.6848±0.1631μg/mL) were found. Furthermore, we provide specific data about the concentration of DBP and DEHP in bottled water attributable to migration of phthalates from respective plastic bottles. PMID:26134297

  4. DNA fluorescence shift sensor: a rapid method for the detection of DNA hybridization using silver nanoclusters.

    PubMed

    Lee, Shin Yong; Hairul Bahara, Nur Hidayah; Choong, Yee Siew; Lim, Theam Soon; Tye, Gee Jun

    2014-11-01

    DNA-templated silver nanoclusters (AgNC) are a class of subnanometer sized fluorophores with good photostability and brightness. It has been applied as a diagnostic tool mainly for deoxyribonucleic acid (DNA) detection. Integration of DNA oligomers to generate AgNCs is interesting as varying DNA sequences can result in different fluorescence spectra. This allows a simple fluorescence shifting effect to occur upon DNA hybridization with the hybridization efficiency being a pronominal factor for successful shifting. The ability to shift the fluorescence spectra as a result of hybridization overcomes the issue of background intensities in most fluorescent based assays. Here we describe an optimized method for the detection of single-stranded and double-stranded synthetic forkhead box P3 (FOXP3) target by hybridization with the DNA fluorescence shift sensor. The system forms a three-way junction by successful hybridization of AgNC, G-rich strand (G-rich) to the target DNA, which generated a shift in fluorescence spectra with a marked increase in fluorescence intensity. The DNA fluorescence shift sensor presents a rapid and specific alternative to conventional DNA detection. PMID:25129336

  5. A Rapid Method to Achieve Aero-Engine Blade Form Detection

    PubMed Central

    Sun, Bin; Li, Bing

    2015-01-01

    This paper proposes a rapid method to detect aero-engine blade form, according to the characteristics of an aero-engine blade surface. This method first deduces an inclination error model in free-form surface measurements based on the non-contact laser triangulation principle. Then a four-coordinate measuring system was independently developed, a special fixture was designed according to the blade shape features, and a fast measurement of the blade features path was planned. Finally, by using the inclination error model for correction of acquired data, the measurement error that was caused by tilt form is compensated. As a result the measurement accuracy of the Laser Displacement Sensor was less than 10 μm. After the experimental verification, this method makes full use of optical non-contact measurement fast speed, high precision and wide measuring range of features. Using a standard gauge block as a measurement reference, the coordinate system conversion data is simple and practical. It not only improves the measurement accuracy of the blade surface, but also its measurement efficiency. Therefore, this method increases the value of the measurement of complex surfaces. PMID:26039420

  6. A rapid method to achieve aero-engine blade form detection.

    PubMed

    Sun, Bin; Li, Bing

    2015-01-01

    This paper proposes a rapid method to detect aero-engine blade form, according to the characteristics of an aero-engine blade surface. This method first deduces an inclination error model in free-form surface measurements based on the non-contact laser triangulation principle. Then a four-coordinate measuring system was independently developed, a special fixture was designed according to the blade shape features, and a fast measurement of the blade features path was planned. Finally, by using the inclination error model for correction of acquired data, the measurement error that was caused by tilt form is compensated. As a result the measurement accuracy of the Laser Displacement Sensor was less than 10 μm. After the experimental verification, this method makes full use of optical non-contact measurement fast speed, high precision and wide measuring range of features. Using a standard gauge block as a measurement reference, the coordinate system conversion data is simple and practical. It not only improves the measurement accuracy of the blade surface, but also its measurement efficiency. Therefore, this method increases the value of the measurement of complex surfaces. PMID:26039420

  7. Development of a rapid method for the detection of biological threats in water

    NASA Astrophysics Data System (ADS)

    Stratis-Cullum, Dimitra N.; Wade, Kellie L.; Pellegrino, Paul M.

    2005-11-01

    In this paper, progress towards the development of real-time sensing of chemical and biological threats in liquid samples will be presented. This overall goal of this work is to combine the selective, molecular recognition of nucleic acid aptamers with a rapid signal transduction using fluorescence resonance energy transfer (FRET) for a single step identify and detect approach. Of particular interest is the application to whole-cell target recognition of biologicals, such as environmental pathogens (e.g., Campylobacter jejuni), without requiring cell lysis or other complex protocols to access biochemical species internal to the organism. An aptamer staining protocol for whole cell targets is developed and applied to the investigation of aptamers against Campylobacter jejuni cells. A comparison of aptamer binding using this method with and without the primer regions utilized in the aptamer selection process is presented and the primer regions were found to have little impact on binding performance. C. jejuni aptamers exhibited strong binding as evidenced through the fluorescence images acquired and little to no background fluorescence was observed from non-specific binding of the streptavidin-dye conjugate used in the staining method. A thrombin targeted molecular aptamer beacon was also studied and a rapid analysis was demonstrated. A 10 nM sample of thrombin was distinguishable from the fluorescence baseline of the probe alone, when using a 40 nM aptamer probe concentration. The fluorescence intensity was found to increase until saturation of the aptamer probe was achieved. These results show promise for the development of single-step identification of whole-cell targets using an aptamer bioreceptor and fluorescence resonance energy transfer transduction signaling scheme.

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

    SciTech Connect

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

    1984-10-01

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

  9. Direct Detection of Nocardia spp. in Clinical Samples by a Rapid Molecular Method

    PubMed Central

    Couble, Andrée; Rodríguez-Nava, Verónica; de Montclos, Michèle Pérouse; Boiron, Patrick; Laurent, Frédéric

    2005-01-01

    We developed a 16S PCR-based assay for the rapid detection of Nocardia spp. directly from human clinical samples. The applicability of the assay was confirmed by using 18 samples from patients with nocardiosis as diagnosed by conventional cultures and 20 clinical samples from patients with confirmed tuberculosis used as negative controls. PMID:15815019

  10. Rapid method to detect duplex formation in sequencing by hybridization methods

    DOEpatents

    Mirzabekov, A.D.; Timofeev, E.N.; Florentiev, V.L.; Kirillov, E.V.

    1999-01-19

    A method for determining the existence of duplexes of oligonucleotide complementary molecules is provided. A plurality of immobilized oligonucleotide molecules, each of a specific length and each having a specific base sequence, is contacted with complementary, single stranded oligonucleotide molecules to form a duplex. Each duplex facilitates intercalation of a fluorescent dye between the base planes of the duplex. The invention also provides for a method for constructing oligonucleotide matrices comprising confining light sensitive fluid to a surface and exposing the light-sensitive fluid to a light pattern. This causes the fluid exposed to the light to coalesce into discrete units and adhere to the surface. This places each of the units in contact with a set of different oligonucleotide molecules so as to allow the molecules to disperse into the units. 13 figs.

  11. Rapid method to detect duplex formation in sequencing by hybridization methods

    DOEpatents

    Mirzabekov, Andrei Darievich; Timofeev, Edward Nikolaevich; Florentiev, Vladimer Leonidovich; Kirillov, Eugene Vladislavovich

    1999-01-01

    A method for determining the existence of duplexes of oligonucleotide complementary molecules is provided whereby a plurality of immobilized oligonucleotide molecules, each of a specific length and each having a specific base sequence, is contacted with complementary, single stranded oligonucleotide molecules to form a duplex so as to facilitate intercalation of a fluorescent dye between the base planes of the duplex. The invention also provides for a method for constructing oligonucleotide matrices comprising confining light sensitive fluid to a surface, exposing said light-sensitive fluid to a light pattern so as to cause the fluid exposed to the light to coalesce into discrete units and adhere to the surface; and contacting each of the units with a set of different oligonucleotide molecules so as to allow the molecules to disperse into the units.

  12. Rapid detection methods for Bacillus anthracis in environmental samples: a review.

    PubMed

    Irenge, Léonid M; Gala, Jean-Luc

    2012-02-01

    Bacillus anthracis is a Gram-positive, spore-forming bacterium, which causes anthrax, an often lethal disease of animals and humans. Although the disease has been well studied since the nineteenth century, it has witnessed a renewed interest during the past decade, due to its use as a bioterrorist agent in the fall of 2001 in the USA. A number of techniques aimed at rapidly detecting B. anthracis, in environmental samples as well as in point-of-care settings for humans suspected of exposure to the pathogen, are now available. These technologies range from culture-based methods to portable DNA amplification devices. Despite recent developments, specific identification of B. anthracis still remains difficult because of its phenotypic and genotypic similarities with other Bacillus species. Accordingly, many efforts are being made to improve the specificity of B. anthracis identification. This mini-review discusses the current challenges around B. anthracis identification, not only in reach-back laboratories but also in the field (in operational conditions). PMID:22262227

  13. A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    PubMed Central

    Nyan, Dougbeh-Chris; Swinson, Kevin L.

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  14. A rapid and label-free SERS detection method for biomarkers in clinical biofluids.

    PubMed

    Kong, Kien Voon; Leong, Weng Kee; Lam, Zhiyong; Gong, Tianxun; Goh, Douglas; Lau, Weber Kam On; Olivo, Malini

    2014-12-29

    A metal carbonyl-functionalized nanostructured substrate can be used in a rapid and simple assay for the detection of A1AT, a potential biomarker for bladder cancer, in clinical urine samples. The assay involves monitoring changes in the carbonyl stretching vibrations of the metal carbonyl via surface-enhanced Raman spectroscopy (SERS). These vibrations lie in the absorption spectral window of 1800-2200 cm(-1), which is free of any spectral interference from biomolecules. PMID:25111592

  15. Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

    PubMed

    Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

    2016-06-01

    Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings. PMID:27063012

  16. A rapid paper chromatographic method for detection of anionic detergent in milk.

    PubMed

    Barui, Amit K; Sharma, Rajan; Rajput, Yudhishthir S; Singh, Smita

    2013-08-01

    A paper chromatographic method for the detection of adulteration of anionic detergent in milk is described. The method is based on the complexing of anionic detergent with methylene blue dye and separation of complex from free dye using simple paper chromatographic method. Since complexing of detergent is with dye, visualization is direct without involvement of subsequent detection of complex. The method is simple and results are available in 10 min. The method is sensitive to detect 0.1 % (w/v) labolene (laboratory grade detergent) or 0.01 % (w/v) sodium dodecylbenzene sulfonate (pure anionic detergent) in milk. The method can be adopted at quality control laboratories in dairies for ascertaining the quality of milk. PMID:24425989

  17. Bench Test for the Detection of Bacterial Contamination in Platelet Concentrates Using Rapid and Cultural Detection Methods with a Standardized Proficiency Panel

    PubMed Central

    Vollmer, Tanja; Knabbe, Cornelius; Geilenkeuser, Wolf-Jochen; Schmidt, Michael; Dreier, Jens

    2015-01-01

    Summary Background The most frequent infectious complication in transfusion therapy in developed countries is related to the bacterial contamination of platelet concentrates (PCs). Rapid and cultural screening methods for bacterial detection in platelets are available, but external performance evaluation, especially of rapid methods, has been difficult to realize so far. Here we summarize the results of three individual collaborative trials using an external quality assessment program (EQAP) for the application of current rapid and cultural screening methods. Methods Three different modules were available for the detection of bacterial contamination: module 1: rapid methods, module 2: culture methods, module 3: bacterial identification methods. The sample set-up included up to six different bacterial strains, 1-2 negative samples and 4-6 positive samples with stabilized bacterial cell counts (approximately 103/104/105 CFU/ml). Time schedule for testing was limited (module 1: 6 h, module 2 and 3: 7 days). Results Samples of module 1 were analyzed with two different rapid methods (BactiFlow, NAT). The results of the three individual collaborative trials showed that all participants detected the negative samples with both assays correctly. Samples spiked with 104 to 105 CFU/ml of bacteria obtained positive results with both rapid screening methods, whereas samples spiked with only 103 CFU/ml disclosed a lower number of correctly identified positive results by NAT (86.6-93.8% sensitivity) compared to BactiFlow (100% sensitivity). The results for modules 2 and 3 revealed a 100% diagnostic sensitivity and specificity in all three collaborative trials. Conclusion This proficiency panel facilitates the verification of the analytical sensitivity of rapid and cultural bacterial detection systems under controlled routine conditions. The concept of samples provided in this EQAP has three main advantages: i) samples can be examined by both rapid and culture methods, ii) the

  18. Rapid concentration and sensitive detection of hookworm ova from wastewater matrices using a real-time PCR method.

    PubMed

    Gyawali, P; Sidhu, J P S; Ahmed, W; Jagals, P; Toze, S

    2015-12-01

    The risk of human hookworm infections from land application of wastewater matrices could be high in regions with high hookworm prevalence. A rapid, sensitive and specific hookworm detection method from wastewater matrices is required in order to assess human health risks. Currently available methods used to identify hookworm ova to the species level are time consuming and lack accuracy. In this study, a real-time PCR method was developed for the rapid, sensitive and specific detection of canine hookworm (Ancylostoma caninum) ova from wastewater matrices. A. caninum was chosen because of its morphological similarity to the human hookworm (Ancylostoma duodenale and Necator americanus). The newly developed PCR method has high detection sensitivity with the ability to detect less than one A. caninum ova from 1 L of secondary treated wastewater at the mean threshold cycle (CT) values ranging from 30.1 to 34.3. The method is also able to detect four A. caninum ova from 1 L of raw wastewater and from ∼4 g of treated sludge with mean CT values ranging from 35.6 to 39.8 and 39.8 to 39.9, respectively. The better detection sensitivity obtained for secondary treated wastewater compared to raw wastewater and sludge samples could be attributed to sample turbidity. The proposed method appears to be rapid, sensitive and specific compared to traditional methods and has potential to aid in the public health risk assessment associated with land application of wastewater matrices. Furthermore, the method can be adapted to detect other helminth ova of interest from wastewater matrices. PMID:26297680

  19. Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

    PubMed Central

    Njiru, Zablon Kithinji; Mikosza, Andrew Stanislaw John; Armstrong, Tanya; Enyaru, John Charles; Ndung'u, Joseph Mathu; Thompson, Andrew Richard Christopher

    2008-01-01

    Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions. PMID:18253475

  20. Rapid detection method for fusaric acid-producing species of Fusarium by PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusaric acid is a mycotoxin produced by species of the fungus Fusarium and can act synergistically with other Fusarium toxins. In order to develop a specific detection method for fusaric acid-producing fungus, PCR prim¬ers were designed to amplify FUB10, a transcription factor gene in fusaric acid ...

  1. A rapid and efficient newly established method to detect COL1A1-PDGFB gene fusion in dermatofibrosarcoma protuberans

    SciTech Connect

    Yokoyama, Yoko; Shimizu, Akira; Okada, Etsuko; Ishikawa, Osamu; Motegi, Sei-ichiro

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer We developed new method to rapidly identify COL1A1-PDGFB fusion in DFSP. Black-Right-Pointing-Pointer New PCR method using a single primer pair detected COL1A1-PDGFB fusion in DFSP. Black-Right-Pointing-Pointer This is the first report of DFSP with a novel COL1A1 breakpoint in exon 5. -- Abstract: The detection of fusion transcripts of the collagen type 1{alpha}1 (COL1A1) and platelet-derived growth factor-BB (PDGFB) genes by genetic analysis has recognized as a reliable and valuable molecular tool for the diagnosis of dermatofibrosarcoma protuberans (DFSP). To detect the COL1A1-PDGFB fusion, almost previous reports performed reverse transcription polymerase chain reaction (RT-PCR) using multiplex forward primers from COL1A1. However, it has possible technical difficulties with respect to the handling of multiple primers and reagents in the procedure. The objective of this study is to establish a rapid, easy, and efficient one-step method of PCR using only a single primer pair to detect the fusion transcripts of the COL1A1 and PDGFB in DFSP. To validate new method, we compared the results of RT-PCR in five patients of DFSP between the previous method using multiplex primers and our established one-step RT-PCR using a single primer pair. In all cases of DFSP, the COL1A1-PDGFB fusion was detected by both previous method and newly established one-step PCR. Importantly, we detected a novel COL1A1 breakpoint in exon 5. The newly developed method is valuable to rapidly identify COL1A1-PDGFB fusion transcripts in DFSP.

  2. Colorimetric method for rapid detection of Oxacillin resistance in Staphylococcus aureus and its comparison with PCR for mec A gene

    PubMed Central

    Ghanwate, Niraj; Thakare, Prashant; Bhise, P. R.; Gawande, Sonali

    2016-01-01

    Rapid and accurate detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important role of clinical microbiology laboratories to avoid treatment failure. The detection of MRSA is based on phenotypic assays which require at least 24 h to perform. Detection of the mecA gene or of PBP 2a is the “gold standard”, but not always available. The aim of this study was to evaluate a rapid method for detection of MRSA by using 3 (4, 5 dimethyl thiazole -2-yl) -2, 5 diphenyl tetrazolium bromide (MTT). Total 126 isolates of MRSA were collected from tertiary healthcare center and were confirmed by oxacillin screening agar test as per CLSI guidelines. Amplification of mecA gene was performed by using PCR. MTT assay was carried out for all the isolates in 96 well Microtitre plate and compared with standard methods of CLSI. Out of 126 isolates, 98 were found to be mecA positive. MTT method was found to be 98.98% sensitive and 96.43% specific. The MTT based colorimetric method is rapid and simple test for screening of oxacillin resistance in Staphylococcus aureus. It significantly shortens the time to just 7 h required to obtained a drug susceptibility test and could be useful to screen MRSA. PMID:26960268

  3. Colorimetric method for rapid detection of Oxacillin resistance in Staphylococcus aureus and its comparison with PCR for mec A gene.

    PubMed

    Ghanwate, Niraj; Thakare, Prashant; Bhise, P R; Gawande, Sonali

    2016-01-01

    Rapid and accurate detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important role of clinical microbiology laboratories to avoid treatment failure. The detection of MRSA is based on phenotypic assays which require at least 24 h to perform. Detection of the mecA gene or of PBP 2a is the "gold standard", but not always available. The aim of this study was to evaluate a rapid method for detection of MRSA by using 3 (4, 5 dimethyl thiazole -2-yl) -2, 5 diphenyl tetrazolium bromide (MTT). Total 126 isolates of MRSA were collected from tertiary healthcare center and were confirmed by oxacillin screening agar test as per CLSI guidelines. Amplification of mecA gene was performed by using PCR. MTT assay was carried out for all the isolates in 96 well Microtitre plate and compared with standard methods of CLSI. Out of 126 isolates, 98 were found to be mecA positive. MTT method was found to be 98.98% sensitive and 96.43% specific. The MTT based colorimetric method is rapid and simple test for screening of oxacillin resistance in Staphylococcus aureus. It significantly shortens the time to just 7 h required to obtained a drug susceptibility test and could be useful to screen MRSA. PMID:26960268

  4. Application of Molecular and Serological Methods for Rapid Detection of Mycoplasma gallisepticum Infection (Avian mycoplasmosis).

    PubMed

    Qasem, Jafar A; Al-Mouqati, Salwa A; Al-Ali, Ebtesam M; Ben-Haji, Ahmad

    2015-02-01

    Mycoplasma infection is a major problem in veterinary medicine and in poultry production. The pathogen has many strains, so that diagnosis of the disease using culture method is not effective. The objective of this work was to evaluate the prevalence of Mycoplasma gallisepticum (MG) in Kuwait poultry farms using serology and molecular methods in comparison to the culture under specific conditions. A total of 50 swab samples from choanal cleft and tracheal samples and blood samples were obtained from three different local farms, the blood samples were processed for an Enzyme Linked Immunosorbent Assay (ELISA) detection and the swab samples for Polymerase Chain Reaction (PCR) and culture methods detection. A PCR diagnostic kit (VenoMGs) and ELISA diagnostic kit (ProFLOK), were used in comparison to the traditional culture method, to study the spread of this disease in samples from broiler and layer flocks. Fifty chicken samples were tested for mycoplasmosis, samples tested with ELISA gave 24 positive (48%) and 29 were positive by PCR (58%) and only seven (14%) were positive with culture methods. Swab samples obtained from the choanal cleft gave more positive (60%) with PCR than tracheal samples (56.6%). The culture gave 20 and 5% positive, respectively for tracheal and choanal samples. The methods reported here are of high sensitivity and specificity for Mycoplasma. Both the PCR and ELISA methods are superior to culture method for detection of avian mycoplasmosis. This study showed that MG infection is prevalent in commercial broiler and layer chickens in Kuwait poultry farms. The use of these methods for surveillance of the disease will establish data concerning the predominant Mycoplasmosis diseases in Kuwait if done on a large scale. PMID:26364358

  5. Microplate-reader method for the rapid analysis of copper in natural waters with chemiluminescence detection

    PubMed Central

    Durand, Axel; Chase, Zanna; Remenyi, Tomas; Quéroué, Fabien

    2013-01-01

    We have developed a method for the determination of copper in natural waters at nanomolar levels. The use of a microplate-reader minimizes sample processing time (~25 s per sample), reagent consumption (~120 μL per sample), and sample volume (~700 μL). Copper is detected by chemiluminescence. This technique is based on the formation of a complex between copper and 1,10-phenanthroline and the subsequent emission of light during the oxidation of the complex by hydrogen peroxide. Samples are acidified to pH 1.7 and then introduced directly into a 24-well plate. Reagents are added during data acquisition via two reagent injectors. When trace metal clean protocols are employed, the reproducibility is generally less than 7% on blanks and the detection limit is 0.7 nM for seawater and 0.4 nM for freshwater. More than 100 samples per hour can be analyzed with this technique, which is simple, robust, and amenable to at-sea analysis. Seawater samples from Storm Bay in Tasmania illustrate the utility of the method for environmental science. Indeed other trace metals for which optical detection methods exist (e.g., chemiluminescence, fluorescence, and absorbance) could be adapted to the microplate-reader. PMID:23335917

  6. A rapid method for detecting specific amplified PCR fragments in microtiter plates.

    PubMed Central

    Ortiz, A; Ritter, E

    1996-01-01

    A simple method is presented to circumvent laborious and time consuming electrophoretic separations of specific PCR amplification products. Specific target DNA is amplified using nucleotides labelled with DIG-dUTP or biotin-dCTP. The labelled PCR products are separated from unincorporated nucleotides or oligonucleotides by using a positively charged DEAE cellulose matrix. Amplification products are visualized directly in the matrix using immunoenzymatic methods or streptavidin-conjugated enzymes. The detection process can be carried out within 2 h, allows the processing of large sample sizes and can potentially be automated. PMID:8774915

  7. Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus.

    PubMed

    Tang, Qing; Tian, Shuguang; Yu, Nong; Zhang, Xi; Jia, Xiaodong; Zhai, Hongyan; Sun, Qun; Han, Li

    2016-04-01

    Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10(2)copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed. PMID:26791368

  8. Rapid detection of bacteria in green tea using a novel pretreatment method in a bioluminescence assay.

    PubMed

    Shinozaki, Yohei; Harada, Yasuhiro

    2014-06-01

    Tea is one of the most popular beverages consumed in the world, and green tea has become a popular beverage in Western as well as Asian countries. A novel pretreatment method for a commercial bioluminescence assay to detect bacteria in green tea was developed and evaluated in this study. Pretreatment buffers with pH levels ranging from 6.0 to 9.0 were selected from MES (morpholineethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), or Tricine buffers. To evaluate the effect of pretreatment and the performance of the assay, serially diluted cultures of Enterobacter cloacae, Escherichia coli, Bacillus subtilis, and Staphylococcus aureus were tested. The improved methods, which consisted of a pretreatment of the sample in alkaline buffer, significantly decreased the background bioluminescence intensity of green tea samples when compared with the conventional method. Pretreatment with alkaline buffers with pH levels ranging from 8.0 to 9.0 increased the bioluminescence intensities of cultures of E. cloacae and S. aureus. Strong log-linear relationships between the bioluminescence intensities and plate counts emerged for the tested strains. Furthermore, the microbial detection limit was 15 CFU in 500 ml of bottled green tea after an 8-h incubation at 35°C and an assay time of 1 h. The results showed that contaminated samples could be detected within 1 h of operation using our improved bioluminescence assay. This method could be used to test for contamination during the manufacturing process as well as for statistical sampling for quality control. PMID:24853516

  9. Safety issues and new rapid detection methods in traditional Chinese medicinal materials

    PubMed Central

    Wang, Lili; Kong, Weijun; Yang, Meihua; Han, Jianping; Chen, Shilin

    2015-01-01

    The safety of traditional Chinese medicine (TCM) is a major strategic issue that involves human health. With the continuous improvement in disease prevention and treatment, the export of TCM and its related products has increased dramatically in China. However, the frequent safety issues of Chinese medicine have become the ‘bottleneck’ impeding the modernization of TCM. It was proved that mycotoxins seriously affect TCM safety; the pesticide residues of TCM are a key problem in TCM international trade; adulterants have also been detected, which is related to market circulation. These three factors have greatly affected TCM safety. In this study, fast, highly effective, economically-feasible and accurate detection methods concerning TCM safety issues were reviewed, especially on the authenticity, mycotoxins and pesticide residues of medicinal materials. PMID:26579423

  10. A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

    SciTech Connect

    Crissman, H.A.; Steinkamp, J.A. )

    1987-11-01

    A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G{sub 1} and G{sub 2}{plus}M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.

  11. A simple and rapid resonance Rayleigh scattering method for detection of indigo carmine in soft drink.

    PubMed

    Li, Qin; Yang, Jidong; Tan, Xuanping; Zhang, Zhan; Hu, Xiaomei; Yang, Menghuan

    2016-08-01

    A novel method that uses acridine orange (AO) to detect indigo carmine (IC) in soft drinks was developed. The method is highly sensitive and is based on a resonance Rayleigh scattering (RRS) technique. In Britton-Robinson (BR) buffer solution, pH 4.3, the weak RRS intensity of AO was greatly enhanced by the addition of IC, with the maximum peak located at 332 nm. Under optimum conditions, it was found that the enhanced RRS intensity was proportional to the concentration of IC over a range of 2-32 × 10(-6)  mol/L. A low detection limit of 2.4 × 10(-8)  mol/L was achieved. The sensitivity and selectivity of the method are high enough to permit the determination of trace amounts of IC without any significant interference from high levels of other components such as common anions and other amino acids. Finally, the concentration of IC in three different soft drinks was determined with satisfactory results. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26791156

  12. Most Probable Number Rapid Viability PCR Method to Detect Viable Spores of Bacillus anthracis in Swab Samples

    SciTech Connect

    Letant, S E; Kane, S R; Murphy, G A; Alfaro, T M; Hodges, L; Rose, L; Raber, E

    2008-05-30

    This note presents a comparison of Most-Probable-Number Rapid Viability (MPN-RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs generated by the Centers for Disease Control and Prevention (CDC) for a multi-center validation study aimed at testing environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were in statistical agreement with the CDC conventional culture method for all three levels of spores tested (10{sup 4}, 10{sup 2}, and 10 spores) even in the presence of dirt. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols.

  13. A rapid detection method of Escherichia coli by surface enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Tao, Feifei; Peng, Yankun; Xu, Tianfeng

    2015-05-01

    Conventional microbiological detection and enumeration methods are time-consuming, labor-intensive, and giving retrospective information. The objectives of the present work are to study the capability of surface enhanced Raman scattering (SERS) to detect Escherichia coli (E. coli) using the presented silver colloidal substrate. The obtained results showed that the adaptive iteratively reweighed Penalized Least Squares (airPLS) algorithm could effectively remove the fluorescent background from original Raman spectra, and Raman characteristic peaks of 558, 682, 726, 1128, 1210 and 1328 cm-1 could be observed stably in the baseline corrected SERS spectra of all studied bacterial concentrations. The detection limit of SERS could be determined to be as low as 0.73 log CFU/ml for E. coli with the prepared silver colloidal substrate. The quantitative prediction results using the intensity values of characteristic peaks were not good, with the correlation coefficients of calibration set and cross validation set of 0.99 and 0.64, respectively.

  14. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification.

    PubMed

    Kikuchi, Taisei; Aikawa, Takuya; Oeda, Yuka; Karim, Nurul; Kanzaki, Natsumi

    2009-12-01

    ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions. PMID:19900002

  15. MLPA: A Rapid, Reliable, and Sensitive Method for Detection and Analysis of Abnormalities of 22q

    PubMed Central

    Vorstman, J.A.S.; Jalali, G.R.; Rappaport, E.F.; Hacker, A.M.; Scott, C.; Emanuel, B.S.

    2010-01-01

    In this study, essential test characteristics of the recently described multiplex ligation-dependent probe amplification (MLPA) method are presented, using chromosome 22 as a model. This novel method allows the relative quantification of ~40–45 different target DNA sequences in a single reaction. For the purpose of this study, MLPA was performed in a blinded manner on a training set containing over 50 samples, including typical 22q11.2 deletions, various atypical deletions, duplications (trisomy and tetrasomy), and unbalanced translocations. All samples in the training set have been previously characterized by fluorescence in situ hybridization (FISH) with cosmid or BAC clones and/or cytogenetic studies. MLPA findings were consistent with cytogenetic and FISH studies, no rearrangement went undetected and repeated tests gave consistent results. At a relative change in comparative signal strength of 30% or more, sensitivity and specificity values were 0.95 and 0.99, respectively. Given that MLPA is likely to be used as an initial screening method, a higher sensitivity, at the cost of a lower specificity, was deemed more appropriate. A receiver operator characteristic (ROC) curve analysis was performed to calculate the most optimal threshold range, with associated sensitivity and specificity values of 0.99 and 0.97, respectively. Finally, performance of each individual probe was analyzed, providing further useful information for the interpretation of MLPA results. In conclusion, MLPA has proven to be a highly sensitive and accurate tool for detecting copy number changes in the 22q11.2 region, making it a fast and economic alternative to currently used methods. The current study provides valuable and detailed information on the characteristics of this novel method. PMID:16791841

  16. Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B.

    PubMed

    Cao, Cuiming; Zhang, Fang; Ji, Mingyu; Pei, Fengyan; Fan, Xiujie; Shen, Hong; Wang, Qingxi; Yang, Weihua; Wang, Yunshan

    2016-07-01

    We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 °C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng/μl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng/μl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries. PMID:27045360

  17. Rapid method to detect duplex formation in sequencing by hybridization methods, a method for constructing containment structures for reagent interaction

    DOEpatents

    Mirzabekov, Andrei Darievich; Yershov, Gennadiy Moiseyevich; Guschin, Dmitry Yuryevich; Gemmell, Margaret Anne; Shick, Valentine V.; Proudnikov, Dmitri Y.; Timofeev, Edward N.

    2002-01-01

    A method for determining the existence of duplexes of oligonucleotide complementary molecules is provided whereby a plurality of immobilized oligonucleotide molecules, each of a specific length and each having a specific base sequence, is contacted with complementary, single stranded oligonucleotide molecules to form a duplex so as to facilitate intercalation of a fluorescent dye between the base planes of the duplex. The invention also provides for a method for constructing oligonucleotide matrices comprising confining light sensitive fluid to a surface, exposing said light-sensitive fluid to a light pattern so as to cause the fluid exposed to the light to polymerize into discrete units and adhere to the surface; and contacting each of the units with a set of different oligonucleotide molecules so as to allow the molecules to disperse into the units.

  18. A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.

    PubMed

    Balakrishnan, Baskar; Barizuddin, Syed; Wuliji, Tumen; El-Dweik, Majed

    2016-08-16

    Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform. PMID:27209618

  19. PLIF: A rapid, accurate method to detect and quantitatively assess protein-lipid interactions.

    PubMed

    Ceccato, Laurie; Chicanne, Gaëtan; Nahoum, Virginie; Pons, Véronique; Payrastre, Bernard; Gaits-Iacovoni, Frédérique; Viaud, Julien

    2016-01-01

    Phosphoinositides are a type of cellular phospholipid that regulate signaling in a wide range of cellular and physiological processes through the interaction between their phosphorylated inositol head group and specific domains in various cytosolic proteins. These lipids also influence the activity of transmembrane proteins. Aberrant phosphoinositide signaling is associated with numerous diseases, including cancer, obesity, and diabetes. Thus, identifying phosphoinositide-binding partners and the aspects that define their specificity can direct drug development. However, current methods are costly, time-consuming, or technically challenging and inaccessible to many laboratories. We developed a method called PLIF (for "protein-lipid interaction by fluorescence") that uses fluorescently labeled liposomes and tethered, tagged proteins or peptides to enable fast and reliable determination of protein domain specificity for given phosphoinositides in a membrane environment. We validated PLIF against previously known phosphoinositide-binding partners for various proteins and obtained relative affinity profiles. Moreover, PLIF analysis of the sorting nexin (SNX) family revealed not only that SNXs bound most strongly to phosphatidylinositol 3-phosphate (PtdIns3P or PI3P), which is known from analysis with other methods, but also that they interacted with other phosphoinositides, which had not previously been detected using other techniques. Different phosphoinositide partners, even those with relatively weak binding affinity, could account for the diverse functions of SNXs in vesicular trafficking and protein sorting. Because PLIF is sensitive, semiquantitative, and performed in a high-throughput manner, it may be used to screen for highly specific protein-lipid interaction inhibitors. PMID:27025878

  20. Development of a loop-mediated isothermal amplification method for rapid detection of porcine boca-like virus.

    PubMed

    Li, Bin; Ma, Jun-jie; Xiao, Shao-bo; Zhang, Xue-han; Wen, Li-bin; Mao, Li; Ni, Yan-xiu; Guo, Rong-li; Zhou, Jun-ming; Lv, Li-xin; He, Kong-wang

    2012-02-01

    The porcine boca-like virus (Pbo-likeV) was recently discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS). In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Pbo-likeV. A set of four primers specific for six regions of Pbo-likeV VP1/2 genes was designed with the online software. The reaction temperature and time were optimized to 65 °C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of Pbo-likeV, and no cross-reaction was observed with other swine viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and classic swine fever virus (CSFV) found commonly in China. The lower detection limit of the LAMP assay was approximately 10 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. Furthermore, the efficiency of LAMP for detection Pbo-likeV in clinical samples was comparable to PCR and sequencing. These results showed that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Pbo-likeV, and the procedure of LAMP does not rely on any special equipment. It has capacity for the detection of Pbo-likeV both in the laboratory and on farms. PMID:22172971

  1. Comparing Rapid and Specific Detection of Brucella in Clinical Samples by PCR-ELISA and Multiplex-PCR Method

    PubMed Central

    Mohammad Hasani, Sharareh; Mirnejad, Reza; Amani, Jafar; Vafadar, Mohamad javad

    2016-01-01

    Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value <0.05. Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis. PMID:27499776

  2. Physical and chemical methods for enhancing rapid detection of viruses and other agents.

    PubMed Central

    Hughes, J H

    1993-01-01

    Viral replication events can be enhanced by physical, chemical, or heat treatment of cells. The centrifugation of cells can stimulate them to proliferate, reduce their generation times, and activate gene expression. Human endothelial cells can be activated to release cyclo-oxygenase metabolites after rocking for 5 min, and mechanical stress can stimulate endothelial cells to proliferate. Centrifugation of virus-infected cultures can increase cytopathic effects (CPE), enhance the number of infected cells, increase viral yields, and reduce viral detection times and may increase viral isolation rates. The rolling of virus-infected cells also has an effect similar to that of centrifugation. The continuous rolling of virus-infected cultures at < or = 2.0 rpm can enhance enterovirus, rhinovirus, reovirus, rotavirus, paramyxovirus, herpesvirus, and vaccinia virus CPE or yields or both. For some viruses, the continuous rolling of infected cell cultures at 96 rpm (1.9 x g) is superior to rolling at 2.0 rpm for viral replication or CPE production. In addition to centrifugation and rolling, the treatment of cells with chemicals or heat can also enhance viral yields or CPE. For example, the treatment of virus-infected cells with dimethyl sulfoxide can enhance viral transformation, increase plaque numbers and plaque size, increase the number of cells producing antigens, and increase viral yields. The infectivity of fowl plague virus is increased by 80-fold when 4% dimethyl sulfoxide is added to culture medium immediately after infection. The heat shocking of virus-infected cells also has been shown to have a stimulatory effect on the replication events of cytomegalovirus, Epstein-Barr virus, and human immunodeficiency virus. The effects of motion, chemicals, or heat treatments on viral replication are not well understood. These treatments apparently activate cells to make them more permissive to viral infection and viral replication. Perhaps heat shock proteins or stress

  3. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening. PMID:24188249

  4. Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions.

    PubMed Central

    Doskeland, S O; Berdal, B P

    1980-01-01

    We sought procedures which would allow a rapid concentration in high yield of bacterial antigens from tissue fluids of patients and which could be applied also to protein-rich fluids like serum. Ethanol precipitation at a subzero temperature with albumin added as an antigen coprecipitant made it possible to achieve a more than 20-fold concentration of antigen in 15 min and a 200-fold concentration in 45 min. Heat-stable antigens could be concentrated from protein-rich fluids (like serum) after the sample had been deproteinized by boiling. Such heating (100 degrees C, 3 min) also liberated bacterial polysaccharides from antibody complexes and elminated the nonspecific interference of serum in enzyme-linked immunosorbent assay. PMID:7372801

  5. Methods for automatized detection of rapid changes in lateral boundary condition fields for NWP limited area models

    NASA Astrophysics Data System (ADS)

    Tudor, M.

    2015-08-01

    Three-hourly temporal resolution of lateral boundary data for limited area models (LAMs) can be too infrequent to resolve rapidly moving storms. This problem is expected to be worse with increasing horizontal resolution. In order to detect intensive disturbances in surface pressure moving rapidly through the model domain, a filtered surface pressure field (MCUF) is computed operationally in the ARPEGE global model of Météo France. The field is distributed in the coupling files along with conventional meteorological fields used for lateral boundary conditions (LBCs) for the operational forecast using limited area model ALADIN (Aire Limitée Adaptation dynamique Développement InterNational) in the Meteorological and Hydrological Service of Croatia (DHMZ). Here an analysis is performed of the MCUF field for the LACE coupling domain for the period from 23 January 2006, when it became available, until 15 November 2014. The MCUF field is a good indicator of rapidly moving pressure disturbances (RMPDs). Its spatial and temporal distribution can be associated with the usual cyclone tracks and areas known to be supporting cyclogenesis. An alternative set of coupling files from the IFS operational run in the European Centre for Medium-Range Weather Forecasts (ECMWF) is also available operationally in DHMZ with 3-hourly temporal resolution, but the MCUF field is not available. Here, several methods are tested that detect RMPDs in surface pressure a posteriori from the IFS model fields provided in the coupling files. MCUF is computed by running ALADIN on the coupling files from IFS. The error function is computed using one-time-step integration of ALADIN on the coupling files without initialization, initialized with digital filter initialization (DFI) or scale-selective DFI (SSDFI). Finally, the amplitude of changes in the mean sea level pressure is computed from the fields in the coupling files. The results are compared to the MCUF field of ARPEGE and the results of same

  6. A new spectroscopy method for in-situ rapid detection and classification of microorganisms.

    SciTech Connect

    Garcia-Rubio, L. H.; Alupoaei, C. E.; Olivares, J. A.; Stark, P. C.; Garcia-Lopez, A.; Stephans, C.; Berg, T.; Klungness, G.; Gennaccaro, A.; Huffman, D. E.

    2004-01-01

    Recent developments in the characterization of particle dispersions have demonstrated that complementary information on the joint particle property distribution (size-shape-chemical composition) of micron and sub-micron particles is available from multiwavelength spectrophotometric measurements. The UV-VIS transmission spectra of the microorganism suspensions reported herein were recorded using a Hewlett-Packard 8453 diode array spectrometer with an acceptance angle smaller than 2 degrees. To eliminate concentration and particle number effects, the transmission spectra were normalized with the average optical density between 230-900 nm. Experimental results demonstrate that microorganisms at various states of growth give rise to spectral differences that can be used for their identification and classification and that this technology can be used for the characterization of the joint particle property distribution for a large variety of continuous, on-line, and in-situ particle characterization applications. An interpretation model has been developed for the quantitative interpretation of spectral patterns resulting from transmission measurements of microorganism suspensions. The interpretation model is based on light scattering theory and spectral deconvolution techniques and yields the quantitative information necessary to define the probability of the detection and identification of microorganisms. A data base of 54 pathogens has been created and demonstrates that the technology can be used in the field for real-time in-situ monitoring applications.

  7. A new rapid method for Clostridium difficile DNA extraction and detection in stool: toward point-of-care diagnostic testing.

    PubMed

    Freifeld, Alison G; Simonsen, Kari A; Booth, Christine S; Zhao, Xing; Whitney, Scott E; Karre, Teresa; Iwen, Peter C; Viljoen, Hendrik J

    2012-01-01

    We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH(+)/toxin(+), n = 48; GDH(-)/toxin(-), n = 81). All 69 stool samples with discordant EIA results (GDH(+)/toxin(-)) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases. PMID:22402170

  8. Heat shock proteins as biomarkers for the rapid detection of brain and spinal cord ischemia: a review and comparison to other methods of detection in thoracic aneurysm repair

    PubMed Central

    McGarvey, Michael

    2010-01-01

    The heat shock proteins (HSPs) are members of highly conserved families of molecular chaperones that have multiple roles in vivo. We discuss the HSPs in general, and Hsp70 and Hsp27 in particular, and their rapid induction by severe stress in the context of tissue and organ expression in physiology and disease. We describe the current state of knowledge of the relationship and interactions between extra- and intracellular HSPs and describe mechanisms and significance of extracellular expression of HSPs. We focus on the role of the heat shock proteins as biomarkers of central nervous system (CNS) ischemia and other severe stressors and discuss recent and novel technologies for rapid measurement of proteins in vivo and ex vivo. The HSPs are compared to other proposed small molecule biomarkers for detection of CNS injury and to other methods of detecting brain and spinal cord ischemia in real time. While other biomarkers may be of use in prognosis and in design of appropriate therapies, none appears to be as rapid as the HSPs; therefore, no other measurement appears to be of use in the immediate detection of ongoing severe ischemia with the intention to immediately intervene to reduce the severity or risk of permanent damage. PMID:20803353

  9. Rapid method for the determination of 14 isoflavones in food using UHPLC coupled to photo diode array detection.

    PubMed

    Shim, You-Shin; Yoon, Won-Jin; Hwang, Jin-Bong; Park, Hyun-Jin; Seo, Dongwon; Ha, Jaeho

    2015-11-15

    A rapid method for the determination of 14 types of isoflavones in food using ultra-high performance liquid chromatography (UHPLC) was validated in terms of precision, accuracy, sensitivity and linearity. The UHPLC separation was performed on a reverse-phase C18 column (particle size 2 μm, i.d. 2 mm, length 100 mm) using a photo diode array detector that was fixed to 260 nm. The limits of detection and quantification of the UHPLC analyses ranged from 0.03 to 0.33 mg kg(-1). The intra-day and inter-day precision of the individual isoflavones were less than 11.77% and calibration curves exhibited good linearity (r(2) = 0.99) within the tested ranges. These results suggest that the rapid method used in this study could be available to determine of 14 types of isoflavones in a variety of food such as soy bean, black bean, red bean and soybean paste. PMID:25977042

  10. Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method.

    PubMed

    Walsh, Helen Ann; Pietersen, Gerhard

    2013-12-01

    Grapevine leafroll disease (GLD) is the most important disease of Grapevines in South Africa. Grapevine leafroll-associated virus type 3 (GLRaV-3) has a close association with the disease and is prevalent in South African vineyards. GLD can be controlled using a combination of virus-free planting material, systemic insecticides to control vector populations and removal of infected vines by roguing. Infected vines are identified each autumn using either symptom display (in red cultivars) or ELISA (in white cultivars). While ELISA is a simple, reliable means of testing for GLRaV-3, it is time consuming, laborious and insensitive and a quicker, more sensitive method of detecting GLRaV-3 in the field is needed. A single-tube one-step reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay combined with a simple RNA extraction protocol was developed for the rapid and easy detection of GLRaV-3. Hydroxy napthol blue was included as an indicator and under isothermal conditions at 60 °C the target viral gene could be amplified in under 2h and positive results could be easily seen by examining the colour change from violet to sky blue. Using this method, 50 samples could be also pooled together with a single positive sample still being detected. A direct comparison of ELISA, nested PCR and RT-LAMP showed that RT-LAMP is as sensitive as nested PCR and could be performed in a much shorter time with less equipment. This assay is may be a possible alternative to ELISA for the detection of GLRaV-3 in the field. PMID:24025344

  11. Rapid detection of pesticides not amenable to multi-residue methods by flow injection-tandem mass spectrometry.

    PubMed

    Mol, Hans G J; van Dam, Ruud C J

    2014-11-01

    Flow injection combined with tandem mass spectrometry (MS/MS) was investigated for the rapid detection of highly polar pesticides that are not amenable to multi-residue methods because they do not partition into organic solvents and require dedicated chromatographic conditions. The pesticides included in this study were amitrole, chlormequat, cyromazine, daminozide, diquat, ethephon, fosetyl-Al, glufosinate, glyphosate and its metabolite aminomethylphosphonic acid, maleic hydrazide, mepiquat and paraquat. The composition of the flow-injection solvent was optimized to achieve maximum MS/MS sensitivity. Instrumental limits of detection varied between <0.05 and 1 pg. Fruit, vegetable, cereal, milk and kidney samples were extracted with water (1% formic acid in case of paraquat/diquat) and ten times diluted in either methanol/0.1% formic acid, methanol/0.1% ammonia or acetonitrile/0.1% ammonia, depending on the pesticide. The ion suppression observed depended strongly on both the matrix and the pesticide. This could be largely compensated for by matrix-matched calibration, but more accurate quantification was obtained by using isotopically labelled standards (commercially available for most of the pesticides studied). The method detection limits ranged from 0.02 mg/kg for chlormequat and mepiquat to 2 mg/kg for maleic hydrazide and were 0.05-0.2 mg/kg for most other pesticide/matrix combinations. This was sufficiently low to test compliance with EU maximum residue limits for many relevant pesticide/commodity combinations. The method substantially reduces the liquid chromatography-MS/MS capacity demand which for many laboratories is prohibitive for inclusion of these pesticides in their monitoring and surveillance programmes. PMID:24518902

  12. A rapid method for peroxide value determination in edible oils based on flow analysis with Fourier transform infrared spectroscopic detection.

    PubMed

    Ruíz, A; Ayora Cañada, M J; Lendl, B

    2001-02-01

    The development of an automated, rapid and highly precise method for determination of the peroxide value in edible oils based on a continuous flow system and Fourier transform infrared (FTIR) spectroscopic detection is described. The sample stream was mixed with a solvent mixture consisting of 25% (v/v) toluene in hexanol which contained triphenylphosphine (TPP). The hydroperoxides present in the sample reacted stoichiometrically with TPP to give triphenylphosphine oxide (TPPO) which has a characteristic and intense absorption band at 542 cm-1. A 10% (m/v) TPP solution in the solvent mixture and a 100 cm reaction coil were necessary for complete reaction. FTIR transmission spectra were recorded using a flow cell equipped with CsI windows having an optical pathlength of 100 microns. By using tert-butyl hydroperoxide spiked oil standards and evaluation of the band formed at 542 cm-1 a linear calibration graph covering the range 1-100 PV (peroxide value; mequiv O2 kg-1 oil) was obtained. The relative standard deviation was 0.23% (n = 11) and the throughput 24 samples h-1. The developed system was also applied to the determination of PV in olive, sunflower and corn oils, showing good agreement with the official reference method of the European Community which is based on titration using organic solvents. The results obtained clearly show that the developed method is superior to the standard wet chemical method, hence suggesting its application in routine analysis and quality control. PMID:11235111

  13. Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E.

    PubMed

    Rasooly, Reuven; Do, Paula; Hernlem, Bradley

    2016-01-01

    Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses through its production of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens to massively activate T cells. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which was detected in 17 of the 38 suspected staphylococcal food poisoning incidents in a British study and was the causative agent in outbreaks in France, UK and USA. The current method for detection of enterotoxin activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals, is impractical to test on a large number of samples, and raises ethical concerns with regard to the use of experimental animals. The purpose of this study is to develop rapid sensitive and quantitative bioassays for detection of active SEE. We apply a genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC (major histocompatibility complex) class II to the engineered T cell line. Exposure of the above mixed culture to SEE induces differential expression of the luciferase gene and bioluminescence is read out in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 10⁸ times more sensitive than the monkey and kitten bioassay. PMID:27187474

  14. Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E

    PubMed Central

    Rasooly, Reuven; Do, Paula; Hernlem, Bradley

    2016-01-01

    Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses through its production of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens to massively activate T cells. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which was detected in 17 of the 38 suspected staphylococcal food poisoning incidents in a British study and was the causative agent in outbreaks in France, UK and USA. The current method for detection of enterotoxin activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals, is impractical to test on a large number of samples, and raises ethical concerns with regard to the use of experimental animals. The purpose of this study is to develop rapid sensitive and quantitative bioassays for detection of active SEE. We apply a genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC (major histocompatibility complex) class II to the engineered T cell line. Exposure of the above mixed culture to SEE induces differential expression of the luciferase gene and bioluminescence is read out in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 109 times more sensitive than the monkey and kitten bioassay. PMID:27187474

  15. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

    PubMed Central

    Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

  16. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

    PubMed

    Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

  17. A rapid method for detection of genetically modified organisms based on magnetic separation and surface-enhanced Raman scattering.

    PubMed

    Guven, Burcu; Boyacı, İsmail Hakkı; Tamer, Ugur; Çalık, Pınar

    2012-01-01

    In this study, a new method combining magnetic separation (MS) and surface-enhanced Raman scattering (SERS) was developed to detect genetically modified organisms (GMOs). An oligonucleotide probe which is specific for 35 S DNA target was immobilized onto gold coated magnetic nanospheres to form oligonucleotide-coated nanoparticles. A self assembled monolayer was formed on gold nanorods using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and the second probe of the 35 S DNA target was immobilized on the activated nanorod surfaces. Probes on the nanoparticles were hybridized with the target oligonucleotide. Optimization parameters for hybridization were investigated by high performance liquid chromatography. Optimum hybridization parameters were determined as: 4 μM probe concentration, 20 min immobilization time, 30 min hybridization time, 55 °C hybridization temperature, 750 mM buffer salt concentration and pH: 7.4. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. The correlation between the target concentration and the SERS signal was found to be linear within the range of 25-100 nM. The analyses were performed with only one hybridization step in 40 min. Real sample analysis was conducted using Bt-176 maize sample. The results showed that the developed MS-SERS assay is capable of detecting GMOs in a rapid and selective manner. PMID:22049365

  18. Comparison of two commercially available rapid detection methods and a conventional culture method to detect naturally occurring salmonellae on broiler carcasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many different screening devices and sampling methods have been used to detect the presence of naturally occurring Salmonella on commercially processed broiler carcasses. The objective of this study was to compare two commercial screening systems (BAX® and Roka®) to a standard cultural procedure use...

  19. Evaluation of Enrichment Method for Detection of Vibrio cholerae O1 using a Rapid Dipstick Test in Bangladesh

    PubMed Central

    George, Christine Marie; Rashid, Mahamud-ur; Sack, David A.; Sack, R. Bradley; Saif-Ur-Rahman, K. M.; Azman, Andrew S; Monira, Shirajum; Bhuyian, Sazzadul Islam; Zillur Rahman, K. M.; Mahmud, M. Toslim; Mustafiz, Munshi; Alam, Munirul

    2014-01-01

    Background Culturing is generally considered to be the gold standard for detecting Vibrio cholerae in stool, though it is not always feasible in resource-limited settings. The Crystal VC dipstick test allows for rapid stool testing for the diagnosis of cholera in the field. However, previous studies have found low specificities (49%–79%) associated with direct testing of stool for cholera using this kit when compared to culturing. Methods In the present study conducted in Dhaka, Bangladesh in 2013, we compare direct testing using the Crystal VC dipstick test and testing after enrichment for 6-hours in Alkaline Peptone Water (APW) to bacterial culture as the gold standard. Samples positive by dipstick but negative by culture were also tested using PCR. Results Stool was collected from 125 patients. The overall specificities of the direct testing and testing after 6-hour enrichment in APW compared to bacterial culture were 91.8% and 98.4% (p=0.125) respectively, and the sensitivities were 65.6% and 75.0% (p=0.07), respectively. Conclusion The increase in the sensitivity of the Crystal VC kit with the use of the 6 hour enrichment step in APW compared to direct testing was marginally significant. The Crystal VC dipstick was found to have a much higher specificity than previously reported (91–98%). Therefore this method provides a promising screening tool for cholera outbreak surveillance in resource limited settings where elimination of false positive results is critical. PMID:24401137

  20. A hydrogel based rapid test method for detection of Escherichia coli (E. coli) in contaminated water samples.

    PubMed

    Gunda, Naga Siva Kumar; Chavali, Ravi; Mitra, Sushanta K

    2016-05-10

    We have formulated a new chemical composition for rapid detection of Escherichia coli (E. coli) with currently available enzymatic substrates. We have evaluated the performance of the new chemical composition with different kinds of bacteria, and metallic and ionic interferences and optimized the chemical composition for rapid and specific detection of E. coli. We used a novel hydrogel based porous matrix to encapsulate the optimized chemical compounds and incorporated it within a readily available plunger-tube assembly. This overall system allows efficient, field deployable, rapid testing of water samples by simultaneously pre-concentrating and detecting E. coli within one integrated unit. We were able to detect E. coli concentrations of 4 × 10(6) CFU mL(-1) to 4 × 10(5) CFU mL(-1) within 5 min and 4 × 10(4) CFU mL(-1) to 400 CFU mL(-1) within 60 min using the integrated plunger-tube assembly containing the hydrogel matrix. PMID:27137782

  1. Development of rapid hemocyte-based extraction methods for detection of hepatitis A virus and murine norovirus in contaminated oysters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The human enteric pathogens, hepatitis A virus and human norovirus, have been shown to contaminate molluscan shellfish and cause foodborne disease in consumers. Rapid viral extraction methods are needed to replace current time consuming methods, which use whole oysters or dissected tissues. In our ...

  2. Rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco

    PubMed Central

    Chaoui, Imane; Atalhi, Naima; Sabouni, Radia; Akrim, Mohammed; Abid, Mohammed; Amzazi, Saaid; ElMzibri, Mohammed

    2014-01-01

    One of the greatest threats to global tuberculosis (TB) control is the growing prevalence of drug resistant strains. In the past decades, considerable efforts have been made upon the development of new molecular technologies and methodologies for detection of drug resistance in Mycobacterium tuberculosis (MTB). A sensitive, specific reverse line blot assay, called rifoligotyping (RIFO), for the detection of genotypic resistance to rifampicin (RIF), was designed and evaluated. RIFO includes oligonucleotide probes specific for wild-type and mutant sequences, allowing specific and sensitive detection of both genotypes in a single assay. The RIFO was applied on 500 MTB isolates from Morocco. The results of the RIFO showed a good sensitivity (90.9%) and high specificity (100%); the positive and negative predictive values were 100% and 96.1%, respectively. This rapid, simple, economical assay provides a practical alternative for RIF genotyping, especially in low-income countries, to improve TB control and management. PMID:26740783

  3. CapE (capture, amplify, extract): A rapid method for detection of low level contamination of water with Verocytotoxigenic Escherichia coli (VTEC).

    PubMed

    Morris, Dearbháile; Kavanagh, Siobhán; Carney, Karen; MacDomhnaill, Brian; Cormican, Martin

    2016-09-01

    Verocytotoxigenic Escherichia coli (VTEC) is associated with a wide spectrum of disease from mild self-limiting diarrhoea to haemolytic uremic syndrome. Contaminated drinking water is accepted as an important route of transmission in Ireland as elsewhere however established methods for detection of VTEC in drinking water have limitations. We describe a sensitive and rapid method for detection of VTEC from large volumes (20 to 30L) of drinking water based on filtration, enrichment culture of filters and real-time PCR detection of VTEC virulence and O antigen determinants from enrichments. The method has potential applications for other waterborne pathogens. PMID:27135590

  4. Rapid detection and differentiation of Listeria monocytogenes and Listeria species in deli meats by a new multiplex PCR method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Listeria monocytogenes is an important foodborne pathogen. To effectively control this pathogen, it is necessary to have a method that can detect and differentiate L. monocytogenes from other Listeria species in food, environmental, and clinical samples. A new multiplex PCR method using new primers ...

  5. Early detection and rapid response

    USGS Publications Warehouse

    Westbrooks, Randy G.; Eplee, Robert E.

    2011-01-01

    Prevention is the first line of defense against introduced invasive species - it is always preferable to prevent the introduction of new invaders into a region or country. However, it is not always possible to detect all alien hitchhikers imported in cargo, or to predict with any degree of certainty which introduced species will become invasive over time. Fortunately, the majority of introduced plants and animals don't become invasive. But, according to scientists at Cornell University, costs and losses due to species that do become invasive are now estimated to be over $137 billion/year in the United States. Early detection and rapid response (EDRR) is the second line of defense against introduced invasive species - EDRR is the preferred management strategy for preventing the establishment and spread of invasive species. Over the past 50 years, there has been a gradual shift away from large and medium scale federal/state single-agency-led weed eradication programs in the United States, to smaller interagency-led projects involving impacted and potential stakeholders. The importance of volunteer weed spotters in detecting and reporting suspected new invasive species has also been recognized in recent years.

  6. A novel method for rapid and non-invasive detection of plants senescence using delayed fluorescence technique

    NASA Astrophysics Data System (ADS)

    Zhang, Lingrui; Xing, Da; Wang, Junsheng; Zeng, Lizhang; Li, Qiang

    2007-05-01

    Plants senescence is a phase of plants ontogeny marked by declining photosynthetic activity that is paralleled by a decline in chloroplast function. The photosystem II ( PSII ) in a plant is considered the primary site where light-induced delayed fluorescence (DF) is produced. With the leaves of Catharanthus roseus (Catharanthus roseus (L.) G.Don) as testing models, we have studied the effects of plants senescence induced by dark and/or exogenous hormones treatments on characteristics of DF by using a home-made portable DF detection system, which can enable various DF parameters, such as DF decay kinetic curve and DF intensity, to be rapidly produced for the plants in a short time. The results show that the changes in DF intensity of green plants can truly reflect the changes in photosynthetic capacity and chlorophyll content. Therefore, DF may be used an important means of evaluating in vivo plants senescence physiology. The changes in DF intensity may provide a new approach for the rapid and early detection of plants senescence caused by age or other senescence-related factors. DF technique could be potential useful for high throughput screening and less time-consuming and automated identifying the interesting mutants with genetic modifications that change plants senescence progress.

  7. Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution.

    PubMed

    Song, Yanting; Takatsuki, Katsuya; Sekiguchi, Tetsushi; Funatsu, Takashi; Shoji, Shuichi; Tsunoda, Makoto

    2016-07-01

    This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field. PMID:27209196

  8. Rapid, potentially automatable, method extract biomarkers for HPLC/ESI/MS/MS to detect and identify BW agents

    SciTech Connect

    White, D.C. |; Burkhalter, R.S.; Smith, C.; Whitaker, K.W.

    1997-12-31

    The program proposes to concentrate on the rapid recovery of signature biomarkers based on automated high-pressure, high-temperature solvent extraction (ASE) and/or supercritical fluid extraction (SFE) to produce lipids, nucleic acids and proteins sequentially concentrated and purified in minutes with yields especially from microeukaryotes, Gram-positive bacteria and spores. Lipids are extracted in higher proportions greater than classical one-phase, room temperature solvent extraction without major changes in lipid composition. High performance liquid chromatography (HPLC) with or without derivatization, electrospray ionization (ESI) and highly specific detection by mass spectrometry (MS) particularly with (MS){sup n} provides the detection, identification and because the signature lipid biomarkers are both phenotypic as well as genotypic biomarkers, insights into potential infectivity of BW agents. Feasibility has been demonstrated with detection, identification, and determination of infectious potential of Cryptosporidium parvum at the sensitivity of a single oocyst (which is unculturable in vitro) and accurate identification and prediction, pathogenicity, and drug-resistance of Mycobacteria spp.

  9. DEVELOPMENT OF A RAPID, QUANTITATIVE METHOD FOR THE DETECTION OF INFECTIVE COXSACKIE AND ECHO VIRUSES IN DRINKING WATER

    EPA Science Inventory

    The objectives of this research are to improve on the current analytical methods for quantitative detection of infective coxsackie and echo viruses in drinking water. The specific objectives of this research are to: (1) Improve the sensitivity and specificity of IMS-PCR for in...

  10. Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method.

    PubMed

    Huang, Xin; Chen, Lili; Xu, Jiangmin; Ji, Hai-Feng; Zhu, Shuifang; Chen, Hongjun

    2014-08-01

    Transgenic maize plant expressing high phytase activity has been reported and approved by Chinese government in 2009. Here, we report a highly specific loop-mediated isothermal amplification (LAMP) method to detect the phytase gene in the GMO maize. The LAMP reaction takes less than 20min and the amplification is visible without gel electrophoresis. The detection sensitivity of the LAMP method is about 30 copies of phytase genomic DNA, which is 33.3 times greater than the conventional PCR method with gel electrophoresis. The quantitative detection results showed that the LAMP method has a good linear correlation between the DNA copy number and the associated Tt values over a large dynamic range of template concentration from 6×10(1) to 6×10(7) copies, with a quantification limit of 60 copies. Therefore, the LAMP method is visual, faster, and more sensitive, and does not need special equipment compared to traditional PCR technique, which is very useful for field tests and fast screening of GMO feeds. PMID:24629956

  11. Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

    PubMed Central

    Bao, Hongmei; Zhao, Yuhui; Wang, Yunhe; Xu, Xiaolong; Shi, Jianzhong; Zeng, Xianying; Wang, Xiurong; Chen, Hualan

    2014-01-01

    A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples. PMID:24689044

  12. Comparison of a fluorogenic assay with a conventional method for rapid detection of Vibrio parahaemolyticus in seafoods.

    PubMed Central

    Venkateswaran, K; Kurusu, T; Satake, M; Shinoda, S

    1996-01-01

    A conventional method and a fluorogenic assay for the detection of Vibrio parahaemolyticus were compared. Among 29 seafood samples examined for the presence of V. parahaemolyticus, 17 samples harbored V. parahaemolyticus, and trypsinlike activity was noticed in 19 seafoods. The added fluorogenic substrate was cleaved in single samples of shrimp, turbo, and cuttlefish from which V. parahaemolyticus could not be isolated by the conventional method. Vibrio alginolyticus, in addition to V. parahaemolyticus, was found to exhibit intracellular trypsinlike activity. Trypsinlike activity in seafoods was observed after the most probable number for the initial density of V. parahaemolyticus-like organisms was found to have reached > 10(2) per g. A V. parahaemolyticus inoculum at 10(4) CFU/ml in arabinose-glucuronate medium was required to attain growth to 10(6) CFU/ml, which is the level necessary for the release of detectable amounts of fluorescent compound from the added substrate. PMID:8795246

  13. A sensitive method for rapid detection of alkyl halides and dehalogenase activity using a multistep enzyme assay.

    PubMed

    Fabritz, Sebastian; Maaß, Franziska; Avrutina, Olga; Heiseler, Tim; Steinmann, Björn; Kolmar, Harald

    2012-01-01

    A method for the detection of haloalkane conversion to the corresponding alcohols by haloalkane dehalogenases is described. It is based on a multistage enzyme reaction which allows for the analysis of alkyl halides in buffered systems. Irreversible hydrolytic dehalogenation catalyzed by haloalkane dehalogenase DhaA from Rhodococcus erythropolis transfers an alkyl halide into a corresponding alcohol that is further oxidized by alcohol oxidase AOX from Pichia pastoris yielding a respective aldehyde and hydrogen peroxide easily detectable via the horseradish peroxidase catalyzed oxidation of chromogenic molecules. Due to its high sensitivity (0.025 mM, 0.43 ppm for 1,3-dibromopropane), low expenditure and the ability of handling a large number of samples in parallel, this method is an attractive alternative to existing procedures for the monitoring of both haloalkanes and dehalogenases. PMID:23006907

  14. Evaluation of an in-house rapid diagnostic method for detection of Salmonella enterica serovar Typhi in fecal specimens.

    PubMed

    Vaishnavi, Chetana; Kaur, Sukhminderjit; Singh, Kartar

    2006-01-01

    Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. Laboratory diagnosis requires isolation and identification of the organism from the patient's blood or feces. Feces is the specimen most commonly submitted to laboratories. Detection of bacterial antigens is an important adjunct to laboratory diagnosis. We carried out an in-house diagnostic method by preparing test reagents comprising of latex beads coated with specific antisera to detect Vi, O9 and H-d antigens of S. typhi. Fecal specimens from one hundred patients with diarrhea and fever as well as from twenty healthy controls were incubated for enrichment in Selenite F broth for 6 hours or overnight. Latex agglutination tests to detect antigens of S. typhi were carried out on centrifuged broth supernatants. Parallel cultures on media selective for S. typhi were also set up. Nine of the supernatants were positive for two or more specific antigens and S. typhi grew in three of the corresponding cultures. None of the samples from 20 healthy controls were positive by either the diagnostic method or by culture. The result of the in-house diagnostic assay can be obtained overnight and may help in directing immediate antimicrobial therapy. PMID:16910055

  15. An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need.

    PubMed

    Wilkes, Rebecca P; Lee, Pei-Yu A; Tsai, Yun-Long; Tsai, Chuan-Fu; Chang, Hsiu-Hui; Chang, Hsiao-Fen G; Wang, Hwa-Tang T

    2015-08-01

    Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2. PMID:25889355

  16. Two novel methods for rapid detection and quantification of DNMT3A R882 mutations in acute myeloid leukemia.

    PubMed

    Mancini, Melissa; Hasan, Syed Khizer; Ottone, Tiziana; Lavorgna, Serena; Ciardi, Claudia; Angelini, Daniela F; Agostini, Francesca; Venditti, Adriano; Lo-Coco, Francesco

    2015-03-01

    DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at amino acid R882 in the methyltransferase domain of the gene. DNMT3A mutations have been reported to be stable during disease progression and are associated with unfavorable outcome in acute myeloid leukemia patients with normal karyotype. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for minimal residual disease monitoring. We describe a new rapid diagnostic RT-PCR assay based on TauI restriction enzyme reaction to identify DNMT3A R882 mutations at diagnosis. In addition, we developed a sensitive and specific test based on peptide nucleic acid real-time PCR technology to monitor DNMT3A R882H mutation. We identified 24 DNMT3A R882H mutated patients out of 134 acute myeloid leukemia screened samples and we analyzed in these patients the kinetics of minimal residual disease after induction and consolidation therapy. This assay may be useful to better assess response to therapy in patients with acute myeloid leukemia bearing the DNMT3A R882H mutation. PMID:25554589

  17. Rapid high-performance liquid chromatographic method for determination of adefovir in plasma using UV detection: application to pharmacokinetic studies.

    PubMed

    Foroutan, Seyed Mohsen; Zarghi, Afshin; Shafaati, Alireza; Movahed, Hooman; Khoddam, Arash

    2011-01-01

    A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of adefovir (CAS 106941-25-7) in human plasma. The separation was achieved on a monolithic silica column (Chromolith Performance RP-18e, 100 x 4.6 mm) using acetonitrile-ammonium dihydrogen phosphate buffer (6:94, v/v), pH 5.2, as the mobile phase at a flow rate of 1.5 ml min(-1). The wavelength was set at 260 nm. The assay enables the measurement of adefovir for therapeutic drug monitoring with a minimum quantification limit of 1 ng ml(-1). The method involves a simple protein precipitation procedure. Analytical recovery was complete. The calibration curve was linear over the concentration range 1-40 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 5%. The method was applied to the determination of adefovir in plasma from 12 subjects dosed with adefovir 2 x 10 mg tablets and pharmacokinetic parameters were evaluated. PMID:21950152

  18. Ascochyta blight: isolation, characterization, and development of a rapid method to detect inhibitors of the chickpea fungal pathogen Ascochyta rabiei.

    PubMed

    Bahr, Luciana; Castelli, María Victoria; Barolo, Melisa Isabel; Ruiz Mostacero, Nathalie; Tosello, María Elena; López, Silvia Noelí

    2016-03-01

    Ascochyta blight is the major disease attacking chickpea (Cicer arietinum) around the world. Since its first time report of isolation in Argentina in 2012, the pathogen has caused severe economic losses and has acquired a great importance. We report here the isolation of Ascochyta rabiei from infected chickpea beans cultivated in Santa Fe, Argentina; its identification by morphological analysis and molecular biology techniques based on internal transcribed spacer (ITS) sequence alignment, its biochemical characterization regarding the capacity to produce proteinase and phospholipase enzymes, and its antifungal susceptibility to common used antifungal agents. In order to detect new inhibitors for A. rabiei from natural sources, a bioautographic method was developed. From the screening method developed, we found that extracts from cultures of Aspergillus parasiticus are active against A. rabiei. PMID:26895871

  19. Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium.

    PubMed

    Qu, Huihua; Zhang, Yue; Qu, Baoping; Cheng, Jinjun; Liu, Shuchen; Feng, Shenglan; Wang, Qingguo; Zhao, Yan

    2016-04-01

    In this work, a novel monoclonal antibody specific for naringin was prepared and characterized. Subsequently, an indirect competitive enzyme-linked immunosorbent assay for naringin was developed, with an effective range from 4.8 to 156 ng/mL naringin. Next, an immunoaffinity column was obtained by coupling anti-naringin monoclonal antibodies to CNBr-activated Sepharose 4B and a rapid immunoaffinity chromatography assay for naringin was developed. The immunoaffinity column was used to separate naringin from Citrus aurantium. The results showed that 1 g of the dry Sepharose 4B can couple 10 mg of immunoglobulin G. And the immunoaffinity column can efficiently and specifically capture approximately 250 μg of naringin without cross reacting with its structurally similar compounds. Moreover, our results indicate that the application of immunoaffinity chromatography can simplify the pretreatment and the isolation process greatly compared to conventional methods, providing a potential method for extracting the target component from structurally similar compounds in natural products. PMID:26864564

  20. Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

    PubMed

    Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

    2015-02-01

    Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications. PMID:25710151

  1. Sensitive, Rapid Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Venkateswaran, Kasthuri; Chen, Fei; Pickett, Molly; Matsuyama, Asahi

    2009-01-01

    A method of sensitive detection of bacterial spores within delays of no more than a few hours has been developed to provide an alternative to a prior three-day NASA standard culture-based assay. A capability for relatively rapid detection of bacterial spores would be beneficial for many endeavors, a few examples being agriculture, medicine, public health, defense against biowarfare, water supply, sanitation, hygiene, and the food-packaging and medical-equipment industries. The method involves the use of a commercial rapid microbial detection system (RMDS) that utilizes a combination of membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and analysis of luminescence images detected by a charge-coupled-device camera. This RMDS has been demonstrated to be highly sensitive in enumerating microbes (it can detect as little as one colony-forming unit per sample) and has been found to yield data in excellent correlation with those of culture-based methods. What makes the present method necessary is that the specific RMDS and the original protocols for its use are not designed for discriminating between bacterial spores and other microbes. In this method, a heat-shock procedure is added prior to an incubation procedure that is specified in the original RMDS protocols. In this heat-shock procedure (which was also described in a prior NASA Tech Briefs article on enumerating sporeforming bacteria), a sample is exposed to a temperature of 80 C for 15 minutes. Spores can survive the heat shock, but nonspore- forming bacteria and spore-forming bacteria that are not in spore form cannot survive. Therefore, any colonies that grow during incubation after the heat shock are deemed to have originated as spores.

  2. Development of a Rapid Soil Water Content Detection Technique Using Active Infrared Thermal Methods for In-Field Applications

    PubMed Central

    Antonucci, Francesca; Pallottino, Federico; Costa, Corrado; Rimatori, Valentina; Giorgi, Stefano; Papetti, Patrizia; Menesatti, Paolo

    2011-01-01

    The aim of this study was to investigate the suitability of active infrared thermography and thermometry in combination with multivariate statistical partial least squares analysis as rapid soil water content detection techniques both in the laboratory and the field. Such techniques allow fast soil water content measurements helpful in both agricultural and environmental fields. These techniques, based on the theory of heat dissipation, were tested by directly measuring temperature dynamic variation of samples after heating. For the assessment of temperature dynamic variations data were collected during three intervals (3, 6 and 10 s). To account for the presence of specific heats differences between water and soil, the analyses were regulated using slopes to linearly describe their trends. For all analyses, the best model was achieved for a 10 s slope. Three different approaches were considered, two in the laboratory and one in the field. The first laboratory-based one was centred on active infrared thermography, considered measurement of temperature variation as independent variable and reported r = 0.74. The second laboratory–based one was focused on active infrared thermometry, added irradiation as independent variable and reported r = 0.76. The in-field experiment was performed by active infrared thermometry, heating bare soil by solar irradiance after exposure due to primary tillage. Some meteorological parameters were inserted as independent variables in the prediction model, which presented r = 0.61. In order to obtain more general and wide estimations in-field a Partial Least Squares Discriminant Analysis on three classes of percentage of soil water content was performed obtaining a high correct classification in the test (88.89%). The prediction error values were lower in the field with respect to laboratory analyses. Both techniques could be used in conjunction with a Geographic Information System for obtaining detailed information on soil

  3. Development of a rapid soil water content detection technique using active infrared thermal methods for in-field applications.

    PubMed

    Antonucci, Francesca; Pallottino, Federico; Costa, Corrado; Rimatori, Valentina; Giorgi, Stefano; Papetti, Patrizia; Menesatti, Paolo

    2011-01-01

    The aim of this study was to investigate the suitability of active infrared thermography and thermometry in combination with multivariate statistical partial least squares analysis as rapid soil water content detection techniques both in the laboratory and the field. Such techniques allow fast soil water content measurements helpful in both agricultural and environmental fields. These techniques, based on the theory of heat dissipation, were tested by directly measuring temperature dynamic variation of samples after heating. For the assessment of temperature dynamic variations data were collected during three intervals (3, 6 and 10 s). To account for the presence of specific heats differences between water and soil, the analyses were regulated using slopes to linearly describe their trends. For all analyses, the best model was achieved for a 10 s slope. Three different approaches were considered, two in the laboratory and one in the field. The first laboratory-based one was centred on active infrared thermography, considered measurement of temperature variation as independent variable and reported r = 0.74. The second laboratory-based one was focused on active infrared thermometry, added irradiation as independent variable and reported r = 0.76. The in-field experiment was performed by active infrared thermometry, heating bare soil by solar irradiance after exposure due to primary tillage. Some meteorological parameters were inserted as independent variables in the prediction model, which presented r = 0.61. In order to obtain more general and wide estimations in-field a Partial Least Squares Discriminant Analysis on three classes of percentage of soil water content was performed obtaining a high correct classification in the test (88.89%). The prediction error values were lower in the field with respect to laboratory analyses. Both techniques could be used in conjunction with a Geographic Information System for obtaining detailed information on soil heterogeneity

  4. Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

    PubMed Central

    2010-01-01

    Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic

  5. Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method.

    PubMed

    Liu, Zhongyuan; Dong, Zhijun; Liu, Dongyan

    2016-07-01

    Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle. PMID:25774948

  6. Establishment and application of a loop-mediated isothermal amplification method for simple, specific, sensitive and rapid detection of Toxoplasma gondii.

    PubMed

    Cao, Lili; Cheng, Ronghua; Yao, Lin; Yuan, Shuxian; Yao, Xinhua

    2014-01-01

    The Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with 6 primers targeting a highly conserved region of the GRA1 gene was developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondii DNA in 423 blood samples and 380 lymph node samples from 10 pig farms, and positive results were obtained for 7.8% and 8.2% of samples, respectively. The results showed that the LAMP method is slightly more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA and is expected to play an important role in clinical detection of T. gondii. PMID:23965849

  7. Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

    PubMed Central

    2013-01-01

    Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina. PMID:23586331

  8. Detection of zinc oxide and cerium dioxide nanoparticles during drinking water treatment by rapid single particle ICP-MS methods.

    PubMed

    Donovan, Ariel R; Adams, Craig D; Ma, Yinfa; Stephan, Chady; Eichholz, Todd; Shi, Honglan

    2016-07-01

    Nanoparticles (NPs) entering water systems are an emerging concern as NPs are more frequently manufactured and used. Single particle inductively coupled plasma-mass spectrometry (SP-ICP-MS) methods were validated to detect Zn- and Ce-containing NPs in surface and drinking water using a short dwell time of 0.1 ms or lower, ensuring precision in single particle detection while eliminating the need for sample preparation. Using this technique, information regarding NP size, size distribution, particle concentration, and dissolved ion concentrations was obtained simultaneously. The fates of Zn- and Ce-NPs, including those found in river water and added engineered NPs, were evaluated by simulating a typical drinking water treatment process. Lime softening, alum coagulation, powdered activated carbon sorption, and disinfection by free chlorine were simulated sequentially using river water. Lime softening removed 38-53 % of Zn-containing and ZnO NPs and >99 % of Ce-containing and CeO2 NPs. Zn-containing and ZnO NP removal increased to 61-74 % and 77-79 % after alum coagulation and disinfection, respectively. Source and drinking water samples were collected from three large drinking water treatment facilities and analyzed for Zn- and Ce-containing NPs. Each facility had these types of NPs present. In all cases, particle concentrations were reduced by a minimum of 60 % and most were reduced by >95 % from source water to finished drinking water. This study concludes that uncoated ZnO and CeO2 NPs may be effectively removed by conventional drinking water treatments including lime softening and alum coagulation. PMID:26960902

  9. Rod Photoreceptors Detect Rapid Flicker

    ERIC Educational Resources Information Center

    Conner, J. D.; MacLeod, Donald I. A.

    1977-01-01

    Rod-isolation techniques show that light-adapted human rods detect flicker frequencies as high as 28 hertz, and that the function relating rod critical flicker frequency to stimulus intensity contains two distinct branches. (MLH)

  10. PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes.

    PubMed

    Janczarek, Monika; Palusińska-Szysz, Marta

    2016-05-01

    Legionella bacteria are organisms of public health interest due to their ability to cause pneumonia (Legionnaires' disease) in susceptible humans and their ubiquitous presence in water supply systems. Rapid diagnosis of Legionnaires' disease allows the use of therapy specific for the disease. L. pneumophila serogroup 1 is the most common cause of infection acquired in community and hospital environments. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this work, simplex and duplex PCR assays with the use of new molecular markers pcs and pmtA involved in phosphatidylcholine synthesis were specified for rapid and cost-efficient identification and distinguishing Legionella species. The sets of primers developed were found to be sensitive and specific for reliable detection of Legionella belonging to the eight most clinically relevant species. Among these, four primer sets I, II, VI, and VII used for duplex-PCRs proved to have the highest identification power and reliability in the detection of the bacteria. Application of this PCR-based method should improve detection of Legionella spp. in both clinical and environmental settings and facilitate molecular typing of these organisms. PMID:26423783

  11. Take Only Photographs, Leave Only Footprints: Novel Applications of Non-Invasive Survey Methods for Rapid Detection of Small, Arboreal Animals.

    PubMed

    Mills, Cheryl A; Godley, Brendan J; Hodgson, David J

    2016-01-01

    The development of appropriate wildlife survey techniques is essential to promote effective and efficient monitoring of species of conservation concern. Here, we demonstrate the utility of two rapid-assessment, non-invasive methods to detect the presence of elusive, small, arboreal animals. We use the hazel dormouse, Muscardinus avellanarius, a rodent of conservation concern, as our focal species. Prevailing hazel dormouse survey methods are prolonged (often taking months to years to detect dormice), dependent on season and habitat, and/or have low detection rates. Alternatives would be of great use to ecologists who undertake dormouse surveys, especially those assessing the need for mitigation measures, as legally required for building development projects. Camera traps and footprint tracking are well-established tools for monitoring elusive large terrestrial mammals, but are rarely used for small species such as rodents, or in arboreal habitats. In trials of these adapted methods, hazel dormice visited bait stations and were successfully detected by both camera traps and tracking equipment at each of two woodland study sites, within days to weeks of installation. Camera trap images and footprints were of adequate quality to allow discrimination between two sympatric small mammal species (hazel dormouse and wood mouse, Apodemus sylvaticus). We discuss the relative merits of these methods with respect to research aims, funds, time available and habitat. PMID:26789632

  12. Take Only Photographs, Leave Only Footprints: Novel Applications of Non-Invasive Survey Methods for Rapid Detection of Small, Arboreal Animals

    PubMed Central

    Mills, Cheryl A.; Godley, Brendan J.; Hodgson, David J.

    2016-01-01

    The development of appropriate wildlife survey techniques is essential to promote effective and efficient monitoring of species of conservation concern. Here, we demonstrate the utility of two rapid-assessment, non-invasive methods to detect the presence of elusive, small, arboreal animals. We use the hazel dormouse, Muscardinus avellanarius, a rodent of conservation concern, as our focal species. Prevailing hazel dormouse survey methods are prolonged (often taking months to years to detect dormice), dependent on season and habitat, and/or have low detection rates. Alternatives would be of great use to ecologists who undertake dormouse surveys, especially those assessing the need for mitigation measures, as legally required for building development projects. Camera traps and footprint tracking are well-established tools for monitoring elusive large terrestrial mammals, but are rarely used for small species such as rodents, or in arboreal habitats. In trials of these adapted methods, hazel dormice visited bait stations and were successfully detected by both camera traps and tracking equipment at each of two woodland study sites, within days to weeks of installation. Camera trap images and footprints were of adequate quality to allow discrimination between two sympatric small mammal species (hazel dormouse and wood mouse, Apodemus sylvaticus). We discuss the relative merits of these methods with respect to research aims, funds, time available and habitat. PMID:26789632

  13. A rapid method of accurate detection and differentiation of Newcastle disease virus pathotypes by demonstrating multiple bands in degenerate primer based nested RT-PCR.

    PubMed

    Desingu, P A; Singh, S D; Dhama, K; Kumar, O R Vinodh; Singh, R; Singh, R K

    2015-02-01

    A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry. PMID:25449112

  14. Simple and rapid method for the determination of the diastereomers of difenacoum in blood and liver using high-performance liquid chromatography with fluorescence detection.

    PubMed

    Kelly, M J; Chambers, J; MacNicoll, A D

    1993-10-22

    A rapid and sensitive high-performance liquid chromatographic method for the analysis of cis and trans diastereomers of the anticoagulant rodenticide difenacoum has been described. The methodology demonstrates potential for the analysis of diastereomers of related 4-hydroxycoumarin anticoagulants. Separations were achieved by reversed-phase chromatography on a Zorbax ODS column with gradient elution using acetonitrile-water, modified with 0.1% acetic acid, as the mobile phase. Detection of the analytes was effected by fluorescence at excitation and emission wavelengths of 310 and 390 nm, respectively. Sample preparation from both plasma and liver has been simplified to reduce preparation time and manipulation. The minimum detectable concentration of each diastereomer was 5 ng/ml. Recoveries of 100% were obtained from plasma and 93% from liver tissue. This method has been used for the investigation of the pharmacokinetics of difenacoum diastereomers in rats, and for investigation of unexplained hypoprothrombinaemic events encountered clinically. PMID:8106576

  15. The Application of Flow Cytometry as a Rapid and Sensitive Screening Method to Detect Contamination of Vitreous Humor Samples and Avoid Miscalculation of the Postmortem Interval.

    PubMed

    Cordeiro, Cristina; Seoane, Rafael; Camba, Ana; Lendoiro, Elena; Rodríguez-Calvo, María S; Vieira, Duarte N; Muñoz-Barús, José I

    2015-09-01

    Research into maximizing the speed, precision, and reliability of estimating the postmortem interval (PMI) has been a recurring object of investigation and methodologies based on the vitreous humor (VH) have provided good results. However, contamination from causes not readily apparent, such as blood, can occur, and thus lead not only to an erroneous estimation of PMI, but also interfere with the correct identification of other substances in the VH. We have developed a flow cytometry method which quantifies blood contamination and is able to detect erythrocytes in 1:750,000 dilution of contaminated VH which affects the results of hypoxanthine. It is an improvement on the previous more complex mass spectrometry method, being faster, more sensitive, and readily available. As such, it could be proposed for the rapid screening of appropriate samples by detecting and eliminating blood contaminated samples from PMI estimation. PMID:25882002

  16. High-performance liquid chromatographic method for rapid and highly sensitive determination of histidine using postcolumn fluorescence detection with o-phthaldialdehyde.

    PubMed

    Tateda, N; Matsuhisa, K; Hasebe, K; Kitajima, N; Miura, T

    1998-11-01

    A high-performance liquid chromatographic method was developed for the rapid and sensitive determination of histidine. The method is based on separation by reversed-phase ion-pair chromatography followed by highly selective fluorescence derivatization of histidine with o-phthaldialdehyde. A linear calibration curve was obtained over the range of 0.25-200 pmol per injection (10 microl) with the coefficient of variation of 0.9% at 2 pmol (n=10) and with the detection limit (SIN=8) of 25 fmol. The method was applicable to the assay of histidine in human serum. Serum histidine values obtained by the present method were in good agreement with values obtained with an amino acid analyzer. PMID:9840433

  17. Development and Application of a Multiplex PCR Method for Rapid Differential Detection of Subgroup A, B, and J Avian Leukosis Viruses

    PubMed Central

    Gao, Qi; Yun, Bingling; Wang, Qi; Jiang, Lili; Zhu, Haibo; Gao, Yanni; Qin, Liting; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei

    2014-01-01

    Avian leukosis virus (ALV) subgroups A, B, and J are very common in poultry flocks and have caused serious economic losses in recent years. A multiplex PCR (mPCR) method for the detection of these three subgroups was developed and optimized in this study. We first designed a common forward primer, PF, and three downstream primers, AR, BR, and JR, which can amplify 715 bp for subgroup A, 515 bp for subgroup B, and 422 bp for subgroup J simultaneously in one reaction. The mPCR method produced neither cross-reactions with other subgroups of ALVs nor nonspecific reactions with other common avian viruses. The detection limit of the mPCR was as low as 1 × 103 viral DNA copies of each of the three subgroups. In animal experiments, the mPCR detected ALVs 2 to 4 days earlier than did virus isolation from whole-blood samples and cloaca swabs. Furthermore, a total of 346 clinical samples (including 127 tissue samples, 86 cloaca swabs, 59 albumen samples, and 74 whole-blood samples) from poultry flocks with suspected ALV infection were examined by mPCR, routine PCR, and virus isolation. The positive sample/total sample ratios for ALV-A, ALV-B, and ALV-J were 48% (166/346) as detected by mPCR and 48% (166/346) as detected by routine PCR. However, the positive sample/total sample ratio detected by virus isolation was 40% (138/346). The results of the mPCR and routine PCR were confirmed by sequencing the specific fragments. These results indicate that the mPCR method is rapid, specific, sensitive, and convenient for use in epidemiological studies of ALV, clinical detection of ALV, and ALV eradication programs. PMID:24131697

  18. Evaluation and comparison of rapid methods for the detection of Salmonella in naturally contaminated pine nuts using different pre enrichment media.

    PubMed

    Wang, Hua; Gill, Vikas S; Cheng, Chorng-Ming; Gonzalez-Escalona, Narjol; Irvin, Kari A; Zheng, Jie; Bell, Rebecca L; Jacobson, Andrew P; Hammack, Thomas S

    2015-04-01

    Foodborne outbreaks, involving pine nuts and peanut butter, illustrate the need to rapidly detect Salmonella in low moisture foods. However, the current Bacteriological Analytical Manual (BAM) culture method for Salmonella, using lactose broth (LB) as a pre enrichment medium, has not reliably supported real-time quantitative PCR (qPCR) assays for certain foods. We evaluated two qPCR assays in LB and four other pre enrichment media: buffered peptone water (BPW), modified BPW (mBPW), Universal Pre enrichment broth (UPB), and BAX(®) MP media to detect Salmonella in naturally-contaminated pine nuts (2011 outbreak). A four-way comparison among culture method, Pathatrix(®) Auto, VIDAS(®) Easy SLM, and qPCR was conducted. Automated DNA extraction techniques were compared with manual extraction methods (boiling or InstaGene™). There were no significant differences (P > 0.05) among the five pre enrichment media for pine nuts using the culture method. While both qPCR assays produced significantly (P ≤ 0.05) higher false negatives in 24 h pre enriched LB than in the other four media, they were as sensitive as the culture method in BPW, mBPW, UPB, and BAX media. The VIDAS Easy and qPCR were equivalent; Pathatrix was the least effective method. The Automatic PrepSEQ™ DNA extraction, using 1000 μL of pre enrichment, was as effective as manual extraction methods. PMID:25475267

  19. Rapid detection of bacteria in water

    NASA Astrophysics Data System (ADS)

    Deininger, Rolf A.; Lee, Ji Y.

    2002-06-01

    A rapid detection of bacteria in water is essential for a timely response. This applies primarily to drinking water, be it bottled water or water from a public supply system, but is equally important for the analysis of water from swimming pools and beaches, and ballast water from oceangoing ships discharging into coastal or inland waters of the US. There are several methods available today for a rapid test including PCR based methods, flow cytometry, and electro chemiluminescence, to name a few. All of the above methods work, but are complicated and/or require expensive equipment and highly trained analysts in a laboratory. The method described here is based on lysing the bacteria after capture on a membrane filter, and measuring the ATP in a luminometer after the addition of luciferin/luciferase. This bioluminescence test can be done onsite, in less than 5 minutes, with equipment that fits onto a clipboard. It is a fast screening test that indicates if there is enough biologically active material in the same to pose a threat to the consumer. If this is the case, an additional step using immunomagnetic separation may be used to identify the responsible organisms. Tests have been done with E. coli 0157:H7, pseudomonas, and logionella. These tests take about 30 minutes each, and allow a quick determination of bacterial threats in a field situation.

  20. A new method to detect rapid oxygen changes around cells: How quickly do calcium channels sense oxygen in cardiomyocytes?

    PubMed Central

    Scaringi, John A.; Rosa, Angelo Oscar; Morad, Martin

    2013-01-01

    Acute hypoxia is thought to trigger protective responses that, in tissues like heart and carotid body, include rapid (5–10 s) suppression of Ca2+ and K+ channels. To gain insight into the mechanism for the suppression of the cardiac l-type Ca2+ channel, we measured O2-dependent fluorescence in the immediate vicinity of voltage-clamped cardiac cells subjected to rapid exchange of solutions with different O2 tensions. This was accomplished with an experimental chamber with a glass bottom that was used as a light guide for excitation of a thin ruthenium-based O2-sensitive ORMOSIL coating. Fluorescence imaging showed that steady-state Po2 was well controlled within the entire stream from an electromagnetically controlled solution “puffer” but that changes were slower at the periphery of the stream (τ1/2 ∼ 500 ms) than immediately around the voltage-clamped myocyte (τ1/2 ∼ 225 ms) where, in turn, firmly attached cells produced an additional local delay of 50–100 ms. Performing simultaneous voltage clamp and O2 measurements, we found that acute hypoxia gradually and reversibly suppressed the Ca2+ channel (CaV1.2). Using Ba2+ as charge carrier, the suppression was significant after 1.5 s, reached ∼10% after 2.5 s, and was nearly completely reversible in 5 s. The described fluorescence measurements provide the means to check and fine tune solution puffers and suggest that changes in Po2 can be accomplished within ∼200 ms. The rapid and reversible suppression of barium current under hypoxia is consistent with the notion that the cardiac Ca2+ channel is directly modulated by O2. PMID:24157525

  1. A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay

    PubMed Central

    Deshpande, Srinidhi; Karim, Farina; Flynn, JoAnne L.; O’Malley, Melanie; Jones, Martin; Nanassy, Oliver; Jeena, Prakash; Alland, David

    2016-01-01

    Background Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available. Methods We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa. Results The assay’s analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6–0.9) and 84% (95% CI 0.6–0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77–1) and 94% (95% CI 0.7–0.99), respectively. Conclusion This novel approach may permit simple and rapid detection of TB using pediatric stool samples. PMID:27007974

  2. Rapid, low cost thin-layer chromatographic screening method for the detection of ochratoxin A in green coffee at a control level of 10 microg/kg.

    PubMed

    Pittet, Alain; Royer, Delphine

    2002-01-16

    A thin-layer chromatographic (TLC) screening method was developed for the detection of ochratoxin A (OTA) in green coffee at a control level of 10 microg/kg. The method is based on extraction of OTA with a mixture of phosphoric acid and dichloromethane, purification by liquid-liquid partition into sodium hydrogen carbonate, separation by normal-phase TLC, and detection by visual estimation of fluorescence intensity under a UV lamp at 366 nm. The method was validated by performing replicate analyses of uncontaminated green coffee material spiked at 3 different levels of OTA (5, 10, and 20 microg/kg), and also by comparing results obtained on a series of test trial green coffees naturally contaminated with OTA (range 0.2 to 136.7 microg/kg) with those measured by a quantitative immunoaffinity/HPLC method. The agreement between the two methods was excellent, and neither false positive nor false negative results were recorded. This screening method is rapid, simple, robust, and very cheap, which makes it particularly well adapted for implementation in coffee-producing countries. PMID:11782189

  3. A rapid, sensitive and automated method for detection of Salmonella species in foods using AG-9600 AmpliSensor Analyzer.

    PubMed

    Chen, S; Yee, A; Griffiths, M; Wu, K Y; Wang, C N; Rahn, K; De Grandis, S A

    1997-09-01

    The AG-9600 AmpliSensor Analyzer is an automated fluorescence-based system for detection of polymerase chain reaction (PCR) products. The principle of the AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to a target sequence within a 284-bp amplified fragment of the Salmonella invA gene. Since the assay is homogenous, the data can be obtained by direct measurement of fluorescence of the amplification mixture. The accumulation of the amplified product, reflected by the fluorescence index, is monitored cycle by cycle by the AG-9600 Analyzer. The detection limit of the assay was less than 2 colony-forming units (cfu) per PCR reaction using a pure culture of Salmonella typhimurium. In post-spiking experiments in which Salmonella was added to the overnight pre-enriched samples (chicken carcass rinses, ground beef, ground pork and raw milk), the detection limit of the assay was 2-6 cfu per PCR reaction. In pre-spiking experiments in which Salmonella was added to the samples prior to overnight pre-enrichment, the detection limit was less than 3 cfu per 25 g or 25 ml of food. The assay was up to 2 orders of magnitude more sensitive than detection by ethidium bromide-stained agarose gel electrophoresis. To further evaluate assay performance, 54 naturally contaminated chicken carcass rinses, 65 raw milk and six ground pork samples were tested in the study. Thirty-eight Salmonella-positive samples confirmed by the Modified Semi-solid Rappaport-Vassiliadis (MSRV) culture assay were found positive using the AmpliSensor assay. Two chicken carcass rinses found positive using the assay were MSRV-negative. In addition, relative quantification using the AmpliSensor assay was linear up to 3 logs of initial target concentration in artificially contaminated food samples. PMID:9351211

  4. A Rapid and Sensitive Next-Generation Sequencing Method to Detect RB1 Mutations Improves Care for Retinoblastoma Patients and Their Families.

    PubMed

    Li, Wenhui L; Buckley, Jonathan; Sanchez-Lara, Pedro A; Maglinte, Dennis T; Viduetsky, Lucy; Tatarinova, Tatiana V; Aparicio, Jennifer G; Kim, Jonathan W; Au, Margaret; Ostrow, Dejerianne; Lee, Thomas C; O'Gorman, Maurice; Judkins, Alexander; Cobrinik, David; Triche, Timothy J

    2016-07-01

    Retinoblastoma is a childhood eye malignancy that can lead to the loss of vision, eye(s), and sometimes life. The tumors are initiated by inactivating mutations in both alleles of the tumor-suppressor gene, RB1, or, rarely, by MYCN amplification. Timely identification of a germline RB1 mutation in blood samples or either somatic RB1 mutation or MYCN amplification in tumors is important for effective care and management of retinoblastoma patients and their families. However, current procedures to thoroughly test RB1 mutations are complicated and lengthy. Herein, we report a next-generation sequencing-based method capable of detecting point mutations, small indels, and large deletions or duplications across the entire RB1 gene and amplification of MYCN gene on a single platform. From DNA extraction to clinical interpretation requires only 3 days, enabling early molecular diagnosis of retinoblastoma and optimal treatment outcomes. This method can also detect low-level mosaic mutations in blood samples that can be missed by routine Sanger sequencing. In addition, it can differentiate between RB1 mutation- and MYCN amplification-driven retinoblastomas. This rapid, comprehensive, and sensitive method for detecting RB1 mutations and MYCN amplification can readily identify RB1 mutation carriers and thus improve the management and genetic counseling for retinoblastoma patients and their families. PMID:27155049

  5. The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster.

    PubMed Central

    Toba, G; Ohsako, T; Miyata, N; Ohtsuka, T; Seong, K H; Aigaki, T

    1999-01-01

    We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome. PMID:9927464

  6. Rapid Corner Detection Using FPGAs

    NASA Technical Reports Server (NTRS)

    Morfopoulos, Arin C.; Metz, Brandon C.

    2010-01-01

    In order to perform precision landings for space missions, a control system must be accurate to within ten meters. Feature detection applied against images taken during descent and correlated against the provided base image is computationally expensive and requires tens of seconds of processing time to do just one image while the goal is to process multiple images per second. To solve this problem, this algorithm takes that processing load from the central processing unit (CPU) and gives it to a reconfigurable field programmable gate array (FPGA), which is able to compute data in parallel at very high clock speeds. The workload of the processor then becomes simpler; to read an image from a camera, it is transferred into the FPGA, and the results are read back from the FPGA. The Harris Corner Detector uses the determinant and trace to find a corner score, with each step of the computation occurring on independent clock cycles. Essentially, the image is converted into an x and y derivative map. Once three lines of pixel information have been queued up, valid pixel derivatives are clocked into the product and averaging phase of the pipeline. Each x and y derivative is squared against itself, as well as the product of the ix and iy derivative, and each value is stored in a WxN size buffer, where W represents the size of the integration window and N is the width of the image. In this particular case, a window size of 5 was chosen, and the image is 640 480. Over a WxN size window, an equidistance Gaussian is applied (to bring out the stronger corners), and then each value in the entire window is summed and stored. The required components of the equation are in place, and it is just a matter of taking the determinant and trace. It should be noted that the trace is being weighted by a constant k, a value that is found empirically to be within 0.04 to 0.15 (and in this implementation is 0.05). The constant k determines the number of corners available to be compared against

  7. Methods of Endotoxin Detection.

    PubMed

    Su, Wenqiong; Ding, Xianting

    2015-08-01

    Endotoxin, present in the outer membrane of all gram-negative bacteria, can pose serious risks to human health, from irreversible shock to death. Therefore, it is essential to develop sensitive, accurate, and rapid methods for its detection. The rabbit pyrogen test is the first standard technique for endotoxin detection and, nowadays, has been replaced by the Limulus Amoebocyte Lysate test, which is the most popular detection technique for endotoxin. With in-depth understanding of endotoxin, biosensors based on endotoxin-sensing components are promising alternatives to pursue in developing low-cost, easy-operation, and fast-response endotoxin detection techniques. This article summarizes the recent advances of endotoxin detection methods with a particular emphasis on optical and electrochemical biosensors based on various sensing elements ranging from nature biomolecules to artificial materials. As the research and technological revolution continues, the highly integrated and miniaturized commercial devices for sensitively and reliably detecting endotoxin will provide a wide range of applications in people's daily life. PMID:25720597

  8. Rapid actinide-separation methods

    SciTech Connect

    Maxwell, S.L. III

    1997-12-31

    New high-speed actinide-separation methods have been developed by the Savannah River Site Central Laboratory that can be applied to nuclear materials process samples, waste solutions and environmental samples. As part of a reengineering effort to improve efficiencies and reduce operating costs, solvent extraction methods (TTA, Hexone, TBP and TIOA) used for over thirty years in the SRS Central Laboratory were replaced with new rapid extraction column methods able to handle a variety of difficult sample matrices and actinide levels. Significant costs savings were realized and costly mixed-waste controls were avoided by using applied vacuum and 50-100 micron particle-size resins from Eichrom Industries. TEVA Resin{reg_sign}, UTEVA Resin{reg_sign}, and TRU Resin{reg_sign} columns are used with flow rates of approximately two to three milliliters per minute to minimize sample turnaround times. Single-column, dual-column and sequential-cartridge methods for plutonium, uranium, neptunium, americium and curium were developed that enable rapid, cost-effective separations prior to alpha-particle counting, thermal ionization and inductively coupled plasma mass spectrometry, and laser phosphorescence measurements.

  9. A Simple and Rapid Method Based on Anti-aggregation of Silver Nanoparticles for Detection of Poly(diallyldimethylammonium chloride) in Tap Water.

    PubMed

    Trisaranakul, Wichaya; Chompoosor, Apiwat; Maneeprakorn, Weerakanya; Nacapricha, Duangjai; Choengchan, Nathawut; Teerasong, Saowapak

    2016-01-01

    A simple and rapid method was developed for the detection of poly(diallyldimethylammonium chloride) (PDADMAC) using citrate-capped silver nanoparticles (AgNPs). Detection was based on anti-aggregation of AgNPs in phosphate buffer caused by PDADMAC. Due to its positive charges, PDADMAC was adsorbed onto AgNPs via electrostatic interaction with citrate, which resulted in the charges at the particle surfaces to become positive and caused repulsion among particles. Furthermore, long-chain PDADMAC provided steric hindrance. These two effects promoted the dispersion of AgNPs in the phosphate buffer. A change in the state of dispersion influenced the surface plasmon resonance (SPR) of AgNPs. Therefore, in this work, the concentration of PDADMAC was determined by monitoring changes in absorbance (at 396 nm) caused by SPR of AgNPs. Under optimal conditions, the calibration was linear over the range of 1 to 100 mg L(-1) with a detection limit of 0.7 mg L(-1). Satisfactory precision was obtained (RSD = 2.8%). This method was successfully applied to the determination of PDADMAC in tap water samples. The recoveries ranged from 86.0 - 107.5%. PMID:27396659

  10. Isolation and identification of Duck tembusu virus strain lH and development of latex-agglutination diagnostic method for rapid detection of antibodies.

    PubMed

    Wang, Quanxi; Wen, Yaping; Yifan Huang; Wu, Yijian; Cai, Yilong; Xu, Lihui; Wang, Changkang; Li, Ang; Wu, Baocheng; Chen, Jilong

    2014-12-01

    SUMMARY. An outbreak of egg-drop syndrome occurred on a Sheldrake duck farm in Longhai in Fujian Province, China, in 2012. The main clinical symptoms were sharply reduced egg production, crooked necks, and death. We isolated the virus from the sick ducks, identified it, and observed the histopathologic changes after viral infection. We detected viral RNA in the blood and feces of the infected ducks and developed a latex-agglutination diagnostic method to detect anti-Tembusu-virus antibodies. Our results show that the pathogenic virus is a Tembusu virus. The histopathologic changes included follicular cell degeneration and necrosis, follicular cavity filled with blood cells, massive necrosis in the brain, and degeneration and necrosis of the nerve and glial cells. When the transmission of the virus in the infected ducks was studied, the duck blood was positive for viral nucleic acid for up to 29 days, and the feces were positive for viral nucleic acid for up to 13 days. We successfully established a simple, rapid, and easy- to-use latex-agglutination diagnostic method for the detection of antibodies against duck Tembusu virus. PMID:25619007

  11. STABILIZATION OF A RACTOPAMINE ENZYME CONJUGATE IN AQUEOUS SOLUTION, A RAPID AND CONVENIENT IMMUNOASSAY METHOD FOR THE DETECTION OF RACTOPAMINE.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is an increasing demand for a sensitive screening method for ractopamine because of the zero tolerance policy in many countries. Most of the commercially available ractopamine ELISA kits require concentrated conjugate to be diluted prior to use. We have observed that the highly diluted racto...

  12. Rapid Detection of Polymyxin Resistance in Enterobacteriaceae

    PubMed Central

    Jayol, Aurélie; Poirel, Laurent

    2016-01-01

    For identification of polymyxin resistance in Enterobacteriaceae, we developed a rapid test that detects glucose metabolization associated with bacterial growth in the presence of a defined concentration of colistin or polymyxin B. Formation of acid metabolites is evidenced by a color change (orange to yellow) of a pH indicator (red phenol). To evaluate the test, we used bacterial colonies of 135 isolates expressing various mechanisms of colistin resistance (intrinsic, chromosomally encoded, and plasmid-mediated MCR-1) and 65 colistin-susceptible isolates. Sensitivity and specificity were 99.3% and 95.4%, respectively, compared with the standard broth microdilution method. This new test is inexpensive, easy to perform, sensitive, specific, and can be completed in <2 hours. It could be useful in countries facing endemic spread of carbapenemase producers and for which polymyxins are last-resort drugs. PMID:27191712

  13. Rapid Detection of Polymyxin Resistance in Enterobacteriaceae.

    PubMed

    Nordmann, Patrice; Jayol, Aurélie; Poirel, Laurent

    2016-06-01

    For identification of polymyxin resistance in Enterobacteriaceae, we developed a rapid test that detects glucose metabolization associated with bacterial growth in the presence of a defined concentration of colistin or polymyxin B. Formation of acid metabolites is evidenced by a color change (orange to yellow) of a pH indicator (red phenol). To evaluate the test, we used bacterial colonies of 135 isolates expressing various mechanisms of colistin resistance (intrinsic, chromosomally encoded, and plasmid-mediated MCR-1) and 65 colistin-susceptible isolates. Sensitivity and specificity were 99.3% and 95.4%, respectively, compared with the standard broth microdilution method. This new test is inexpensive, easy to perform, sensitive, specific, and can be completed in <2 hours. It could be useful in countries facing endemic spread of carbapenemase producers and for which polymyxins are last-resort drugs. PMID:27191712

  14. A Rapid, Novel Method of Volumetric Assessment of MRI-Detected Subchondral Bone Marrow Lesions in Knee Osteoarthritis

    PubMed Central

    Ratzlaff, C.; Guermazi, A.; Collins, J.; Katz, J.N.; Losina, E.; Vanwyngaarden, C.; Russell, R.; Iranpour, T.; Duryea, J.

    2013-01-01

    Purpose To assess reliability and validity of a semi-automated quantitative method for osteoarthritis (OA)-related bone marrow lesion (BML) assessment in the femur and tibia. Methods In a cross-sectional study of subjects with knee OA, we examined concurrent criterion and clinical validation of a novel method of semi-automated quantitative BML measurement. The primary outcome was total segmented BML volume in femoral and tibial medial and lateral knee compartments. Criterion validation was examined through comparison of BML volumes with Whole-Organ Magnetic Resonance Imaging Score (WORMS) scoring. Clinical validation was examined via associations of tibial and femoral BML volume with the Western Ontario and McMaster University OA Index weight-bearing pain questions. Results Among the 115 subjects, mean age was 62 years, mean BMI 30.4 (kg/m2), 84% were white and 52% male. The ICC for intra-reader reliability was 0.96 and 0.97 for inter-reader reliability. Significant Spearman's correlations were found between segmented BML volume and WORMS BML scoring for tibial medial (0.75) and lateral (0.73) compartments, and for femoral medial (0.72) and lateral (0.88) compartments. Significant positive associations were found between weight-bearing pain and total femoral BML volume (p<0.003), but not total tibial BML (p<0.101) Conclusion We have documented a moderately strong correlation between a novel measurement method of femoral and tibial BML volume and semi-quantitative WORMS scores, providing evidence of criterion validity. The hypothesis that weight-bearing pain was associated with BML volume was confirmed for total femoral BML volume but not total tibial BML volume. The lack of association between tibial BML volume and pain requires further investigation. PMID:23518154

  15. Nanostructured bioluminescent sensor for rapidly detecting thrombin.

    PubMed

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

    2016-03-15

    Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470 nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10 min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8 nM to 8 μM in both buffer and human urine spiked samples. The limit of detection is as low as 80 pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine. PMID:26397418

  16. The Development of a Novel, Validated, Rapid and Simple Method for the Detection of Sarcocystis fayeri in Horse Meat in the Sanitary Control Setting.

    PubMed

    Furukawa, Masato; Minegishi, Yasutaka; Izumiyama, Shinji; Yagita, Kenji; Mori, Hideto; Uemura, Taku; Etoh, Yoshiki; Maeda, Eriko; Sasaki, Mari; Ichinose, Kazuya; Harada, Seiya; Kamata, Yoichi; Otagiri, Masaki; Sugita-Konishi, Yoshiko; Ohnishi, Takahiro

    2016-01-01

    Sarcocystis fayeri (S. fayeri) is a newly identified causative agent of foodborne disease that is associated with the consumption of raw horse meat. The testing methods prescribed by the Ministry of Health, Labour and Welfare of Japan are time consuming and require the use of expensive equipment and a high level of technical expertise. Accordingly, these methods are not suitable for use in the routine sanitary control setting to prevent outbreaks of foodborne disease. In order to solve these problems, we have developed a new, rapid and simple testing method using LAMP, which takes only 1 hour to perform and which does not involve the use of any expensive equipment or expert techniques. For the validation of this method, an inter-laboratory study was performed among 5 institutes using 10 samples infected with various concentrations of S. fayeri. The results of the inter-laboratory study demonstrated that our LAMP method could detect S. fayeri at concentrations greater than 10(4) copies/g. Thus, this new method could be useful in screening for S. fayeri as a routine sanitary control procedure. PMID:27350431

  17. Computational analysis of open loop handwriting movements in Parkinson's disease: a rapid method to detect dopamimetic effects.

    PubMed

    Eichhorn, T E; Gasser, T; Mai, N; Marquardt, C; Arnold, G; Schwarz, J; Oertel, W H

    1996-05-01

    We used a computational analysis of open loop handwriting movements and a clinical rating scale for monitoring the effect of apomorphine in 16 patients with early untreated parkinsonism [subsequently L-DOPA responsive, probable Parkinson's disease (PD)], six patients with long-standing PD with L-DOPA associated motor fluctuations, and seven patients with known L-DOPA unresponsive parkinsonism. Subjects were instructed to write fluently concentric circles of approximately 12 mm in diameter. Movements were recorded for two periods of 3 s each, using a digitizing tablet. Mean peak velocity (Vmax) and mean peak acceleration (Amax) were determined. In addition, two sensitive indices describing the degree of automation of handwriting were derived: (a) NCV, calculated as the mean Number of Changes in direction of Velocity per half circle, and (b) NCA, the mean Number of Changes in the direction of Acceleration. Clinical rating was performed according to the Unified Parkinson's Disease Rating Scale part III (UPDRS III). After apomorphine injection, the patients with early untreated probable PD showed significant improvement of Vmax, Amax, NCV, NCA, and UPDRS III scores. Likewise, the patients with long-standing PD improved significantly in all kinematic parameters and UPDRS III scores. Patients with L-DOPA unresponsive parkinsonism failed to change significantly in any of the parameters tested. These observations suggest that the computer-assisted analysis of automated handwriting movements can be used as an objective quick method for quantifying dopamimetic effects on the kinematics of handwriting movements in parkinsonian patients. PMID:8723147

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system.

    PubMed

    Goh, Khye Seng; Rastogi, Nalin

    2010-05-01

    Mycobacterium tuberculosis reduces nitrate very strongly as compared to Mycobacterium bovis and M. bovis BCG. Nitrate reductase, in conjunction with niacin accumulation, constitutes one of the major biochemical tests used in clinical microbiology laboratories to differentiate M. tuberculosis from other members of the M. tuberculosis complex, as well as nontuberculous Mycobacteria. Determination of nitrate reductase activity is currently performed using cultures grown on solid media with a slow detection time and the need for large quantities of bacilli, as otherwise the test is not reliable. Hereby, we propose a nitrate reduction test coupled to Bactec MGIT960 system as a simple, rapid and economic method with a total gain of time of about 3 to 4weeks over the conventional solid medium. In our study, almost all the M. tuberculosis and Mycobacterium canettii strains gave a strongly positive nitrate reductase result within 1day of positive detection by the MGIT960 system. In contrast, M. bovis, M. bovis BCG and M. africanum strains remained negative even after 14days of incubation. The possibility to detect nitrate reductase within 1 to 3days of a positive culture using MGIT960 opens new perspectives with the possibility of confirming M. tuberculosis - starting directly from pathological specimens. PMID:20298726

  20. Development of the molecular methods for rapid detection and differentiation of Fusarium oxysporum and F. oxysporum f. sp. niveum in Taiwan.

    PubMed

    Lin, Ying-Hong; Chen, Kan-Shu; Chang, Jing-Yi; Wan, Yu-Ling; Hsu, Ching-Chi; Huang, Jenn-Wen; Chang, Pi-Fang Linda

    2010-09-30

    Fusarium wilt, caused by Fusarium oxysporum (Fo), is one of the most important fungal diseases worldwide. Like other plant pathogens, Fo displays specialized forms in association with its hosts. For example, F. oxysporum f. sp. niveum (Fon) is the damaging pathogen causing Fusarium wilt disease on watermelon, whereas F. oxysporum f. sp. cubense is the pathogen that infects banana. A rapid and reliable pathogen identification or disease diagnosis is essential for the integrated disease management practices in many crops. In this study, two new primer sets, Fon-1/Fon-2 and FnSc-1/FnSc-2, were developed to differentiate Fon and Fo, respectively. The PCR method using the novel primer sets has high sensitivity to detect Fon when the DNA concentration was as low as 0.01 pg or when the conidia number was as few as 5. In comparison with the published primer set, the Fon-1/Fon-2 primer set, derived from the sequence of OP-M12 random primer-amplified fragment, produced a 174 bp DNA fragment, and was more specific to Fon in Taiwan. In addition, with optimized PCR parameters, the molecular method using the Fon-1/Fon-2 primer set could directly detect Fon even when watermelon samples were collected in its early stage of disease development. PMID:20471505

  1. An on-line normal-phase high performance liquid chromatography method for the rapid detection of radical scavengers in non-polar food matrixes.

    PubMed

    Zhang, Qiang; van der Klift, Elbert J C; Janssen, Hans-Gerd; van Beek, Teris A

    2009-10-23

    An on-line method for the rapid pinpointing of radical scavengers in non-polar mixtures like vegetable oils was developed. To avoid problems with dissolving the sample, normal-phase chromatography on bare silica gel was used with mixtures of hexane and methyl tert-butyl ether as the eluent. The high performance liquid chromatography-separated analytes are mixed post-column with a solution of stable free radicals in hexane. Reduced levels of the radical as a result of a reaction with a radical scavenger are detected as negative peaks by an absorbance detector. After investigating a number of different reagents, solvents, concentrations and solution flow rates an optimized instrumental set-up incorporating a superloop for pulse-free delivery of the reagent solution is presented. Both 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,6-di-tert-butyl-alpha-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy (galvinoxyl) were used as stable free radicals. The method is suitable for both isocratic and gradient HPLC operation. The method has a simple experimental protocol, uses standard instruments and inexpensive and stable reagents, and accepts any hexane-soluble sample. It can also be used for semi-quantitative analysis. The method was applied to several pure, non-polar natural antioxidants, vegetable oils and lipid-soluble rosemary extract. The limits of detection varied from 0.2 to 176 microg/ml, depending on the compound tested. PMID:19726044

  2. Rapid auditory learning of temporal gap detection.

    PubMed

    Mishra, Srikanta K; Panda, Manasa R

    2016-07-01

    The rapid initial phase of training-induced improvement has been shown to reflect a genuine sensory change in perception. Several features of early and rapid learning, such as generalization and stability, remain to be characterized. The present study demonstrated that learning effects from brief training on a temporal gap detection task using spectrally similar narrowband noise markers defining the gap (within-channel task), transfer across ears, however, not across spectrally dissimilar markers (between-channel task). The learning effects associated with brief training on a gap detection task were found to be stable for at least a day. These initial findings have significant implications for characterizing early and rapid learning effects. PMID:27475211

  3. Rapid detection and identification of infectious agents

    SciTech Connect

    Kingsbury, D.T.; Falkow, S.

    1985-01-01

    This book contains papers divided among five sections. Some of the paper titles are: Aspects of Using Nucleic Acid Filter Hybridization to Characterize and Detect Enteroviral RNAs; Rapid Identification of Lesihmania Species using Specific Hybridization of Kinetoplast DNA Sequences; Selection of DNA Probes for use in the Diagnosis of Infectious Disease; and Summary of DNA Probes.

  4. Rapid photometric detection of thymine residues partially flipped out of double helix as a method for direct scanning of point mutations and apurinic DNA sites.

    PubMed

    Logvina, N A; Yakubovskaya, M G; Dolinnaya, N G

    2011-02-01

    A spectroscopic assay for detection of extrahelical thymine residues in DNA heteroduplexes under their modification by potassium permanganate has been developed. The assay is based on increase in absorbance at 420 nm due to accumulation of thymidine oxidation intermediates and soluble manganese dioxide. The analysis was carried out using a set of 19-bp DNA duplexes containing unpaired thymidines opposite tetrahydrofuranyl derivatives mimicking a widespread DNA damage (apurinic (AP) sites) and a library of 50-bp DNA duplexes containing all types of base mismatches in different surroundings. The relation between the selectivity of unpaired T oxidation and the thermal stability of DNA double helix was investigated. The method described here was shown to discriminate between DNA duplexes with one or two AP sites and to reveal thymine-containing mismatches and all noncanonical base pairs in AT-surroundings. Comparative results of CCM analysis and the rapid photometric assay for mismatch detection are demonstrated for the first time in the same model system. The chemical reactivity of target thymines was shown to correlate with local disturbance of double helix at the mismatch site. As the spectroscopic assay does not require the DNA cleavage reaction and gel electrophoresis, it can be easily automated and used for primary screening of somatic mutations. PMID:21568858

  5. Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

    PubMed

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-06-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. PMID:24648561

  6. Sensitive and Rapid HPLC Method for Determination of Memantine in Human Plasma Using OPA Derivatization and Fluorescence Detection: Application to Pharmacokinetic Studies

    PubMed Central

    Zarghi, Afshin; Shafaati, Alireza; Foroutan, Seyed Mohsen; Khoddam, Arash; Madadian, Babak

    2010-01-01

    A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of memantine in human plasma after derivatization with o-phthaldialdehyde (OPA) and fluorescence detection. Amantadine was used as internal standard. The derivatized memantine and amantadine were eluted in less than 10 min with no interference from endogenous plasma peaks. The analysis was carried out on a monolithic silica column (Chromolith Performance RP-18e, 100×4.6 mm). The mobile phase was composed of a mixture of acetonitrile and 0.025 M phosphate buffer (50:50, v/v, pH=4.6) with a flow rate of 2.5 mLmin−1. The excitation and emission wavelengths were set at 335 nm and 440 nm respectively. The assay enables the measurement of memantine for therapeutic drug monitoring with a lower quantification limit of 2 ngmL−1. The method involves simple extraction procedure and analytical recovery was 82.8± 0.9%. The calibration curve was linear over the concentration range 2–80 ngmL−1. The coefficients of variation for inter-day and intra-day assay were found to be less than 8%. The method was successfully applied to pharmacokinetic studies in humans. PMID:21179320

  7. A RAPID METHOD FOR THE EXTRACTION OF FUNGAL DNA FROM ENVIRONMENTAL SAMPLES: EVALUATION IN THE QUANTITATIVE ANALYSIS OF MEMNONIELLA ECHINATA CONIDIA USING REAL TIME DETECTION OF PCR PRODUCTS

    EPA Science Inventory

    New technologies are creating the potential for using nucleic acid sequence detection to perform routine microbiological analyses of environmental samples. Our laboratory has recently reported on the development of a method for the quantitative detection of Stachybotrys chartarum...

  8. Rapid methods and automation in dairy microbiology.

    PubMed

    Vasavada, P C

    1993-10-01

    The importance of microbiology to the dairy industry has been demonstrated by recent outbreaks of foodborne illness associated with consumption of milk and dairy products that had been contaminated with pathogenic organisms or toxins. Undesirable microorganisms constitute the primary hazard to safety, quality, and wholesomeness of milk and dairy foods. Consequently, increased emphasis has been placed on the microbiological analysis of milk and dairy products designed to evaluate quality and to ensure safety and regulatory compliance. The focus of dairy microbiology, however, remains largely on conventional methods: plate counts, most probable numbers, and dye reduction tests. These methods are slow, tedious, intensive in their requirements for material and labor, and often not suitable for assessing the quality and shelf-life of perishable dairy foods. With the exception of coliforms, Salmonella, and Staphylococcus aureus, isolation and characterization of various organisms occurring in milk and milk products are seldom a part of the routine microbiological analysis in the dairy industry. Recent emphasis on the programs based on HACCP (Hazard Analysis and Critical Control Points) for total quality management in the dairy industry and increased demand for microbiological surveillance of products, process, and environment have led to increased interest in rapid methods and automation in microbiology. Several methods for rapid detection, isolation, enumeration, and characterization of microorganisms are being adapted by the dairy industry. This presentation reviews rapid methods and automation in microbiology for microbiological analysis of milk and dairy products. PMID:8227634

  9. Nucleic acid detection methods

    DOEpatents

    Smith, C.L.; Yaar, R.; Szafranski, P.; Cantor, C.R.

    1998-05-19

    The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3{prime}-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated. 18 figs.

  10. Nucleic Acid Detection Methods

    DOEpatents

    Smith, Cassandra L.; Yaar, Ron; Szafranski, Przemyslaw; Cantor, Charles R.

    1998-05-19

    The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.

  11. The Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Salmonella enterica serovar Typhi

    PubMed Central

    Fan, Fenxia; Du, Pengcheng; Kan, Biao; Yan, Meiying

    2015-01-01

    Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol. PMID:25910059

  12. Rapid detection of irradiated frozen hamburgers

    NASA Astrophysics Data System (ADS)

    Delincée, Henry

    2002-03-01

    DNA comet assay can be employed as a rapid and inexpensive screening test to check whether frozen ground beef patties (hamburgers) have been irradiated as a means to increase their safety by eliminating pathogenic bacteria, e.g. E. coli O157:H7. Such a detection procedure will provide an additional check on compliance with existing regulations, e.g. enforcement of labelling and rules in international trade. Frozen ready prepared hamburgers from the market place were `electron irradiated' with doses of 0, 1.3, 2.7, 4.5 and 7.2kGy covering the range of potential commercial irradiation. DNA fragmentation in the hamburgers was made visible within a few hours using the comet assay, and non-irradiated hamburgers could be easily discerned from the irradiated ones. Even after 9 months of frozen storage, irradiated hamburgers could be identified. Since DNA fragmentation may also occur with other food processes (e.g. temperature abuse), positive screening tests shall be confirmed using a validated method to specifically prove an irradiation treatment, e.g. EN 1784 or EN 1785.

  13. Laboratory evaluation on the sensitivity and specificity of a novel and rapid detection method for malaria diagnosis based on magneto-optical technology (MOT)

    PubMed Central

    2010-01-01

    Background This study describes the laboratory evaluation of a novel diagnostic platform for malaria. The Magneto Optical Test (MOT) is based on the bio-physical detection of haemozoin in clinical samples. Having an assay time of around one minute, it offers the potential of high throughput screening. Methods Blood samples of confirmed malaria patients from different regions of Africa, patients with other diseases and healthy non-endemic controls were used in the present study. The samples were analysed with two reference tests, i.e. an histidine rich protein-2 based rapid diagnostic test (RDT) and a conventional Pan-Plasmodium PCR, and the MOT as index test. Data were entered in 2 × 2 tables and analysed for sensitivity and specificity. The agreement between microscopy, RDT and PCR and the MOT assay was determined by calculating Kappa values with a 95% confidence interval. Results The observed sensitivity/specificity of the MOT test in comparison with clinical description, RDT or PCR ranged from 77.2 - 78.8% (sensitivity) and from 72.5 - 74.6% (specificity). In general, the agreement between MOT and the other assays is around 0.5 indicating a moderate agreement between the reference and the index test. However, when RDT and PCR are compared to each other, an almost perfect agreement can be observed (k = 0.97) with a sensitivity and specificity of >95%. Conclusions Although MOT sensitivity and specificity are currently not yet at a competing level compared to other diagnostic test, such as PCR and RDTs, it has a potential to rapidly screen patients for malaria in endemic as well as non-endemic countries. PMID:20642834

  14. Electrochemical-surface enhanced Raman spectroscopy (E-SERS) of uric acid: a potential rapid diagnostic method for early preeclampsia detection.

    PubMed

    Goodall, Barbara L; Robinson, Ashley M; Brosseau, Christa L

    2013-02-01

    An increased level of uric acid in urine and plasma is indicative of the development of preeclampsia, a hypertensive disorder that can occur during pregnancy. The preliminary steps towards developing a rapid tool for early diagnosis of preeclampsia using electrochemical SERS (E-SERS) for the detection of uric acid in urine are presented herein. Characterization of the uric acid species was completed using cyclic voltammetry, UV spectroscopy, Raman spectroscopy and electrochemical surface-enhanced Raman spectroscopy (E-SERS). E-SERS was capable of easily detecting uric acid directly at concentrations <1 mM in urine simulant, without the need for costly enzymes and bulky equipment, and thus demonstrates promise as a rapid point-of-care diagnostic tool for detection of early onset preeclampsia in developing nation settings. PMID:23187309

  15. A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma.

    PubMed

    Peer, Cody J; Spencer, Shawn D; VanDenBerg, Dustin A H; Pacanowski, Michael A; Horenstein, Richard B; Figg, William D

    2012-01-01

    A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix(®)) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2 μm-C(18) column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically relevant concentration range of 0.01-50 ng/mL for parent clopidogrel and 0.1-150 ng/mL (r(2)=0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) <±12% and precision (%CV) of <±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range. PMID:22169056

  16. A rapid method for preparation of nucleic acid extracts from potato psyllids for detection of 'Candidatus Liberibacter solancearum' and molecular analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid method has been developed and validated for PCR analysis of potato psyllids for Candidatus Liberibacter solanacearum (Lso), the causal agent of zebra chip disease of potatoes. The method is also suitable for PCR amplification and high resolution melting analysis of the cytochrome oxidase I ...

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

    PubMed

    Aswani, Shankar; Lauer, Matthew

    2014-06-01

    When sudden catastrophic events occur, it becomes critical for coastal communities to detect and respond to environmental transformations because failure to do so may undermine overall ecosystem resilience and threaten people's livelihoods. We therefore asked how capable of detecting rapid ecological change following massive environmental disruptions local, indigenous people are. We assessed the direction and periodicity of experimental learning of people in the Western Solomon Islands after a tsunami in 2007. We compared the results of marine science surveys with local ecological knowledge of the benthos across 3 affected villages and 3 periods before and after the tsunami. We sought to determine how people recognize biophysical changes in the environment before and after catastrophic events such as earthquakes and tsunamis and whether people have the ability to detect ecological changes over short time scales or need longer time scales to recognize changes. Indigenous people were able to detect changes in the benthos over time. Detection levels differed between marine science surveys and local ecological knowledge sources over time, but overall patterns of statistically significant detection of change were evident for various habitats. Our findings have implications for marine conservation, coastal management policies, and disaster-relief efforts because when people are able to detect ecological changes, this, in turn, affects how they exploit and manage their marine resources. PMID:24528101

  18. Weld leaks rapidly and safely detected

    NASA Technical Reports Server (NTRS)

    1965-01-01

    Test method detects leaks that occur during hydrostatic pressure testing of welded joints in metal tanks. A strip of aluminum foil and a strip of water-soluble paper are placed over the weld. A voltage applied between the tank wall and the foil strip is monitored to detect a decrease in ohmic resistance caused by water leakage into the paper layer.

  19. Rapid Detection of the Varicella Zoster Virus

    NASA Technical Reports Server (NTRS)

    Lewis, Michelle P.; Harding, Robert

    2011-01-01

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

  20. Rapid electrochemical detection on a mobile phone.

    PubMed

    Lillehoj, Peter B; Huang, Ming-Chun; Truong, Newton; Ho, Chih-Ming

    2013-08-01

    We present a compact mobile phone platform for rapid, quantitative biomolecular detection. This system consists of an embedded circuit for signal processing and data analysis, and disposable microfluidic chips for fluidic handling and biosensing. Capillary flow is employed for sample loading, processing, and pumping to enhance operational portability and simplicity. Graphical step-by-step instructions displayed on the phone assists the operator through the detection process. After the completion of each measurement, the results are displayed on the screen for immediate assessment and the data is automatically saved to the phone's memory for future analysis and transmission. Validation of this device was carried out by detecting Plasmodium falciparum histidine-rich protein 2 (PfHRP2), an important biomarker for malaria, with a lower limit of detection of 16 ng mL(-1) in human serum. The simple detection process can be carried out with two loading steps and takes 15 min to complete each measurement. Due to its compact size and high performance, this device offers immense potential as a widely accessible, point-of-care diagnostic platform, especially in remote and rural areas. In addition to its impact on global healthcare, this technology is relevant to other important applications including food safety, environmental monitoring and biosecurity. PMID:23689554

  1. A rapid method for airborne tritium analysis

    SciTech Connect

    Hofstetter, K.J.; Wilson, H.T. )

    1991-11-01

    Tritium is one of the principal radionuclides released to the environment from nuclear fuel and target reprocessing, heavy-water production, and other nuclear industry operations. For example, the majority of the off-site dose to the public at the Savannah River site (SRS) in 1988 was from tritium oxide (HTO). The absorbed dose is highly dependent on chemical form; HTO is 10,000 more hazardous than the elemental form (HT). Commercially available tritium monitors do not discriminate between chemical form and have high detection limits. Consequently, tedious laboratory methods must be used to analyze HTO in air. Desiccants are used to remove all the water from an air sample. The tritiated water is then desorbed and analyzed by liquid scintillation spectrometry. The method is complex and takes several hours to complete. During an unplanned release, present-time atmospheric tritium concentrations are never available. To improve emergency response capabilities, a rapid sampling and analysis method was developed for measuring low-level HTO concentrations in air. Standard desiccant sampling and water desorption procedure was modified for use in the SRS mobile laboratory, which is equipped with a liquid scintillation counter. These tests indicate that an HTO concentration of 0.2% DCG (7 Bq/m{sup 3}) can be detected by this method with a 10-min sample collection time and a 10-min count.

  2. Optical sensor for rapid microbial detection

    NASA Astrophysics Data System (ADS)

    Al-Adhami, Mustafa; Tilahun, Dagmawi; Rao, Govind; Kostov, Yordan

    2016-05-01

    In biotechnology, the ability to instantly detect contaminants is key to running a reliable bioprocess. Bioprocesses are prone to be contaminated by cells that are abundant in our environment; detection and quantification of these cells would aid in the preservation of the bioprocess product. This paper discusses the design and development of a portable kinetics fluorometer which acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye, and plots it. Resazurin is used as an indicator dye since the viable contaminant cells reduce Resazurin toResorufin, the latter being strongly fluorescent. A photodiode detects fluorescence change by generating current proportional to the intensity of the light that reached it, and a trans-impedance differential op-amp ensures amplification of the photodiodes' signal. A microfluidic chip was designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the Resazurin reduction rate. E. coli in LB media, along with Resazurin were injected into the microfluidic chip. The optical sensor detected the presence of E. coli in the media based on the fluorescence change that occurred in the indicator dye in concentrations as low as 10 CFU/ml. A method was devised to detect and determine an approximate amount of contamination with this device. This paper discusses application of this method to detect and estimate sample contamination. This device provides fast, accurate, and inexpensive means to optically detect the presence of viable cells.

  3. A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on label-free amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogen...

  4. Rapid method for the simultaneous detection of boar taint compounds by means of solid phase microextraction coupled to gas chromatography/mass spectrometry.

    PubMed

    Verplanken, Kaat; Wauters, Jella; Van Durme, Jim; Claus, Dirk; Vercammen, Joeri; De Saeger, Sarah; Vanhaecke, Lynn

    2016-09-01

    Because of animal welfare issues, the voluntary ban on surgical castration of male piglets, starting January 2018 was announced in a European Treaty. One viable alternative is the fattening of entire male pigs. However, this can cause negative consumer reactions due to the occurrence of boar taint and possibly lead to severe economic losses in pig husbandry. In this study, headspace solid phase microextraction (HS-SPME) coupled to GC-MS was used in the development and optimization of a candidate method for fast and accurate detection of the boar taint compounds. Remarkably fast extraction (45s) of the boar taint compounds from adipose tissue was achieved by singeing the fat with a soldering iron while released volatiles were extracted in-situ using HS-SPME. The obtained method showed good performance characteristics after validation according to CD 2002/657/EC and ISO/IEC 17025 guidelines. Moreover, cross-validation with an in-house UHPLC-HR-Orbitrap-MS method showed good agreement between an in-laboratory method and the new candidate method for the fast extraction and detection of skatole and androstenone, which emphasizes the accuracy of this new SPME-GC-MS method. Threshold detection of the boar taint compounds on a portable GC-MS could not be achieved. However, despite the lack of sensitivity obtained on the latter instrument, a very fast method with run-to-run time of 3.5min for the detection of the boar taint compounds was developed. PMID:27492596

  5. Rapid onsite detection of bacterial spores of biothreat importance by paper-based colorimetric method using erbium-pyrocatechol violet complex.

    PubMed

    Shivakiran, M S; Venkataramana, M; Lakshmana Rao, P V

    2016-01-01

    Dipicolinic acid (DPA) is an important chemical marker for the detection of bacterial spores. In this study, complexes of lanthanide series elements such as erbium, europium, neodymium, and terbium were prepared with pyrocatechol violet and effectively immobilized the pyrocatechol violet (PV)-metal complex on a filter paper using polyvinyl alcohol. These filter paper strips were employed for the onsite detection of bacterial spores. The test filter papers were evaluated quantitatively with different concentrations of DPA and spores of various bacteria. Among the four lanthanide ions, erbium displayed better sensitivity than the other ions. The limit of detection of this test for DPA was 60 μM and 5 × 10(6) spores. The effect of other non-spore-forming bacteria and interfering chemicals on the test strips was also evaluated. The non-spore-forming bacteria did not have considerable effect on the test strip whereas chemicals such as EDTA had significant effects on the test results. The present test is rapid and robust, capable of providing timely results for better judgement to save resources on unnecessary decontamination procedures during false alarms. PMID:26603759

  6. The effect of sample size and matrix on the ability of rapid methods to detect E. coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective. E. coli O157:H7 testing schemes and test methods are constantly evolving areas and as better ways of testing come about, there is a tendency to implement these changes while not recognizing potential problems that may result later. For example, most detection kits are validated accordin...

  7. Rapid aneuploidy detection or karyotyping? Ethical reflection

    PubMed Central

    de Jong, Antina; Dondorp, Wybo J; Timmermans, Daniëlle RM; van Lith, Jan MM; de Wert, Guido MWR

    2011-01-01

    No consensus exists whether women at increased risk for trisomy 21, 13, and 18 should be offered stand-alone rapid aneuploidy detection (RAD) or karyotyping. In this paper, the ethical implications of a fast, relatively cheap and targeted RAD are examined. The advantages of RAD seem less robust than its proponents suggest. Fast test results only give a short-term psychological benefit. The cost advantage of RAD is apparent, but must be weighed against consequences like missed abnormalities, which are evaluated differently by professionals and pregnant women. Since pre-test information about RAD will have to include telling women about karyotyping as a possible alternative, the advantage of RAD in terms of the quantity of information that needs to be given may also be smaller than suggested. We conclude that none of the supposed arguments in favour of RAD is decisive in itself. Whether the case for RAD may still be regarded as convincing when taking these arguments together seems to depend on one's implicit view of what prenatal screening is about. Are we basically dealing with a test for trisomy 21 and a few conditions more? Or are there good grounds for also testing for the wider range of abnormalities that karyotyping can detect? As professionals and pregnant women may have different views about this, we suggest that the best approach is to offer women a choice between RAD and karyotyping. This approach is most in line with the general aim of prenatal screening: providing opportunities for autonomous reproductive choice. PMID:21629296

  8. Rapid detection of methanol in artisanal alcoholic beverages

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  9. Rapid Method Using Two Microbial Enzymes for Detection of l-Abrine in Food as a Marker for the Toxic Protein Abrin

    PubMed Central

    Dodge, Anthony G.; Carrasquillo, Kelvin; Rivera, Luis; Xu, Lei; Wackett, Lawrence P.

    2014-01-01

    Abrin is a toxic protein produced by the ornamental plant Abrus precatorius, and it is of concern as a biothreat agent. The small coextracting molecule N-methyl-l-tryptophan (l-abrine) is specific to members of the genus Abrus and thus can be used as a marker for the presence or ingestion of abrin. Current methods for the detection of abrin or l-abrine in foods and other matrices require complex sample preparation and expensive instrumentation. To develop a fast and portable method for the detection of l-abrine in beverages and foods, the Escherichia coli proteins N-methyltryptophan oxidase (MTOX) and tryptophanase were expressed and purified. The two enzymes jointly degraded l-abrine to products that included ammonia and indole, and colorimetric assays for the detection of those analytes in beverage and food samples were evaluated. An indole assay using a modified version of Ehrlich's/Kovac's reagent was more sensitive and less subject to negative interferences from components in the samples than the Berthelot ammonia assay. The two enzymes were added into food and beverage samples spiked with l-abrine, and indole was detected as a degradation product, with the visual lower detection limit being 2.5 to 10.0 μM (∼0.6 to 2.2 ppm) l-abrine in the samples tested. Results could be obtained in as little as 15 min. Sample preparation was limited to pH adjustment of some samples. Visual detection was found to be about as sensitive as detection with a spectrophotometer, especially in milk-based matrices. PMID:25527549

  10. Rapid method using two microbial enzymes for detection of L-abrine in food as a marker for the toxic protein abrin.

    PubMed

    Dodge, Anthony G; Carrasquillo, Kelvin; Rivera, Luis; Xu, Lei; Wackett, Lawrence P; Sadowsky, Michael J

    2015-03-01

    Abrin is a toxic protein produced by the ornamental plant Abrus precatorius, and it is of concern as a biothreat agent. The small coextracting molecule N-methyl-l-tryptophan (l-abrine) is specific to members of the genus Abrus and thus can be used as a marker for the presence or ingestion of abrin. Current methods for the detection of abrin or l-abrine in foods and other matrices require complex sample preparation and expensive instrumentation. To develop a fast and portable method for the detection of l-abrine in beverages and foods, the Escherichia coli proteins N-methyltryptophan oxidase (MTOX) and tryptophanase were expressed and purified. The two enzymes jointly degraded l-abrine to products that included ammonia and indole, and colorimetric assays for the detection of those analytes in beverage and food samples were evaluated. An indole assay using a modified version of Ehrlich's/Kovac's reagent was more sensitive and less subject to negative interferences from components in the samples than the Berthelot ammonia assay. The two enzymes were added into food and beverage samples spiked with l-abrine, and indole was detected as a degradation product, with the visual lower detection limit being 2.5 to 10.0 μM (∼0.6 to 2.2 ppm) l-abrine in the samples tested. Results could be obtained in as little as 15 min. Sample preparation was limited to pH adjustment of some samples. Visual detection was found to be about as sensitive as detection with a spectrophotometer, especially in milk-based matrices. PMID:25527549

  11. SEARCHING FOR RAPID METHODS IN ENVIRONMENTAL BACTERIOLOGY

    EPA Science Inventory

    The search for rapid methods in sanitary bacteriology is more urgent today than ever before because of increased necessity for processing poorer quality source waters and controlling quality of sewage effluent discharges. Selection of criteria for rapid tests involving either mod...

  12. Rapid methods for identification of yeasts.

    PubMed Central

    Huppert, M; Harper, G; Sun, S H; Delanerolle, V

    1975-01-01

    Opportunistic infections by yeasts have been implicated as one of the major causes of complications in the compromised patient. Rapid recognition and identification of these yeasts is essential for patient management, but conventional liquid medium methods for completing identification tests are cumbersome and time consuming. Rapid tests have been devised based on modifications of methods commonly used in bacteriology. These rapid methods included tests for carbohydrate and nitrate assimilation, fermentation, and urease production. These were compared with several current methods for accuracy of results, for time to final identification, and for economy of time and reagents. In addition, the usual tests for pseudogerm tube formation, for production of hyphae or pseudohyphae, and for growth temperatures were included. The rapid tests achieved 96% or better accuracy compared with expected results, and 46 species of yeasts were identified in 1 to 2 days compared with the 10 to 14 days required by conventional liquid culture methods. Images PMID:1241586

  13. Rapid transdermal bloodless and reagent-free malaria detection

    NASA Astrophysics Data System (ADS)

    Lukianova-Hleb, Ekaterina Y.; Campbell, Kelly M.; Constantinou, Pamela E.; Braam, Janet; Olson, John S.; Ware, Russell E.; Sullivan, David S.; Lapotko, Dmitri

    2014-02-01

    Successful diagnosis, screening, and elimination of malaria critically depend on rapid and sensitive detection of this dangerous infection, preferably transdermally and without sophisticated reagents or blood drawing. Such diagnostic methods are not currently available. Here we show that the high optical absorbance and nanosize of endogenous heme nanoparticles called hemozoin, a unique component of all blood-stage malaria parasites, generate a transient vapor nanobubble around hemozoin in response to a short and safe near-infrared picosecond laser pulse. The acoustic signals of these malaria-specific nanobubbles provided the first transdermal non-invasive and rapid detection of a malaria infection as low as 0.00034% in animals without using any reagents or drawing blood. These on-demand transient events have no analogs among current malaria markers and probes, can detect and screen malaria in seconds and can be realized as a compact, easy to use, inexpensive and safe field technology.

  14. Detection of coliforms in drinking water using skin patches: a rapid, reliable method that does not require an external energy source.

    PubMed

    Nam, Sehee; Kim, Min-jeong; Park, MinSun; Kim, Nuri; Lee, Yu-jin; Lee, Gyu-Cheol

    2014-02-01

    The detection of coliforms requires incubation in a laboratory, generally powered using electricity. In many parts of the developing world, however, external energy sources such as electricity are not readily available. To develop a fast, reliable method for detecting coliforms in water without an external energy source, we assessed the efficacy of six test kits for the identification of coliforms in water samples. To assess the possibility of using body temperature as the sole source of heat for incubation, bacterial samples were then mixed with the enzymatic test kit reagent and attached to the human body surface using a patch system. The patches were attached to the bodies of volunteers for 24 hours and the practicality and accuracy of the patches were assessed. Coliforms were detected within 24 hours in all patches. This innovation will facilitate the testing of water quality by researchers and by economically disadvantaged people without electricity. PMID:24420783

  15. Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification.

    PubMed

    Kato, Haru; Yokoyama, Toshiyuki; Kato, Hideaki; Arakawa, Yoshichika

    2005-12-01

    We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A(+)B(+)) and TcdA-negative, TcdB-positive (A(-)B(+)) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A(+)B(+) or A(-)B(+) C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus. PMID:16333105

  16. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

    PubMed

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  17. Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  18. A rapid and sensitive method for hydroxyl radical detection on a microfluidic chip using an N-doped porous carbon nanofiber modified pencil graphite electrode.

    PubMed

    Ouyang, Jun; Li, Zhong-Qiu; Zhang, Jing; Wang, Chen; Wang, Jiong; Xia, Xing-Hua; Zhou, Guo-Jun

    2014-07-01

    Hydroxyl radicals (˙OH) play an important role in human diseases. Traditional detection methods are time consuming and require expensive instruments. Here, we present a simple and sensitive method for the detection of hydroxyl radicals on a microfluidic chip using an electrochemical technique. Aniline monomer is electrochemically polymerized on the surface of a pencil graphite electrode and carbonized at 800 °C. The resulting N-doped porous carbon nanofiber-modified pencil graphite electrode is embedded into a microfluidic chip directly as a working electrode. 4-Hydroxybenzoic acid (4-HBA) is selected as the trapping agent owing to its unique 3,4-DHBA product and high trapping efficiency. A low detection limit of 1.0 × 10(-6) M is achieved on the microfluidic chip. As a demonstration, the microfluidic chip is successfully utilized for the detection of ˙OH in cigarette smoke. The strong π-π stacking and hydrophobic interactions between the nitrogen-doped carbon materials and the pencil graphite make the modified electrode well-suited for the microfluidic chip. PMID:24834984

  19. Multigrid contact detection method

    NASA Astrophysics Data System (ADS)

    He, Kejing; Dong, Shoubin; Zhou, Zhaoyao

    2007-03-01

    Contact detection is a general problem of many physical simulations. This work presents a O(N) multigrid method for general contact detection problems (MGCD). The multigrid idea is integrated with contact detection problems. Both the time complexity and memory consumption of the MGCD are O(N) . Unlike other methods, whose efficiencies are influenced strongly by the object size distribution, the performance of MGCD is insensitive to the object size distribution. We compare the MGCD with the no binary search (NBS) method and the multilevel boxing method in three dimensions for both time complexity and memory consumption. For objects with similar size, the MGCD is as good as the NBS method, both of which outperform the multilevel boxing method regarding memory consumption. For objects with diverse size, the MGCD outperform both the NBS method and the multilevel boxing method. We use the MGCD to solve the contact detection problem for a granular simulation system based on the discrete element method. From this granular simulation, we get the density property of monosize packing and binary packing with size ratio equal to 10. The packing density for monosize particles is 0.636. For binary packing with size ratio equal to 10, when the number of small particles is 300 times as the number of big particles, the maximal packing density 0.824 is achieved.

  20. Staph ID/R: a Rapid Method for Determining Staphylococcus Species Identity and Detecting the mecA Gene Directly from Positive Blood Culture

    PubMed Central

    Pasko, Chris; Dunn, John; Jaeckel, Heidi; Nieuwlandt, Dan; Weed, Diane; Woodruff, Evelyn; Zheng, Xiaotian

    2012-01-01

    Rapid diagnosis of staphylococcal bacteremia directs appropriate antimicrobial therapy, leading to improved patient outcome. We describe herein a rapid test (<75 min) that can identify the major pathogenic strains of Staphylococcus to the species level as well as the presence or absence of the methicillin resistance determinant gene, mecA. The test, Staph ID/R, combines a rapid isothermal nucleic acid amplification method, helicase-dependent amplification (HDA), with a chip-based array that produces unambiguous visible results. The analytic sensitivity was 1 CFU per reaction for the mecA gene and was 1 to 250 CFU per reaction depending on the staphylococcal species present in the positive blood culture. Staph ID/R has excellent specificity as well, with no cross-reactivity observed. We validated the performance of Staph ID/R by testing 104 frozen clinical positive blood cultures and comparing the results with rpoB gene or 16S rRNA gene sequencing for species identity determinations and mecA gene PCR to confirm mecA gene results. Staph ID/R agreed with mecA gene PCR for all samples and agreed with rpoB/16S rRNA gene sequencing in all cases except for one sample that contained a mixture of two staphylococcal species, one of which Staph ID/R correctly identified, for an overall agreement of 99.0% (P < 0.01). Staph ID/R could potentially be used to positively affect patient management for Staphylococcus-mediated bacteremia. PMID:22170912

  1. Rapid determination of letrozole, citalopram and their metabolites by high performance liquid chromatography-fluorescence detection in urine: Method validation and application to real samples.

    PubMed

    Rodríguez, J; Castañeda, G; Muñoz, L

    2013-01-15

    This work reports the validation of a high precision and accuracy method for the simultaneous determination of letrozole, citalopram and their metabolites in urine by high performance liquid chromatography with fluorescence detection. Dilution (urine:mobile phase, 1:2, v/v) was the only sample preparation step. The separation was carried out in a Kromasil C(18) (150mm×4.6mm) column, and the mobile phase was phosphate buffer 80mM (pH 3.0) and acetonitrile (65:35, v/v) at a flow rate of 1.0mL/min. The analytes were detected at 295nm after excitation at 230nm. Linearity was observed in the range of 1.0-1000ng/mL for letrozole and its metabolite and 2.5-1000ng/mL for citalopram and their metabolites, with limits of detection and quantification between 0.09-1.0 and 0.27-1.65ng/mL, respectively. The precisions were satisfactory with RSDs between 0.17 and 5.71%. The accuracy was studied by spiking three urines from healthy female volunteers, and the recoveries were from 85 to 103%. The method was applied to urine samples from women under treatment for breast cancer and depression diseases. PMID:23262245

  2. PHOTOACTIVATED LUMINESCENCE METHOD FOR RAPID SCREENING OF POLYCHLORINATED BIPHENYLS

    EPA Science Inventory

    The U.S. Department of Energy and the U.S. Environmental Protection Agency have a strong need for screening capabilities for hazardous materials. his paper describes a new method based on enhanced photoactivated luminescence (ELP) for rapid detection of PCBs. he EPL method descri...

  3. Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms and Escherichia coli

    PubMed Central

    Tryland, I.; Fiksdal, L.

    1998-01-01

    Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria. PMID:9501441

  4. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  5. Integrated optical biosensor for rapid detection of bacteria

    NASA Astrophysics Data System (ADS)

    Mathesz, Anna; Valkai, Sándor; Újvárosy, Attila; Aekbote, Badri; Sipos, Orsolya; Stercz, Balázs; Kocsis, Béla; Szabó, Dóra; Dér, András

    2016-02-01

    In medical diagnostics, rapid detection of pathogenic bacteria from body fluids is one of the basic issues. Most state-of-the-art methods require optical labeling, increasing the complexity, duration and cost of the analysis. Therefore, there is a strong need for developing selective sensory devices based on label-free techniques, in order to increase the speed, and reduce the cost of detection. In a recent paper, we have shown that an integrated optical Mach-Zehnder interferometer, a highly sensitive all-optical device made of a cheap photopolymer, can be used as a powerful lab-on-a-chip tool for specific, labelfree detection of proteins. By proper modifications of this technique, our interferometric biosensor was combined with a microfluidic system allowing the rapid and specific detection of bacteria from solutions, having the surface of the sensor functionalized by bacterium-specific antibodies. The experiments proved that the biosensor was able to detect Escherichia coli bacteria at concentrations of 106 cfu/ml within a few minutes, that makes our device an appropriate tool for fast, label-free detection of bacteria from body fluids such as urine or sputum. On the other hand, possible applications of the device may not be restricted to medical microbiology, since bacterial identification is an important task in microbial forensics, criminal investigations, bio-terrorism threats and in environmental studies, as well.

  6. Integrated optical biosensor for rapid detection of bacteria

    NASA Astrophysics Data System (ADS)

    Mathesz, Anna; Valkai, Sándor; Újvárosy, Attila; Aekbote, Badri; Sipos, Orsolya; Stercz, Balázs; Kocsis, Béla; Szabó, Dóra; Dér, András

    2015-12-01

    In medical diagnostics, rapid detection of pathogenic bacteria from body fluids is one of the basic issues. Most state-of-the-art methods require optical labeling, increasing the complexity, duration and cost of the analysis. Therefore, there is a strong need for developing selective sensory devices based on label-free techniques, in order to increase the speed, and reduce the cost of detection. In a recent paper, we have shown that an integrated optical Mach-Zehnder interferometer, a highly sensitive all-optical device made of a cheap photopolymer, can be used as a powerful lab-on-a-chip tool for specific, labelfree detection of proteins. By proper modifications of this technique, our interferometric biosensor was combined with a microfluidic system allowing the rapid and specific detection of bacteria from solutions, having the surface of the sensor functionalized by bacterium-specific antibodies. The experiments proved that the biosensor was able to detect Escherichia coli bacteria at concentrations of 106 cfu/ml within a few minutes, that makes our device an appropriate tool for fast, label-free detection of bacteria from body fluids such as urine or sputum. On the other hand, possible applications of the device may not be restricted to medical microbiology, since bacterial identification is an important task in microbial forensics, criminal investigations, bio-terrorism threats and in environmental studies, as well.

  7. Rapid High Performance Liquid Chromatographic Method for Determination of Clarithromycin in Human Plasma Using Amperometric Detection: Application in Pharmacokinetic and Bioequivalence Studies

    PubMed Central

    Foroutan, Seyed Mohsen; Zarghi, Afshin; Shafaati, Alireza; Madadian, Babak; Abolfathi, Farshid

    2013-01-01

    A rapid, sensitive and reproducible HPLC method using amperometric detector was developed and validated for the analysis of clarithromycin in human plasma. The separation was achieved on a monolithic silica column (MZ- C8 125×4.0 mm) using acetonitrile-methanol-potassium dihydrogen phosphate buffer (40:6:54,v/v), with pH of 7.5, as the mobile phase at a flow rate of 1.5 mL/min. The assay enables the measurement of clarithromycin for therapeutic drug monitoring with a minimum quantification limit of 20 ng/mL. The method involves simple, protein precipitation procedure and analytical recovery was complete. The calibration curve was linear over the concentration range of 0.1-6 μg/mL. The coefficients of variation for inter-day and intra-day assay were found to be less than 6%. This method was used in bioequivalency and pharmacokinetic studies of the test (generic) product 2 × 500 mg clarithromycin tablets, with respect to the reference product. PMID:24250673

  8. Rapid high performance liquid chromatographic method for determination of clarithromycin in human plasma using amperometric detection: application in pharmacokinetic and bioequivalence studies.

    PubMed

    Foroutan, Seyed Mohsen; Zarghi, Afshin; Shafaati, Alireza; Madadian, Babak; Abolfathi, Farshid

    2013-01-01

    A rapid, sensitive and reproducible HPLC method using amperometric detector was developed and validated for the analysis of clarithromycin in human plasma. The separation was achieved on a monolithic silica column (MZ- C8 125×4.0 mm) using acetonitrile-methanol-potassium dihydrogen phosphate buffer (40:6:54,v/v), with pH of 7.5, as the mobile phase at a flow rate of 1.5 mL/min. The assay enables the measurement of clarithromycin for therapeutic drug monitoring with a minimum quantification limit of 20 ng/mL. The method involves simple, protein precipitation procedure and analytical recovery was complete. The calibration curve was linear over the concentration range of 0.1-6 μg/mL. The coefficients of variation for inter-day and intra-day assay were found to be less than 6%. This method was used in bioequivalency and pharmacokinetic studies of the test (generic) product 2 × 500 mg clarithromycin tablets, with respect to the reference product. PMID:24250673

  9. Rapid visual tests: fast and reliable detection of ochratoxin A.

    PubMed

    Bazin, Ingrid; Nabais, Elodie; Lopez-Ferber, Miguel

    2010-09-01

    This paper reviews the early detection strategies that have been employed for the rapid monitoring of ochratoxin A (OTA) contamination of food. OTA, a mycotoxin mainly produced by some Aspergillus and Penicillium species, is found in cereals, coffee, wine, pork and grapes. To minimize the entry of this mycotoxin into the food chain, rapid diagnostic tools are required. To this end, the potential use of lateral flow devices has also been developed. In this study, we analyze the robustness of test strips using published methods for colorimetric detection. Different test formats are discussed, and challenges in the development of lateral flow devices for on-site determination of OTA, with requirements such as robustness, speed, and cost-effectiveness, are discussed. PMID:22069682

  10. A rapid and simple method for the determination of 3,4-dihydroxyphenylacetic acid, norepinephrine, dopamine, and serotonin in mouse brain homogenate by HPLC with fluorimetric detection.

    PubMed

    De Benedetto, Giuseppe Egidio; Fico, Daniela; Pennetta, Antonio; Malitesta, Cosimino; Nicolardi, Giuseppe; Lofrumento, Dario Domenico; De Nuccio, Francesco; La Pesa, Velia

    2014-09-01

    A fast and simple isocratic high-performance liquid chromatography method for the determination of 3,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in homogenate samples of mouse striatum employing the direct fluorescence of the neurotransmitters is described. The method has been optimized and validated. The analytes were separated in 15min on a reversed-phase column (C18) with acetate buffer (pH 4.0, 12mM)-methanol (86:14, v/v) as mobile phase; the flow rate was 1ml/min. The fluorescence measurements were carried out at 320nm with excitation at 279nm. The calibration curve for DA was linear up to about 2.5μg/ml, with a coefficient of determination (r(2)) of 0.9995 with a lower limit of quantification of 0.031μg/ml. Since the procedure does not involve sample pre-purification or derivatisation, the recovery ranged from 97% to 102% and relative standard deviation (RSD) was better than 2.9%, the use of the internal standard is not mandatory, further simplifying the method. Similar performance was obtained for the other analytes. As a result, thanks to its simplicity, rapidity and adequate working range, the method can be used for the determination of 3,4-dihydroxyphenylacetic acid, dopamine, norepinephrine and serotonin in animal tissues. An experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson-like disease has been used to demonstrate the method is fit-for-purpose. PMID:24971521

  11. Rapid Detection of Carbapenemase-producing Enterobacteriaceae

    PubMed Central

    Poirel, Laurent; Dortet, Laurent

    2012-01-01

    To rapidly identify carbapenemase producers in Enterobacteriaceae, we developed the Carba NP test. The test uses isolated bacterial colonies and is based on in vitro hydrolysis of a carbapenem, imipenem. It was 100% sensitive and specific compared with molecular-based techniques. This rapid (<2 hours), inexpensive technique may be implemented in any laboratory. PMID:22932472

  12. Rapid method for the determination and confirmation of fluoroquinolone residues in catfish using liquid chromatography/fluorescence detection and liquid chromatography-tandem mass spectrometry.

    PubMed

    McMullen, Sarah E; Schenck, Frank J; Vega, Victor A

    2009-01-01

    A simplified method for the extraction and determination of four fluoroquinolone (FQ) residues (ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin) in catfish is presented. In this method, the FQ residues were extracted with acidified acetonitrile, and the extract was defatted with dispersive C18 solid-phase extraction (SPE) sorbent or hexane. A portion of the extract was evaporated and reconstituted in the mobile phase. The quantitative determination was accomplished with LC-fluorescence detection (FLD), and the confirmation was by LC-MS/MS. Fortifications of catfish tissue were carried out at 0.5x, x, 2x, and 4x, where x = 5 ppb (U.S. Food and Drug Administration current regulatory target level). Recoveries for the LC/FLD determination of five replicates (for both cleanup routes) at each level ranged from 64 to 98%, with RSD values <8%. The method quantitation limits for all residues were <1 ng/g. The LC-MS/MS analysis of the same extracts confirmed all FQ residues at all levels. This method is an improvement over existing methodologies since additional cleanup steps, such as cation exchange SPE column cleanup, are not utilized. The C18 dispersive SPE method represents a novel cleanup approach for FQs in fish tissue. PMID:19714995

  13. Development of a rapid method based on solid-phase extraction and liquid chromatography with ultraviolet absorbance detection for the determination of polyphenols in alcohol-free beers.

    PubMed

    García, A Alonso; Grande, B Cancho; Gándara, J Simal

    2004-10-29

    An analytical method based on solid-phase extraction (SPE) and followed by liquid chromatographic separation and ultraviolet detection (HPLC-UV) is proposed for the determination of 10 phenolic compounds which participate on beer stability and sensory properties in alcohol-free beers. Acetonitrile was found to be the most appropriate solvent for the elution of polyphenolic compounds adsorbed on C18 cartridges. The performance of the method was assessed by the evaluation of parameters such as absolute recovery (generally higher than 60%), repeatability (lower than 10%), linearity (r2 higher than 0.993) and limits of quantitation (ranging from 1 to 37 microg/L); no matrix effects were observed. The polyphenol content of different Spanish alcohol-free beers is presented. Five phenolic compounds such as protocatechuic, p-coumaric, ferulic, caffeic acids, and (+)-catechin were identified at levels lower than 10 mg/L. PMID:15553142

  14. METHODS FOR RAPID IDENTIFICATION OF VIRUSES

    EPA Science Inventory

    The progress made in the last few years in methods for direct detection of viruses in clinical samples, without the need for in vitro culture, suggests that they will be applicable to at least some types of environmental samples. The methods for detecting viral antigen include: r...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. Methods of Melanoma Detection.

    PubMed

    Leachman, Sancy A; Cassidy, Pamela B; Chen, Suephy C; Curiel, Clara; Geller, Alan; Gareau, Daniel; Pellacani, Giovanni; Grichnik, James M; Malvehy, Josep; North, Jeffrey; Jacques, Steven L; Petrie, Tracy; Puig, Susana; Swetter, Susan M; Tofte, Susan; Weinstock, Martin A

    2016-01-01

    Detection and removal of melanoma, before it has metastasized, dramatically improves prognosis and survival. The purpose of this chapter is to (1) summarize current methods of melanoma detection and (2) review state-of-the-art detection methods and technologies that have the potential to reduce melanoma mortality. Current strategies for the detection of melanoma range from population-based educational campaigns and screening to the use of algorithm-driven imaging technologies and performance of assays that identify markers of transformation. This chapter will begin by describing state-of-the-art methods for educating and increasing awareness of at-risk individuals and for performing comprehensive screening examinations. Standard and advanced photographic methods designed to improve reliability and reproducibility of the clinical examination will also be reviewed. Devices that magnify and/or enhance malignant features of individual melanocytic lesions (and algorithms that are available to interpret the results obtained from these devices) will be compared and contrasted. In vivo confocal microscopy and other cellular-level in vivo technologies will be compared to traditional tissue biopsy, and the role of a noninvasive "optical biopsy" in the clinical setting will be discussed. Finally, cellular and molecular methods that have been applied to the diagnosis of melanoma, such as comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), will be discussed. PMID:26601859

  17. Improving the Enrichment and Plating Methods for Rapid Detection of Non-O157 Shiga Toxin-Producing Escherichia coli in Dairy Compost.

    PubMed

    Wang, Hongye; Chen, Zhao; Jiang, Xiuping

    2016-03-01

    A culture method to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) was optimized in this study. The finished dairy compost with 30% moisture content was inoculated with a cocktail of six non-O157 STEC serovars at initial concentrations of 1 to 100 CFU/g. Afterward, non-O157 STEC cells in the inoculated dairy compost were enriched by four methods, followed by plating onto cefixime-tellurite sorbitol MacConkey agar supplemented with 5 mg/liter novobiocin (CTNSMAC) and modified Rainbow agar containing 5 mg/liter novobiocin, 0.05 mg/liter cefixime trihydrate, and 0.15 mg/liter potassium tellurite (mRBA). Immunomagnetic bead separation (IMS) was used to compare the cell concentration of individual non-O157 STEC serotypes after enrichment. There was no significant difference (P > 0.05) between CTN-SMAC and mRBA for non-O157 STEC enumeration. The single-step selective enrichment recovered ca. 0.54 log CFU/g more cells (ca. 0.41 log CFU/g for compost-adapted cells) (P < 0.05) compared with the two-step enrichment. Furthermore, the duration of the process to detect non-O157 STEC from dairy compost by selective enrichment, followed by IMS, was optimized. Among six non-O157 STEC serotypes, serotypes O111, O45, and O145 reached the highest cell density after enrichment in dairy compost, and the cell populations reached 7.3, 7.4, and 7.8 log CFU/g within 16 h of incubation, respectively. In contrast, without an enrichment step, the IMS detection limit of individual non-O157 STEC serovars ranged from 3.15 to 4.15 log CFU/g in dairy compost. These results demonstrate that low levels of non-O157 STEC can be detected within 2 days from dairy compost by using a culture method with an optimized enrichment procedure followed by IMS. PMID:26939651

  18. 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

  19. Rapid detection of bacteria in foods and biological fluids

    NASA Technical Reports Server (NTRS)

    Fealey, R. D.; Renner, W.

    1973-01-01

    Simple and inexpensive apparatus, called "redox monitoring cell," rapidly detects presence of bacteria. Bacteria is detected by measuring drop in oxygen content in test solution. Apparatus consists of vial with two specially designed electrodes connected to sensitive voltmeter.

  20. Detection of endocervical anti-Chlamydia trachomatis immunoglobulin A in pregnant women by a rapid, 6-minute enzyme-linked immunosorbent assay: comparison with PCR and chlamydial antigen detection methods.

    PubMed Central

    Witkin, S S; Bongiovanni, A M; Inglis, S R

    1997-01-01

    There is a need for a rapid, uncomplicated, and inexpensive test for Chlamydia trachomatis infection in women. We evaluated the ability of a 6-min enzyme-linked immunosorbent assay (ELISA) that requires no laboratory equipment (IgA Rapid SeroTest; Savyon Diagnostics) to detect C. trachomatis immunoglobulin A (IgA) in the endocervices of 167 inner-city pregnant women and compared the results with DNA amplification (Amplicor PCR; Roche Diagnostics) and antigen detection (Chlamydiazyme; Abbott Laboratories) performed on the same women. Anti-C. trachomatis IgA was detected in the cervices of 32 women (19.2%). Samples from 23 women (13.8%) were PCR positive, while chlamydial antigen was present in 20 women (12.0%). There was only 1 sample (4.3%) that was positive by PCR but negative by ELISA; 10 samples were ELISA positive and PCR negative. In contrast, seven samples (30.4%) were PCR positive but Chlamydiazyme negative and four were Chlamydiazyme positive and PCR negative. Compared to PCR, the IgA ELISA had a sensitivity of 95.7%, a specificity of 93.1%, a positive predictive value of 68.8%, and a negative predictive value of 99.3%. The antigen assay had a sensitivity of only 69.6%, a specificity of 97.2%, a positive predictive value of 80.0%, and a negative predictive value of 95.2%. In high-risk groups where laboratory testing is not available, or where the patient might not return to obtain her testing result and be treated, the Rapid IgA SeroTest is a viable alternative for detection of cervical C. trachomatis in pregnant women. PMID:9196193

  1. Rapid screening method for quinolone residues in livestock and fishery products using immobilised metal chelate affinity chromatographic clean-up and liquid chromatography-fluorescence detection.

    PubMed

    Takeda, N; Gotoh, M; Matsuoka, T

    2011-09-01

    An efficient LC method was developed for screening the presence of quinolones (QLs)--comprising fluoroquinolones (FQs) and acidic quinolones (AQs)--residues in various livestock and fishery products. Targeted analytes were for nine FQs of marbofloxacin (MAR), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), danofloxacin (DAN), orbifloxacin (ORB), difloxacin (DIF) and sarafloxacin (SAR), and three AQs of oxolinic acid (OXA), nalidixic acid (NAL) and flumequine (FMQ). Samples comprised ten different food products covering five matrices: muscle (cattle, swine and chicken), liver (chicken), raw fish (shrimp and salmon), egg (chicken), and processed food (ham, sausage and fish sausage). This method involved a simple extraction with (1:1) acetonitrile-methanol, a highly selective clean-up with an immobilised metal chelate affinity column charged with Fe(3+), a fast isocratic LC analysis using a short column (20 mm × 4.6 mm, 3 µm) with a mobile phase of (15:85:0.1) methanol/water/formic acid, and fluorescence detection (excitation/emission wavelengths of 295 nm/455 nm for FQs (495 nm for MAR), and 320 nm/365 nm for AQs). Among FQs, pairs of NOR/OFL, ORB/DIF and ENR/DAN were incompletely resolved. A confirmatory LC run with a Mg(2+) containing methanolic mobile phase was also proposed for the samples suspected of being positive. The optimised method gave satisfactory recoveries of 88.5% (56.1-108.6%) and 78.7% (44.1-99.5%) for intra- and inter-day assays with relative standard deviations of 7.2% (0.7-18.4%) and 6.8% (1.4-16.6%), respectively. Limits of quantitation ranged from 0.8 µg kg(-1) (DAN) to 6.5 µg kg(-1) (SAR). This method was successfully employed to analyse 113 real samples and two positive samples were found: fish sausage (CIP 990 µg kg(-1)) and shrimp (ENR 20 µg kg(-1)). PMID:21749230

  2. Methods for Doping Detection.

    PubMed

    Ponzetto, Federico; Giraud, Sylvain; Leuenberger, Nicolas; Boccard, Julien; Nicoli, Raul; Baume, Norbert; Rudaz, Serge; Saugy, Martial

    2016-01-01

    Over the past few years, the World Anti-Doping Agency (WADA) has focused its efforts on detecting not only small prohibited molecules, but also larger endogenous molecules such as hormones, in the view of implementing an endocrinological module in the Athlete Biological Passport (ABP). In this chapter, the detection of two major types of hormones used for doping, growth hormone (GH) and endogenous anabolic androgenic steroids (EAASs), will be discussed: a brief historical background followed by a description of state-of-the-art methods applied by accredited anti-doping laboratories will be provided and then current research trends outlined. In addition, microRNAs (miRNAs) will also be presented as a new class of biomarkers for doping detection. PMID:27348309

  3. Error detection method

    DOEpatents

    Olson, Eric J.

    2013-06-11

    An apparatus, program product, and method that run an algorithm on a hardware based processor, generate a hardware error as a result of running the algorithm, generate an algorithm output for the algorithm, compare the algorithm output to another output for the algorithm, and detect the hardware error from the comparison. The algorithm is designed to cause the hardware based processor to heat to a degree that increases the likelihood of hardware errors to manifest, and the hardware error is observable in the algorithm output. As such, electronic components may be sufficiently heated and/or sufficiently stressed to create better conditions for generating hardware errors, and the output of the algorithm may be compared at the end of the run to detect a hardware error that occurred anywhere during the run that may otherwise not be detected by traditional methodologies (e.g., due to cooling, insufficient heat and/or stress, etc.).

  4. Rapid, Culture-Free Detection of Staphylococcus aureus Bacteremia

    PubMed Central

    Burghardt, Elliot L.; Flenker, Katie S.; Clark, Karen C.; Miguel, Jeff; Ince, Dilek; Winokur, Patricia; Ford, Bradley; McNamara, James O.

    2016-01-01

    S. aureus bacteremia (SAB) is a common condition with high rates of morbidity and mortality. Current methods used to diagnose SAB take at least a day, and often longer. Patients with suspected bacteremia must therefore be empirically treated, often unnecessarily, while assay results are pending. In this proof-of-concept study, we describe an inexpensive assay that detects SAB via the detection of micrococcal nuclease (an enzyme secreted by S. aureus) in patient plasma samples in less than three hours. In total, 17 patient plasma samples from culture-confirmed S. aureus bacteremic individuals were tested. 16 of these yielded greater nuclease assay signals than samples from uninfected controls or individuals with non-S. aureus bacteremia. These results suggest that a nuclease-detecting assay may enable the rapid and inexpensive diagnosis of SAB, which is expected to substantially reduce the mortality and morbidity that result from this condition. PMID:27305148

  5. Comment on “Rapid visual detection of blood cyanide” by C. Männel-Croisé and F. Zelder, Analytical Methods, 2012, 4, 2632

    PubMed Central

    Kadjo, Akinde F.; Boss, Gerry R

    2015-01-01

    Cyanide poisoning from Inhaled HCN is all too common in victims of smoke inhalation in fires. While the toxic effects arise primarily from its inhibitory effects on cytochrome c oxidase, the majority of the cyanide binds to methemoglobin (metHb) in the blood. It can be considered as the detoxification mechanism: one of the antidotes used earlier was nitrite which primarily works by converting hemoglobin to metHb (normally present to the extent of ~1% of the total hemoglobin). Vitamin B12 (hydroxocobalamin) and related analogs have long been known to have high affinity for cyanide and has been used as antidotes – the binding of cyanide to many compounds in this general family also results in a significant change in color that can be used for analytical purposes. Männel Croisé and Zelder (Anal. Methods, 2012, 4, 2632) have advocated direct addition of a related compound to blood samples and isolating the colored measurand on a solid phase extraction cartridge. While they demonstrated attractive rapid measurement of cyanide in spiked blood samples, we believe that this is not a practically usable procedure regardless of the exact chromogenic reagent used. Cyanide bound to metHb dissociates too slowly for a 1 min reaction to work as suggested – we believe for reasons unknown (eg., metHb levels in their blood samples unusually low), cyanide added to their blood samples did not (have time to) bind to metHb and these samples may not resemble real situations where significant amount of the cyanide will be bound to metHb. PMID:26640525

  6. Rapid assessment of assignments using plagiarism detection software.

    PubMed

    Bischoff, Whitney R; Abrego, Patricia C

    2011-01-01

    Faculty members most often use plagiarism detection software to detect portions of students' written work that have been copied and/or not attributed to their authors. The rise in plagiarism has led to a parallel rise in software products designed to detect plagiarism. Some of these products are configurable for rapid assessment and teaching, as well as for plagiarism detection. PMID:22024673

  7. Method for detecting biomolecules

    DOEpatents

    Huo, Qisheng; Liu, Jun

    2008-08-12

    A method for detecting and measuring the concentration of biomolecules in solution, utilizing a conducting electrode in contact with a solution containing target biomolecules, with a film with controllable pore size distribution characteristics applied to at least one surface of the conducting electrode. The film is functionalized with probe molecules that chemically interact with the target biomolecules at the film surface, blocking indicator molecules present in solution from diffusing from the solution to the electrode, thereby changing the electrochemical response of the electrode

  8. RAPID METHOD FOR DETERMINATION OF RADIOSTRONTIUM IN EMERGENCY MILK SAMPLES

    SciTech Connect

    Maxwell, S.; Culligan, B.

    2008-07-17

    A new rapid separation method for radiostrontium in emergency milk samples was developed at the Savannah River Site (SRS) Environmental Bioassay Laboratory (Aiken, SC, USA) that will allow rapid separation and measurement of Sr-90 within 8 hours. The new method uses calcium phosphate precipitation, nitric acid dissolution of the precipitate to coagulate residual fat/proteins and a rapid strontium separation using Sr Resin (Eichrom Technologies, Darien, IL, USA) with vacuum-assisted flow rates. The method is much faster than previous method that use calcination or cation exchange pretreatment, has excellent chemical recovery, and effectively removes beta interferences. When a 100 ml sample aliquot is used, the method has a detection limit of 0.5 Bq/L, well below generic emergency action levels.

  9. Rapid and quantitative detection of hepatitis B virus

    PubMed Central

    Liu, Yue-Ping; Yao, Chun-Yan

    2015-01-01

    Despite availability of a universal vaccine, hepatitis B virus (HBV) infection has a huge impact on public health worldwide. Accurate and timely diagnosis of HBV infection is needed. Rapid developments have been made in the diagnostic and monitoring methods for HBV infection, including serological and molecular assays. In clinical practice, qualitative hepatitis B surface antigen (HBsAg) testing has long served as a diagnostic marker for individuals infected with HBV. More recently, HBsAg level has been used to predict treatment outcome when determined early during treatment or at baseline. However, identification of HBV DNA positive cases that do not have detectable HBsAg has encouraged the application of molecular tests. Hence, combination of quantitative detection of HBV DNA and HBsAg can be used to discriminate patients during the course of HBV infection and to monitor therapy. This article reviews the most commonly used quantitative methods for HBsAg and HBV DNA. PMID:26576084

  10. New reversed phase dispersive liquid-liquid microextraction method for the determination of phenolic compounds in virgin olive oil by rapid resolution liquid chromathography with ultraviolet-visible and mass spectrometry detection.

    PubMed

    Godoy-Caballero, M P; Acedo-Valenzuela, M I; Galeano-Díaz, T

    2013-10-25

    The determination of phenolic compounds in virgin olive oil using a new reversed phase dispersive liquid-liquid microextraction (RP-DLLME) procedure coupled with rapid resolution liquid chromatography-diode array and mass spectrometry detection (RRLC-DAD-MS) have been performed. A rapid resolution Zorbax Eclipse XDB-C18 column (4.6 mm × 50 mm, 1.8 μm particle size) has been employed and eighteen phenolic compounds belonging to different families have been identified and quantified spending a total time of 26 and 13 min with UV-visible and MS detection, respectively. Response surface methodology has been applied by means of a central composite design for the optimization of the variables affecting the extraction procedure searching for the best recovery. The validation of the methods was performed through the establishment of the external standard calibration curves and the analytical figures of merit. Limits of detection ranging from 10 to 400 ng mL(-1) and 1 to 200 ng mL(-1) were achieved using UV-visible and MS detection, respectively. The extraction of phenolic compounds from virgin olive oil was performed in a simple and rapid way by RP-DLLME with ethanol:water 60:40 (v/v) as extracting solvent and 1,4-dioxane as disperser solvent. The quantification of the phenolic compounds in virgin olive oils from different olive varieties was carried out by means of the standard addition method and, finally the procedure for the sample treatment was validated using the well established solid phase extraction procedure with Diol cartridges. PMID:23895920

  11. Rapid detection of Yersinia pestis recombinant fraction 1 capsular antigen.

    PubMed

    Tsui, Pei-Yi; Tsai, Hui-Ping; Chiao, Der-Jiang; Liu, Cheng-Che; Shyu, Rong-Hwa

    2015-09-01

    Yersinia pestis, an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10(5) CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10(5) CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge. PMID:25994256

  12. Rapid leak detection with liquid crystals

    NASA Technical Reports Server (NTRS)

    Heisman, R. M.; Iceland, W. F.; Ruppe, E. P.

    1978-01-01

    Small leaks in vacuum lines are detected by applying liquid-crystal coating, warming suspected area, and observing color change due to differential cooling by leak jet. Technique is used on inside or outside walls of vacuum-jacketed lines.

  13. Rapid Detection of Subtype H10N8 Influenza Virus by One-Step Reverse Transcription–Loop-Mediated Isothermal Amplification Methods

    PubMed Central

    Bao, Hongmei; Feng, Xiaoxiao; Ma, Yong; Shi, Jianzhong; Zhao, Yuhui; Gu, Linlin

    2015-01-01

    We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription–loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets. PMID:26378283

  14. Rapid detection of Porcine circovirus 2 by recombinase polymerase amplification.

    PubMed

    Wang, Jianchang; Wang, Jinfeng; Liu, Libing; Li, Ruiwen; Yuan, Wanzhe

    2016-09-01

    Porcine circovirus-associated disease, caused primarily by Porcine circovirus 2 (PCV-2), has become endemic in many pig-producing countries and has resulted in significant economic losses to the swine industry worldwide. Tests for PCV-2 infection include PCR, nested PCR, competitive PCR, and real-time PCR (rtPCR). Recombinase polymerase amplification (RPA) has emerged as an isothermal gene amplification technology for the molecular detection of infectious disease agents. RPA is performed at a constant temperature and therefore can be carried out in a water bath. In addition, RPA is completed in ~30 min, much faster than PCR, which usually takes >60 min. We developed a RPA-based method for the detection of PCV-2. The detection limit of RPA was 10(2) copies of PCV-2 genomic DNA. RPA showed the same sensitivity as rtPCR but was 10 times more sensitive than conventional PCR. Successful amplification of PCV-2 DNA, but not other viral templates, demonstrated high specificity of the RPA assay. This method was also validated using clinical samples. The results showed that the RPA assay had a diagnostic agreement rate of 93.7% with conventional PCR and 100% with rtPCR. These findings suggest that the RPA assay is a simple, rapid, and cost-effective method for PCV-2 detection, which could be potentially applied in clinical diagnosis and field surveillance of PCV-2 infection. PMID:27493138

  15. Rapid detection of autosomal aneuploidy using microsatellite markers

    SciTech Connect

    Ray, P.N.; Teshima, I.E.; Winsor, E.J.T.

    1994-09-01

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

  16. Rapid detection of tuberculosis using droplet-based microfluidics

    NASA Astrophysics Data System (ADS)

    Rosenfeld, Liat; Cheng, Yunfeng; Rao, Jianghong; Tang, Sindy K. Y.

    2014-03-01

    Tuberculosis is one of the most deadly diseases that kills over one million people each year and infects one-third of the world's population. The disease is spread by infection with Mycobacterium tuberculosis (Mtb). Owing to its airborne transmission, early diagnosis is critical to the prevention and control of TB. Standard diagnostic methods, acid-fast smear from sputum, often do not become positive until after transmission occurs, which allows the spread of the disease. Culture-based techniques are more sensitive, but take weeks to obtain results because of the extremely slow growth rate of Mtb. In this study a new method to detect indicator enzyme based on the isolation of tubercle bacillus in a large number of picoliter droplets combined with a fluorescent probe has been developed. We use BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and a designed BlaC-specific fluorogenic substrates as probes for Mtb detection. We present here a new method to detect the indicator enzyme based on the isolation, digitization and concentration of bacteria samples in a large number of picoliter drops. We show that by controlling the size of the droplets we can control the rate of conversion. Hence rapid increase in signal has been observed as the size of the drops has been decreased. Our vision is that this tool will be able to detect tubercle bacilli in a sensitive, rapid, specific and quantitative manner in vitro at a low cost, particularly in resource limited settings where TB is the most prevalent.

  17. Inoculation of beef with low concentrations of Escherichia coli O157:H7 and examination of factors that interfere with its detection by culture isolation and rapid methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reliable detection of Escherichia coli O157:H7 in test-and-hold programs requires the detection of as little as 1 CFU E. coli O157:H7 in a sample of beef trim or ground beef that is up to 375 grams in size. We present a reliable protocol for generating a control inoculum for verification testing at...

  18. Olfactory processing: detection of rapid changes.

    PubMed

    Croy, Ilona; Krone, Franziska; Walker, Susannah; Hummel, Thomas

    2015-06-01

    Changes in the olfactory environment have a rather poor chance of being detected. Aim of the present study was to determine, whether the same (cued) or different (uncued) odors can generally be detected at short inter stimulus intervals (ISI) below 2.5 s. Furthermore we investigated, whether inhibition of return, an attentional phenomenon facilitating the detection of new stimuli at longer ISI, is present in the domain of olfaction. Thirteen normosmic people (3 men, 10 women; age range 19-27 years; mean age 23 years) participated. Stimulation was performed using air-dilution olfactometry with 2 odors: phenylethylalcohol and hydrogen disulfide. Reaction time to target stimuli was assessed in cued and uncued conditions at ISIs of 1, 1.5, 2, and 2.5 s. There was a significant main effect of ISI, indicating that odors presented only 1 s apart are missed frequently. Uncued presentation facilitated detection at short ISIs, implying that changes of the olfactory environment are detected better than presentation of the same odor again. Effects in relation to "olfactory inhibition of return," on the other hand, are not supported by our results. This suggests that attention works different for the olfactory system compared with the visual and auditory systems. PMID:25911421

  19. Methods of Recording Rapid Wind Changes

    NASA Technical Reports Server (NTRS)

    Magnan, A

    1932-01-01

    The purpose of our research was to determine the rapid changes of air currents which impose varying stresses on the wings of airplanes. We attempted to express in figures the turbulence of the air, which perhaps plays some role in the behavior of airplanes in flight, as well as in the realization of certain methods of gliding flight. This is the reason which led us to conceive and develop the experimental equipment (hot-wire anemometer) described herein.

  20. RAPID DETECTION OF ALGAL TOXINS - PHASE I

    EPA Science Inventory

    The proposed program will demonstrate the ability of a detector based on measuring the conductance of nicotinic acetylcholine receptor (nAChR) ion channels to detect and quantify anatoxin-a contamination in drinking water systems.  The nAChR is extremely sensitive to the p...

  1. Monoclonal antibody technologies and rapid detection assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  2. Rapid test for the detection of hazardous microbiological material

    NASA Astrophysics Data System (ADS)

    Mordmueller, Mario; Bohling, Christian; John, Andreas; Schade, Wolfgang

    2009-09-01

    After attacks with anthrax pathogens have been committed since 2001 all over the world the fast detection and determination of biological samples has attracted interest. A very promising method for a rapid test is Laser Induced Breakdown Spectroscopy (LIBS). LIBS is an optical method which uses time-resolved or time-integrated spectral analysis of optical plasma emission after pulsed laser excitation. Even though LIBS is well established for the determination of metals and other inorganic materials the analysis of microbiological organisms is difficult due to their very similar stoichiometric composition. To analyze similar LIBS-spectra computer assisted chemometrics is a very useful approach. In this paper we report on first results of developing a compact and fully automated rapid test for the detection of hazardous microbiological material. Experiments have been carried out with two setups: A bulky one which is composed of standard laboratory components and a compact one consisting of miniaturized industrial components. Both setups work at an excitation wavelength of λ=1064nm (Nd:YAG). Data analysis is done by Principal Component Analysis (PCA) with an adjacent neural network for fully automated sample identification.

  3. Neurally inspired rapid detection of sparse objects in videos

    NASA Astrophysics Data System (ADS)

    Khosla, Deepak; Huber, David J.; Bhattacharyya, Rajan; Daily, Mike; Tasinga, Penn

    2010-04-01

    In this paper, we describe COGNIVA, a closed-loop Cognitive-Neural method and system for image and video analysis that combines recent technological breakthroughs in bio-vision cognitive algorithms and neural signatures of human visual processing. COGNIVA is an "operational neuroscience" framework for intelligent and rapid search and categorization of Items Of Interest (IOI) in imagery and video. The IOI could be a single object, group of objects, specific image regions, specific spatio-temporal pattern/sequence or even the category that the image itself belongs to (e.g., vehicle or non-vehicle). There are two main types of approach for rapid search and categorization of IOI in imagery and video. The first approach uses conventional machine vision or bio-inspired cognitive algorithms. These usually need a predefined set of IOI and suffer from high false alarm rates. The second class of algorithms is based on neural signatures of target detection. These algorithms usually break the entire image into sub-images and process EEG data from these images and classify them based on it. This approach may suffer from high false alarms and is slow because the entire image is chipped and presented to the human observer. The proposed COGNIVA overcomes the limitations of both methods by combining them resulting in a low false alarm rate and high detection with high throughput making it applicable to both image and video analysis. In the most basic form, COGNIVA first uses bioinspired cognitive algorithms for deciding potential IOI in a sequence of images/video. These potential IOI are then shown to a human and neural signatures of visual detection of IOI are collected and processed. The resulting signatures are used to categorize and provide final IOI. We will present the concept and typical results of COGNIVA for detecting Items of interest in image data.

  4. Rapid diagnosis of mumps virus infections by immunofluorescence methods.

    PubMed

    Lennette, D A; Emmons, R W; Lennette, E H

    1976-08-01

    Mumps and its complications, particularly meningoencephalitis, is an important disease problem, and more rapid diagnostic methods are desirable. A study was made of immunofluorescence methods for the early detection of mumps virus isolated in cell cultures, or adsorbed directly from clinical specimens onto guinea pig erythrocytes. A specific diagnosis could be made in hours to 2 or 3 days utilizing immunofluorescence methods, in contrast to about 6 days by standard methods. Details of the direct immunofluorescence methods are presented, to encourage wider application in clinical virology laboratories. PMID:787002

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

    PubMed

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

    2012-03-01

    Solid phase microextraction (SPME) coupled with either gas chromatography-ionization flame detector (CG-FID) or multidimensional gas chromatography-mass spectrometry (MDGC-MS) was evaluated for its ability to detect volatile hydrocarbons produced during the irradiation of cooked ham. The chromatogram of an irradiated sample obtained using GC-FID showed a complex pattern of peaks, with several co-eluting peaks superimposed, indicating that the method was unlikely to resolve adequately the volatile hydrocarbons formed during irradiation. Using SPME-MDGC-MS 1-tetradecene (C(1-14:1)), n-pentadecane (C(15:0)), 1-hexadecene (C(1-16:1)), n-heptadecane (C(17:0)) and 8-heptadecene (C(8-17:1)) were detected in cooked ham irradiated at 0.5, 2, 4 and 8kGy. This method allows the detection of most n-alkanes and n-alkenes produced during the irradiation of the majority of fatty acids in cooked ham, namely oleic acid, stearic acid and palmitic acid. SPME is rapid and inexpensive and does not require organic solvents. The proposed SPME-MDGC-MS method allows the determination of radiolytic markers in cooked ham in less than 115min. PMID:22100714

  6. Rapid Detection and Identification of Biogenic Aerosol Releases and Sources

    NASA Astrophysics Data System (ADS)

    Wagner, J.; Macher, J.; Ghosal, S.; Ahmed, K.; Hemati, K.; Wall, S.; Kumagai, K.

    2011-12-01

    Biogenic aerosols can be important contributors to aerosol chemistry, cloud droplet and ice nucleation, absorption and scattering of radiation, human health and comfort, and plant, animal, and microbial ecology. Many types of bioaerosols, e.g., fungal spores, are released into the atmosphere in response to specific climatological and meteorological conditions. The rapid identification of bioaerosol releases is thus important for better characterization of the above phenomena, as well as enabling public officials to respond quickly and appropriately to releases of infectious agents or biological toxins. One approach to rapid and accurate bioaerosol detection is to employ sequential, automated samples that can be fed directly into an image acquisition and data analysis device. Raman spectroscopy-based identification of bioaerosols, automated analysis of microscopy images, and automated detection of near-monodisperse peaks in aerosol size-distribution data were investigated as complementary approaches to traditional, manual methods for the identification and counting of fungal and actinomycete spores. Manual light microscopy is a widely used analytical technique that is compatible with a number of air sample formats and requires minimal sample preparation. However, a major drawback is its dependence on a human analyst's ability to distinguish particles and accurately count, size, and identify them. Therefore, automated methods, such as those evaluated in this study, have the potential to provide cost-effective and rapid alternatives if demonstrated to be accurate and reliable. An exploratory examination of individual spores for several macro- and microfungi (those with and without large fruiting bodies) by Raman microspectroscopy found unique spectral features that were used to identify fungi to the genus level. Automated analyses of digital spore images accurately recognized and counted single fungal spores and clusters. An automated procedure to discriminate near

  7. A one-step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primers designed against conserved regions of segment 6 (s6) gene were used for the detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 subtype. The entire amplification could be completed within 40 min at 62...

  8. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40 - 50 million annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal a...

  9. LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine

    PubMed Central

    Kogovšek, P; Hodgetts, J; Hall, J; Prezelj, N; Nikolić, P; Mehle, N; Lenarčič, R; Rotter, A; Dickinson, M; Boonham, N; Dermastia, M; Ravnikar, M

    2015-01-01

    In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive. PMID:26146413

  10. Rapid method for monitoring N-nitrosodimethylamine in drinking water at the ng/L level without pre-concentration using high-performance liquid chromatography-chemiluminescence detection.

    PubMed

    Kodamatani, Hitoshi; Yamasaki, Hitomi; Sakaguchi, Takeru; Itoh, Shinya; Iwaya, Yoshimi; Saga, Makoto; Saito, Keiitsu; Kanzaki, Ryo; Tomiyasu, Takashi

    2016-08-19

    As a contaminant in drinking water, N-nitrosodimethylamine (NDMA) is of great concern because of its carcinogenicity; it has been limited to levels of ng/L by regulatory bodies worldwide. Consequently, a rapid and sensitive method for monitoring NDMA in drinking water is urgently required. In this study, we report an improvement of our previously proposed HPLC-based system for NDMA determination. The approach consists of the HPLC separation of NDMA, followed by NDMA photolysis to form peroxynitrite and detection with a luminol chemiluminescence reaction. The detection limit for the improved HPLC method was 0.2ng/L, which is 10 times more sensitive than our previously reported system. For tap water measurements, only the addition of an ascorbic acid solution to eliminate residual chlorine and passage through an Oasis MAX solid-phase extraction cartridge are needed. The proposed NDMA determination method requires a sample volume of less than 2mL and a complete analysis time of less than 15min per sample. The method was utilized for the long-term monitoring of NDMA in tap water. The NDMA level measured in the municipal water survey was 4.9ng/L, and a seasonal change of the NDMA concentration in tap water was confirmed. The proposed method should constitute a useful NDMA monitoring method for protecting drinking water quality. PMID:27443252