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Sample records for rapid detection method

  1. Advances in rapid detection methods for foodborne pathogens.

    PubMed

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

    2014-03-28

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

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

    PubMed

    March-Rosselló, Gabriel Alberto

    2017-03-01

    The most widely used antibiotic susceptibility testing methods in Clinical Microbiology are based on the phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. These conventional methods take typically 24hours to obtain results. Here we review the main techniques for rapid determination of antibiotic susceptibility. Data obtained with different methods such as molecular techniques, microarrays, commercial methods used in work routine, immunochromatographic methods, colorimetric methods, image methods, nephelometry, MALDI-TOF mass spectrometry, flow cytometry, chemiluminescence and bioluminescence, microfluids and methods based on cell disruption are analysed in detail.

  3. Rapid qualitative method for detecting staphylococcal nuclease in foods.

    PubMed Central

    Koupal, A; Deibel, R H

    1978-01-01

    A rapid method for the detection of heat-stable staphylococcal nuclease in foods is described. The procedure consists of an acid precipitation, boiling, and centrifugation followed by enzyme detection in an agar plate containing deoxyribonucleic acid. To test the efficacy of the procedure, purified Staphylococcus aureus nuclease was added to various foods and recovery experiments were performed. Additionally, foods were inoculated and incubated with S. aureus, and the staphylococcal counts were compared with nuclease activity. The results indicate that the procedure possesses merit for a rapid method that can be incorporated into quality control programs. The procedure requires approximately 2.5 h, and it will detect nuclease levels as low as 10 ng/g of food. Images PMID:677882

  4. A new ultrasound based method for rapid microorganism detection

    NASA Astrophysics Data System (ADS)

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

    2012-05-01

    A new method for rapid detection of catalase positive microorganisms by using an ultrasonic measuring method is proposed in this work. The developed technique is based on the detection of oxygen bubbles produced by the hydrolysis of hydrogen peroxide induced by the enzyme catalase which is present in many microorganisms. The bubbles are trapped in a media based on agar gel which was especially developed for microbiological evaluation. It is found that microorganism concentrations of the order of 105 c.f.u./ml can be detected by using this method. The results obtained show up that the proposed method is competitive with other modern commercial methods like luminescence by ATP system. The method can also be used for characterization of enzyme activity.

  5. 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.

  6. Detection of urinary tract infections by rapid methods.

    PubMed Central

    Pezzlo, M

    1988-01-01

    A review of rapid urine screens for detection of bacteriuria and pyuria demonstrates a number of available alternatives to the culture method. Selection of one or more of these systems for routine use is dependent upon the laboratory and the patient population being tested. The laboratory approach to the diagnosis of urinary tract infection should consider the clinical diagnosis of the patient whenever possible. Keeping in mind that quantitative urine cultures alone cannot be used to detect infection in some patient populations unless lower colony counts are considered, a rapid screen may be a more practical approach. It has become accepted that 10(5) CFU/ml can no longer be used as the standard for all patient groups, that pyuria often is important in making the diagnosis of a urinary tract infection, and that most of the rapid screens are more sensitive than the culture method at 10(5) CFU/ml. Presently, no one approach can be recommended for all laboratories and all patient groups. However, each diagnostic laboratory should select one approach which is best for its situation. It is not practical, efficient, or cost effective to define a protocol for each possible clinical condition; however, all should be considered when developing a protocol. This protocol should be compatible with the patient population and communicated to the physicians. Use of a rapid screen should be beneficial to the patient, the physician, and the laboratory. PMID:3058296

  7. 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

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

    PubMed

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

    2014-04-01

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

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

    PubMed

    Bystryak, Simon; Ossina, Natalya

    2017-08-24

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

  10. 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.

  11. Rapid and robust detection methods for poison and microbial contamination.

    PubMed

    Hoehl, Melanie M; Lu, Peter J; Sims, Peter A; Slocum, Alexander H

    2012-06-27

    Real-time on-site monitoring of analytes is currently in high demand for food contamination, water, medicines, and ingestible household products that were never tested appropriately. Here we introduce chemical methods for the rapid quantification of a wide range of chemical and microbial contaminations using a simple instrument. Within the testing procedure, we used a multichannel, multisample, UV-vis spectrophotometer/fluorometer that employs two frequencies of light simultaneously to interrogate the sample. We present new enzyme- and dye-based methods to detect (di)ethylene glycol in consumables above 0.1 wt % without interference and alcohols above 1 ppb. Using DNA intercalating dyes, we can detect a range of pathogens ( E. coli , Salmonella , V. Cholera, and a model for Malaria) in water, foods, and blood without background signal. We achieved universal scaling independent of pathogen size above 10(4) CFU/mL by taking advantage of the simultaneous measurement at multiple wavelengths. We can detect contaminants directly, without separation, purification, concentration, or incubation. Our chemistry is stable to ± 1% for >3 weeks without refrigeration, and measurements require <5 min.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Simple and Rapid Method for Detecting Biofilm Forming Bacteria.

    PubMed

    Kalia, Vipin Chandra; Prakash, Jyotsana; Koul, Shikha; Ray, Subhasree

    2017-03-01

    Biofilm forming bacteria play a vital role in causing infectious diseases and for enhancing the efficiency of the bioremediation process through immobilization. Different media and conditions have been reported for detecting biofilm forming bacteria, however, they are not quite rapid. Here, we propose the use of a simple medium which can be used for detecting biofilm former, and also provide a mechanism to regulate the expression of biofilm formation process.

  14. 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

  15. 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

  16. Rapid detection of malaria parasite by toluidine blue method: a new staining method.

    PubMed

    Annam, Vamseedhar; Mohan, Chakkirala Nalini; Mrinalini, Vazhayil Ramunny

    2013-10-01

    Malaria is a commonest mosquito-borne infectious disease worldwide. Early identification and management of malaria prevents complications and mortality. Identification of the malaria mainly relies on detection of the parasite on blood smears. The present study was conducted to compare Toluidine blue method with Leishman method for detection of malaria parasite and also to study the efficacy and advantages of using Toluidine blue method. In 540 consecutive patients with clinical suspicion of malaria, peripheral smears were prepared. Smears were processed for both conventional Leishman method and Toluidine blue method simultaneously. The significance of Toluidine blue method over Leishman method was analyzed using Chi-square (χ(2)) test. Out of 540 smears, 28.3% (153/540) were positive for malaria parasite on conventional Leishman method, while the smear positivity was more by Toluidine blue method to 33.3% (180/540) [P value < 0.01]. The remaining 66.67% (360/540) were negative by both Toluidine blue method and conventional Leishman method. The Toluidine blue method is simple, rapid, inexpensive, and easily available. The implementation of Toluidine blue method clearly improves microscopic detection of malaria parasite and can be a useful contribution to routine hematology even at rural health sectors.

  17. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

  20. Rapid Quantitative Method for Salmonella Detection in Polluted Waters

    PubMed Central

    Cheng, Chu Ming; Boyle, William C.; Goepfert, J. M.

    1971-01-01

    A procedure has been developed for the enumeration of salmonellae in polluted waters using several modifications of existing techniques. Confirmation of salmonellae is achieved within 48 hr. This procedure includes selective enrichment in m-Tetrathionate Broth (22 ± 1 hr), plating on Brilliant Green Sulfa Agar (20 ± 1 hr), and confirmation by flagellar (H) agglutination of the growth in a mannosecontaining medium (6 ± 1 hr). An incubation temperature of 41.5 C was used throughout this procedure. Dilution to extinction techniques (most probable number) were employed to enumerate salmonellae. Large sample volumes were concentrated through the use of membrane filters. This technique proved to be rapid and reliable for the enumeration of salmonellae in water, waste water, and waste-water sludges. PMID:4930275

  1. 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

  2. A Nucleic Acid Probe and Method for the Rapid Detection of Typhoid Fever Bacteria.

    DTIC Science & Technology

    This invention relates to a nucleic acid probe and method for the rapid detection of typhoid fever bacteria by use of a nucleic acid hybridization...a nucleic acid hybridization reaction with a clinical specimen containing typhoid fever bacteria.

  3. Assessment criteria and approaches for rapid detection methods to be used in the food industry.

    PubMed

    Wiedmann, Martin; Wang, Siyun; Post, Laurie; Nightingale, Kendra

    2014-04-01

    The number of commercially available kits and methods for rapid detection of foodborne pathogens continues to increase at a considerable pace, and the diversity of methods and assay formats is reaching a point where it is very difficult even for experts to weigh the advantages and disadvantages of different methods and to decide which methods to choose for a certain testing need. Although a number of documents outline quantitative criteria that can be used to evaluate different detection methods (e.g., exclusivity and inclusivity), a diversity of criteria is typically used by industry to select specific methods that are used for pathogen detection. This article is intended to provide an overall outline of criteria that the food industry can use to evaluate new rapid detection methods, with a specific focus on nucleic acid-based detection methods.

  4. Rapid detection of human fecal Eubacterium species and related genera by nested PCR method.

    PubMed

    Kageyama, A; Benno, Y

    2001-01-01

    PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.

  5. Microwave-Accelerated Method for Ultra-Rapid Extraction of Neisseria gonorrhoeae DNA for Downstream Detection

    PubMed Central

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

    2016-01-01

    Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by the detection of the genomic target often involving 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 (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 present 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 minutes 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 towards the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

  6. 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.

  7. Rapid method for the detection and quantification of N-nitrosodibutylamine in rubber products.

    PubMed

    Zwickenpflug, W; Richter, E

    1987-11-01

    A rapid method for the detection and quantification of N-nitrosodibutylamine (NDBA) migrating from rubber products into a neutral buffer solution is described. The extraction, clean up, and concentration is done by a one-step procedure using C18 cartridges. NDBA in the ppb range is identified and quantitated by gas chromatography (GC) with a thermal energy analyzer (TEA) as detector.

  8. 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.

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

    DOEpatents

    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.

  10. Rapid detection and identification of beer-spoilage lactic acid bacteria by microcolony method.

    PubMed

    Asano, Shizuka; Iijima, Kazumaru; Suzuki, Koji; Motoyama, Yasuo; Ogata, Tomoo; Kitagawa, Yasushi

    2009-08-01

    We evaluated a microcolony method for the detection and identification of beer-spoilage lactic acid bacteria (LAB). In this approach, bacterial cells were trapped on a polycarbonate membrane filter and cultured on ABD medium, a medium that allows highly specific detection of beer-spoilage LAB strains. After short-time incubation, viable cells forming microcolonies were stained with carboxyfluorescein diacetate (CFDA) and counted with muFinder Inspection System. In our study, we first investigated the growth behavior of various beer-spoilage LAB by traditional culture method, and Lactobacillus lindneri and several L. paracollinoides strains were selected as slow growers on ABD medium. Then the detection speeds were evaluated by microcolony method, using these slowly growing strains. As a result, all of the slowly growing beer-spoilage LAB strains were detected within 3 days of incubation. The specificity of this method was found to be exceptionally high and even discriminated intra-species differences in beer-spoilage ability of LAB strains upon detection. These results indicate that our microcolony approach allows rapid and specific detection of beer-spoilage LAB strains with inexpensive CFDA staining. For further confirmation of species status of detected strains, subsequent treatment with species-specific fluorescence in situ hybridization (FISH) probes was shown as effective for identifying the CFDA-detected microcolonies to the species level. In addition, no false-positive results arising from noise signals were recognized for CFDA staining and FISH methods. Taken together, the developed microcolony method was demonstrated as a rapid and highly specific countermeasure against beer-spoilage LAB, and compared favorably with the conventional culture methods.

  11. A loop-mediated isothermal amplification method for rapid detection of NDM-1 gene.

    PubMed

    Qi, Jing; Du, Yijun; Zhu, Xiaoling; Bai, Hua; Luo, Yanbo; Liu, Yuqing

    2012-08-01

    A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.

  12. Rapid, Direct Fluorescent-Antibody Method for the Detection of Salmonellae in Food and Feeds

    PubMed Central

    Insalata, N. F.; Mahnke, C. W.; Dunlap, W. G.

    1972-01-01

    An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies. PMID:4564047

  13. 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.

  14. 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

  15. Rapid methods for detection and enumeration of Campylobacter spp. in foods.

    PubMed

    Wang, Haiyan

    2002-01-01

    Campylobacter spp. are the most commonly reported bacterial cause of acute diarrheal disease in humans throughout the world. Traditional cultural methods for the detection and quantitation of Campylobacterspp. are slow and tedious; therefore, specific, sensitive, and rapid methods for campylobacters are needed to collect sufficient data for risk assessment and food safety policy development. We developed several rapid methods based on polymerase chain reaction (PCR), DNA hybridization, hydrophobic grid membrane filters (HGMFs), and enzyme immunoassays (EIAs). A PCR assay targeting C. jejuni, combined with a simple sample preparation procedure, detects as few as 0.3 most probable number (MPN)/mL C. jejuni in naturally contaminated chicken rinses after 20-24 h enrichment. An HGMF-EIA method using a commercial polyclonal antibody for Campylobacter detects and enumerates thermophilic Campylobacter spp. from spiked chicken rinse and milk, and naturally contaminated chicken rinses. A C. jejuni-specific probe in an HGMF-DNA hybridization protocol specifically detects and quantitates C. jejuni in food samples. A dot-blot EIA combined with an MPN procedure quantitates thermophilic campylobacters from samples that might be difficult to filter through HGMFs.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. Rapid and label-free bioanalytical method of alpha fetoprotein detection using LSPR chip

    NASA Astrophysics Data System (ADS)

    Kim, Dongjoo; Kim, Jinwoon; Kwak, Cheol Hwan; Heo, Nam Su; Oh, Seo Yeong; Lee, Hoomin; Lee, Go-Woon; Vilian, A. T. Ezhil; Han, Young-Kyu; Kim, Woo-Sik; Kim, Gi-bum; Kwon, Soonjo; Huh, Yun Suk

    2017-07-01

    Alpha fetoprotein (AFP) is a cancer marker, particularly for hepatocellular carcinoma. Normal levels of AFP are less than 20 ng/mL; however, its levels can reach more than 400 ng/mL in patients with HCC. Enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been employed for clinical diagnosis of AFP; however, these methods are time consuming and labor intensive. In this study, we developed a localized surface plasmon resonance (LSPR) based biosensor for simple and rapid detection of AFP. This biosensor consists of a UV-Vis spectrometer, a cuvette cell, and a biosensor chip nanopatterned with gold nanoparticles (AuNPs). In our LSPR biosensor, binding of AFP to the surface of the sensor chip led to an increasing magnitude of the LSPR signals, which was measured by an ultraviolet-visible (UV-Vis) spectrometer. Our LSPR biosensor showed sufficient detectability of AFP at concentrations of 1 ng/mL to 1 μg/mL. Moreover, the overall procedure for detection of AFP was completed within 20 min. This biosensor could also be utilized for a point of care test (POCT) by employing a portable UV-Vis spectrometer. Owing to the simplicity and rapidity of the detection process, our LSPR biosensor is expected to replace traditional diagnostic methods for the early detection of diseases.

  1. Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method

    PubMed Central

    Fischbach, Jens; Loh, Qiuting; Bier, Frank F.; Lim, Theam Soon; Frohme, Marcus; Glökler, Jörn

    2017-01-01

    We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate. PMID:28338022

  2. 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

  3. A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins.

    PubMed

    Majerus, P; Zakaria, Z

    1992-10-01

    A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins is described. Aflatoxins B1, B2, G1, and G2, are extracted by methanol/water (85 + 15) and partitioned into methylene dichloride. The methylene dichloride solution is cleaned up on a polypropylene column, filled with 0.5 g silica gel 60. The aflatoxins are eluted with chloroform-acetone (90:10) and are detected using bidirectional thin-layer chromatography (TLC) with aluminium silica gel foil. The mean recovery of aflatoxins B1, B2, G1, and G2 in corn samples was 73, 78, 80, and 64%, respectively; the limit of detection was 0.5 micrograms/kg. The results can also be confirmed by derivative formation using trifluoroacetic acid on the TLC plate. The method has been applied to a wide range of foods with good results.

  4. High Resolution Melting Analysis: A Rapid and Accurate Method to Detect CALR Mutations

    PubMed Central

    Moreno, Melania; Torres, Laura; Santana-Lopez, Gonzalo; Rodriguez-Medina, Carlos; Perera, María; Bellosillo, Beatriz; de la Iglesia, Silvia; Molero, Teresa; Gomez-Casares, Maria Teresa

    2014-01-01

    Background The recent discovery of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN). We tested the feasibility of high-resolution melting (HRM) as a screening method for rapid detection of CALR mutations. Methods CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET. Results Nineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34), 14% of persistent thrombocytosis suggestive of MPN (3/21) and none of the secondary thrombocytosis (0/98). Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%. Conclusions This study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations. PMID:25068507

  5. Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection.

    PubMed

    Balachandran, Priya; Friberg, Maria; Vanlandingham, V; Kozak, K; Manolis, Amanda; Brevnov, Maxim; Crowley, Erin; Bird, Patrick; Goins, David; Furtado, Manohar R; Petrauskene, Olga V; Tebbs, Robert S; Charbonneau, Duane

    2012-02-01

    Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

  6. A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis.

    PubMed

    Sun, Yisuo; Li, Shufen; Zhou, Lin; Zhou, Lin; Zhong, Qiu; Fang, Shisong; Chen, Tao; Bi, Lijun; Mat, Wai-Kin; Zhao, Cunyou; Xue, Hong

    2014-05-01

    Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9 %, 95.5 %, and 92.4 % with ropB in rifampin resistance, 67.3 %, 97.1 %, and 84.3 % with rpsL and rrs in streptomycin resistance, 60.0 %, 96.0 %, and 91.4 % with embB in ethambutol resistance, 68.4 %, 94.9 %, and 86.3 % with inhA and katG in isoniazid resistance, and 74.1 %, 98.9 %, and 93.2 % in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.

  7. 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.

  8. Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Ito, Akira

    2009-01-01

    Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.

  9. Simple, Rapid, and Highly Sensitive Detection of Diphosgene and Triphosgene by Spectrophotometric Methods

    PubMed Central

    Joy, Abraham; Anim-Danso, Emmanuel; Kohn, Joachim

    2009-01-01

    Methods for the detection and estimation of diphosgene and triphosgene are described. These compounds are widely used phosgene precursors which produce an intensely colored purple pentamethine oxonol dye when reacted with 1,3-dimethylbarbituric acid (DBA) and pyridine (or a pyridine derivative). Two quantitative methods are described, based on either UV absorbance or fluorescence of the oxonol dye. Detection limits are ~ 4 µmol/L by UV and <0.4 µmol/L by fluorescence. The third method is a test strip for the simple and rapid detection and semi-quantitative estimation of diphosgene and triphosgene, using a filter paper embedded with dimethylbarbituric acid and poly(4-vinylpyridine). Addition of a test solution to the paper causes a color change from white to light blue at low concentrations and to pink at higher concentrations of triphosgene. The test strip is useful for quick on-site detection of triphosgene and diphosgene in reaction mixtures. The test strip is easy to perform and provides clear signal readouts indicative of the presence of phosgene precursors. The utility of this method was demonstrated by the qualitative determination of residual triphosgene during the production of poly(Bisphenol A carbonate). PMID:19782219

  10. 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

  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. [On-site detection of bioterrorism-relevant agents : Rapid detection methods for viruses, bacteria and toxins - capabilities and limitations].

    PubMed

    Stern, Daniel; Richter, Martin; Schrick, Livia; Lasch, Peter; Keeren, Kathrin; Polleichtner, Angela; Lemmer, Karin; Nitsche, Andreas; Grunow, Roland; Herzog, Christian; Dorner, Brigitte G; Schaade, Lars

    2016-12-01

    In Europe, besides the threat of terrorist attacks involving conventional methods such as explosive devices and automatic weapons, there is also a potential threat of terrorist groups using non-conventional material like biological agents in the scope of future attacks. Consequently, rapid and reliable detection systems for biological agents are being developed and tested continuously to inform crisis management. For environmental detection, a broad spectrum of different laboratory-based techniques has been developed for relevant biological agents. However for environmental samples, fast and reliable on-site detection methods are desired by first responders for rapid assessment.Based on different functional principles, generic, immunological and nucleic-acid-based on-site detection methods can be distinguished. Those should be facile, fast, sensitive, and specific. However, commercially available kits usually have limited sensitivity and often have not been validated independently. Furthermore in this context, the multitude of relevant biological agents that potentially have to be considered present in complex environmental matrices poses a serious challenge for reliable detection. Therefore, detailed knowledge of the specific scope of applications and the limitations of different analytical systems is necessary to evaluate the results obtained purposefully.The aim of this article is to provide an overview of the analytical principles, benefits and limitations of prevailing on-site environmental detection systems for bioterrorism-relevant viruses, bacteria and toxins. Despite promising developments the informative value of currently available on-site tests is still limited. Thus, expert laboratories have to conduct confirmatory testing.

  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. A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

    PubMed Central

    Govan, V. A.; Brözel, V.; Allsopp, M. H.; Davison, S.

    1998-01-01

    Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae. PMID:9572987

  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. Crown Copyright © 2015. Published by Elsevier

  17. [Research on rapid and quantitative detection method for organophosphorus pesticide residue].

    PubMed

    Sun, Yuan-Xin; Chen, Bing-Tai; Yi, Sen; Sun, Ming

    2014-05-01

    The methods of physical-chemical inspection is adopted in the traditional pesticide residue detection, which require a lot of pretreatment processes, are time-consuming and complicated. In the present study, the authors take chlorpyrifos applied widely in the present agricultural field as the research object and propose a rapid and quantitative detection method for organophosphorus pesticide residues. At first, according to the chemical characteristics of chlorpyrifos and comprehensive chromogenic effect of several colorimetric reagents and secondary pollution, the pretreatment of the scheme of chromogenic reaction of chlorpyrifos with resorcin in a weak alkaline environment was determined. Secondly, by analyzing Uv-Vis spectrum data of chlorpyrifos samples whose content were between 0. 5 and 400 mg kg-1, it was confirmed that the characteristic information after the color reaction mainly was concentrated among 360 approximately 400 nm. Thirdly, the full spectrum forecasting model was established based on the partial least squares, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 995 6, standard deviation of calibration (RMSEC) was 2. 814 7 mg kg-1, and standard deviation of verification (RMSEP) was 8. 012 4 mg kg-1. Fourthly, the wavelengths whose center wavelength is 400 nm was extracted as characteristic region to build a forecasting model, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 999 3, standard deviation of calibration (RMSEC) was 2. 566 7 mg kg-1 , standard deviation of verification (RMSEP) was 4. 886 6 mg kg-1, respectively. At last, by analyzing the near infrared spectrum data of chlorpyrifos samples with contents between 0. 5 and 16 mg kg-1, the authors found that although the characteristics of the chromogenic functional group are not obvious, the change of absorption peaks of resorcin itself in the neighborhood of 5 200 cm

  18. Evaluation of a rapid PCR-based method for the detection of animal material.

    PubMed

    Yancy, Haile F; Mohla, Anuja; Farrell, Dorothy E; Myers, Michael J

    2005-12-01

    A rapid PCR-based analytical method for detection of animal-derived materials in complete feed was developed. Using a commercially available DNA forensic kit for the extraction of DNA from animal feed, a sensitive method was developed that was capable of detecting as little as 0.03% bovine meat and bone meal in complete feed in under 8 h of total assay time. The reduction in assay time was accomplished by reducing the DNA extraction time to 2 h and using the simpler cleanup procedure of the kit. Assay sensitivity can be increased to 0.006% by increasing the DNA extraction time to an overnight incubation of approximately 16 h. Examination of dairy feed samples containing either bovine meat and bone meal, porcine meat and bone meal, or lamb meal at a level of 0.1% (wt/wt basis) suggested that this method may be suitable for regulatory uses. The adoption of this commercially available kit for use with animal feeds yields an assay that is quicker and simpler to perform than a previously validated assay for the detection of animal proteins in animal feed.

  19. 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.

  20. A rapid and sensitive method for the detection of aromatic amines in cosmetics.

    PubMed

    Hailong, Xiao; Fen, Qian; Ying, Xu; Jianhong, Pan; Haiyun, Tu; Hongqing, Wang; Saijun, Lin; Jichun, Han

    2014-02-01

    Aromatic amines (AAs) are common chemical pollutants and banned ingredients in cosmetics. In this study, a rapid, simple and stable method for the detection of nine AAs in cosmetics was established based on the optimization of cation exchange solid-phase extraction and liquid chromatography tandem mass spectrometry. The method displayed good linearity within a range of 2-1,000 µg/kg, with limits of quantitation at the level of µg/kg for cosmetic samples. The recoveries obtained for all analyzed amines ranged between 83.6 and 97.8%, and the repeatability (r) and reproducibility (R) values indicated that all nine AAs showed good precision (r ≤ 4.5% and R ≤ 7.7%). The method was applied for the detection of 36 cosmetic samples. It was found that the primary pollutants of AAs were 3, 3'-dichlorobenzidine and 4-aminoazobenzene. The total amine concentration in cosmetic samples ranged from 880 to 5,200 µg/kg. The proposed method is applicable for the analysis of most cosmetic samples.

  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.

  2. A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement

    PubMed Central

    Valuchova, Sona; Fulnecek, Jaroslav; Petrov, Alexander P.; Tripsianes, Konstantinos; Riha, Karel

    2016-01-01

    Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions. PMID:28008962

  3. Rapid Method for Detection, Identification, and Susceptibility Testing of Enteric Pathogens

    PubMed Central

    Stager, Charles E.; Erikson, Eric; Davis, James R.

    1983-01-01

    Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35°C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion results

  4. Rapid method for detection, identification, and susceptibility testing of enteric pathogens.

    PubMed

    Stager, C E; Erikson, E; Davis, J R

    1983-01-01

    Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35 degrees C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion

  5. A rapid and specific method for the detection of indole in complex biological samples.

    PubMed

    Darkoh, Charles; Chappell, Cynthia; Gonzales, Christopher; Okhuysen, Pablo

    2015-12-01

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. 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.

  8. A novel method for rapid detection of Streptococcus pneumoniae antigens in blood.

    PubMed

    Fukushima, Kiyoyasu; Kubo, Toru; Ehara, Naomi; Nakano, Reiji; Matsutake, Toyoshi; Ishimatu, Yuji; Tanaka, Yumi; Akamatsu, Suguru; Izumikawa, Koichi; Kohno, Shigeru

    2016-03-01

    In this study, we used "RAPIRUN(®)Streptococcus pneumoniae HS (otitis media/sinusitis) (RAPIRUN-HS)," a rapid S. pneumoniae antigen detection kit, to investigate methods for detecting S. pneumoniae antigens in blood of 32 bacterial pneumonia patients. We simultaneously performed PCR to detect S. pneumoniae in blood samples. The results of these tests were compared based on pneumonia severity, determined using the Pneumonia Severity Index (PSI) score classification. Four S. pneumoniae PCR-positive patients of the six severe pneumococcal pneumonia patients (PSI risk class IV/V) also tested positive using RAPIRUN-HS. Twenty-four mild to moderate pneumonia patients (PSI risk class I-III) were S. pneumoniae PCR-negative; of these, 21 tested negative using RAPIRUN-HS. The pneumococcal pneumonia patients testing positive using RAPIRUN-HS had low leukocyte counts and elevated C-reactive protein and procalcitonin levels, indicating that RAPIRUN-HS results were correlated with pneumonia severity. The time course evaluations of the laboratory tests for severe pneumococcal pneumonia patients showed that RAPIRUN-HS and S. pneumoniae PCR yielded positive results earlier than the changes in procalcitonin and IL-6. Thus, concomitant pneumococcal bacteremia was strongly suspected in patients testing positive using RAPIRUN-HS. In conclusion, RAPIRUN-HS may be useful for determining whether to admit patients into hospitals and selecting the appropriate antimicrobial agents.

  9. Method for rapid detection and identification of chaetomium and evaluation of resistance to peracetic acid.

    PubMed

    Nakayama, Motokazu; Hosoya, Kouichi; Tomiyama, Daisuke; Tsugukuni, Takashi; Matsuzawa, Tetsuhiro; Imanishi, Yumi; Yaguchi, Takashi

    2013-06-01

    In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the β-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

  10. Resazurin tube method: rapid, simple, and inexpensive method for detection of drug resistance in the clinical isolates of mycobacterium tuberculosis.

    PubMed

    Patil, Santosh S; Mohite, Shivajirao T; Kulkarni, Sunanda A; Udgaonkar, Usha S

    2014-10-01

    Tuberculosis (TB) remains a serious public health problem worldwide. The emergence of drug resistance and multidrug resistance (MDR) has become the main threat to TB treatment and control programs. Rapid detection is critical for the effective treatment of patients. In recent times, a new method using the colorimetric indicator resazurin has been proposed for drug susceptibility of Mycobacterium tuberculosis. In this study, the resazurin reduction assay was adapted to screw cap tubes. Using the Resazurin Tube Method (RTM), a total of 100 clinical isolates were tested against Rifampicin (RIF) and Isoniazide (INH). By visual reading, the minimum inhibitory concentrations (MICs) were obtained after eight days. The results obtained were compared with the gold standard proportion method. Excellent results were obtained for RTM with a sensitivity of 100% for both RIF and INH, with a specificity of 98.7 and 95.3%, respectively. Kappa is the measure of agreement between the RTM and proportion method (PM) for RIF and INH, which was found to be 0.972 and 0.935 for RIF and INH, respectively. The RTM appears to be a reliable method for the rapid and simultaneous detection of MDR-TB and drug susceptibility testing (DST) of M. tuberculosis. It is simple, inexpensive, and with no biohazard risk involved.

  11. Novel Sample Preparation Method for Safe and Rapid Detection of Bacillus anthracis Spores in Environmental Powders and Nasal Swabs

    PubMed Central

    Luna, Vicki A.; King, Debra; Davis, Carisa; Rycerz, Tony; Ewert, Matthew; Cannons, Andrew; Amuso, Philip; Cattani, Jacqueline

    2003-01-01

    Bacillus anthracis spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects B. anthracis in samples containing <10 spores. PMID:12624060

  12. 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

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

    PubMed

    Sun, Bin; Li, Bing

    2015-06-01

    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.

  14. Radial immunodiffusion: a simple and rapid method for detection of Marek's disease antigen(s).

    PubMed

    Marquardt, W W

    1972-05-01

    A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek's disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek's disease.

  15. Development of a fluorescence in situ hybridization (FISH) method for rapid detection of Ulva prolifera

    NASA Astrophysics Data System (ADS)

    Zhang, Qing-Chun; Liu, Qing; Kang, Zhen-Jun; Yu, Ren-Cheng; Yan, Tian; Zhou, Ming-Jiang

    2015-09-01

    Large-scale green tides have occurred consecutively since 2007 in the Yellow Sea (YS), China. The dominant causative species of the green tides has been identified as Ulva prolifera. The origin of green tides in the YS has been traced back to the Subei Shoal based on the results of remote-sensing, numerical simulations and field investigations. However, it is difficult to study the early development of green tides in the Subei Shoal because of the mixture of multiple green algae and the morphological diversity of U. prolifera when under variable environmental conditions. In this study, a rapid and accurate fluorescence in situ hybridization (FISH) method was developed to detect U. prolifera from the community of green algae targeting the 5S rDNA spacer region of U. prolifera. Two specific probes, 5S-1 and 5S-2, were designed based on the sequences of the 5S rDNA spacer regions of U. prolifera, Ulva linza and Ulva flexuosa. Specificity of the FISH method was tested using the six species of green algae commonly occurring in the Subei Shoal, including U. prolifera, U. linza, U. flexuosa, Ulva compressa, Ulva pertusa and Blidingia sp. The results showed that only U. prolifera could be labeled with both probes. Probe 5S-1, which showed a much higher labeling efficiency on U. prolifera, was ultimately selected as the probe for the FISH detection. The sample preparation method was optimized, particularly for the mature green algae, by the addition of cellulase and proteinase K in the pre-hybridization solution. Labeling efficiency with the probe 5S-1 reached 96% on average under the optimized conditions. The successful development of the FISH method has been applied to qualitative and quantitative analysis of field samples collected from the YS, and the results indicate a potential use in future green algae studies.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

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

    PubMed

    Nyan, Dougbeh-Chris; Swinson, Kevin L

    2015-12-08

    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.

  2. 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.

  3. A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model.

    PubMed

    Tu, Shih-Hsin; Hsieh, Yi-Chen; Huang, Li-Chi; Lin, Chun-Yu; Hsu, Kai-Wen; Hsieh, Wen-Shyang; Chi, Wei-Ming; Lee, Chia-Hwa

    2017-06-23

    As cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models. In this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D-imaging on orthotropic-developed breast-tumor-bearing mice. In this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x10(11) cell numbers with great correlation (R(2) = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R(2) > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0

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

    PubMed

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

    2017-02-26

    Sexually transmitted diseases easily spread among sexually active people and often have no symptoms. Rapid and accurate method for detecting these infections are necessary in early stages. The traditional detection methods of them are difficult and time-consuming. In this study, multiplex real time PCR was optimized for rapid identification of Chlamydia trachomatis and Mycoplasma hominis in a single tube and was performed with our designed primers. The sensitivity test was carried out to designed primers with diluted genomic DNA. To defined the specificity, non STD bacteria were used as DNA template. This study indicated that the developed multiplex real time PCR can be an effective alternative procedure to the conventional methods for rapid and accurate identification of C Chlamydia trachomatis and Mycoplasma hominis. Multiplex real-time PCR Results of them were checked with melting curves. The sensitivity of our designed primer by multiplex real time PCR for Chlamydia trachomatis and Mycoplasma hominis were 4.78×10(10) and 8.35×10(10) , respectively, Which the primers did not amplify any product from a non-STD species. Multiplex real time PCR by our new primers and analysis of melting curves were successfully usable for rapid and accurate detection of Chlamydia trachomatis and Mycoplasma hominis. This assay instead of traditional culture method, has considerable potential to be rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous and direct detection. © 2017 Wiley Periodicals, Inc.

  5. Comparing rapid methods for detecting Listeria in seafood and environmental samples using the most probably number (MPN) technique.

    PubMed

    Cruz, Cristina D; Win, Jessicah K; Chantarachoti, Jiraporn; Mutukumira, Anthony N; Fletcher, Graham C

    2012-02-15

    The standard Bacteriological Analytical Manual (BAM) protocol for detecting Listeria in food and on environmental surfaces takes about 96 h. Some studies indicate that rapid methods, which produce results within 48 h, may be as sensitive and accurate as the culture protocol. As they only give presence/absence results, it can be difficult to compare the accuracy of results generated. We used the Most Probable Number (MPN) technique to evaluate the performance and detection limits of six rapid kits for detecting Listeria in seafood and on an environmental surface compared with the standard protocol. Three seafood products and an environmental surface were inoculated with similar known cell concentrations of Listeria and analyzed according to the manufacturers' instructions. The MPN was estimated using the MPN-BAM spreadsheet. For the seafood products no differences were observed among the rapid kits and efficiency was similar to the BAM method. On the environmental surface the BAM protocol had a higher recovery rate (sensitivity) than any of the rapid kits tested. Clearview™, Reveal®, TECRA® and VIDAS® LDUO detected the cells but only at high concentrations (>10(2) CFU/10 cm(2)). Two kits (VIP™ and Petrifilm™) failed to detect 10(4) CFU/10 cm(2). The MPN method was a useful tool for comparing the results generated by these presence/absence test kits. There remains a need to develop a rapid and sensitive method for detecting Listeria in environmental samples that performs as well as the BAM protocol, since none of the rapid tests used in this study achieved a satisfactory result. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. A rapid and simple method of detection of Blepharisma japonicum using PCR and immobilisation on FTA paper

    PubMed Central

    Hide, Geoff; Hughes, Jacqueline M; McNuff, Robert

    2003-01-01

    Background The rapid expansion in the availability of genome and DNA sequence information has opened up new possibilities for the development of methods for detecting free-living protozoa in environmental samples. The protozoan Blepharisma japonicum was used to investigate a rapid and simple detection system based on polymerase chain reaction amplification (PCR) from organisms immobilised on FTA paper. Results Using primers designed from the α-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved. Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper. Conclusion This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms. PMID:14516472

  7. Rapid, Potentially Automatable, Method Extract Biomarkers for HPLC/ESI/MS/MS to Detect and Identify BW Agents

    DTIC Science & Technology

    1997-11-01

    status can sometimes be reflected in the infectious potential or drug resistance of those pathogens. For example, in Mycobacterium tuberculosis ... Mycobacterium tuberculosis , its antibiotic resistance and prediction of pathogenicity amongst Mycobacterium spp. based on signature lipid biomarkers ...TITLE AND SUBTITLE Rapid, Potentially Automatable, Method Extract Biomarkers for HPLC/ESI/MS/MS to Detect and Identify BW Agents 5a. CONTRACT NUMBER 5b

  8. 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.

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

    PubMed

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

    2015-07-01

    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. 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 10(3)/10(4)/10(5) CFU/ml). Time schedule for testing was limited (module 1: 6 h, module 2 and 3: 7 days). 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 10(4) to 10(5) CFU/ml of bacteria obtained positive results with both rapid screening methods, whereas samples spiked with only 10(3) 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. 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 provided material is matrix

  10. A simple, rapid, cost-effective and sensitive method for detection of Salmonella in environmental and pecan samples.

    PubMed

    Dobhal, S; Zhang, G; Rohla, C; Smith, M W; Ma, L M

    2014-10-01

    PCR is widely used in the routine detection of foodborne human pathogens; however, challenges remain in overcoming PCR inhibitors present in some sample matrices. The objective of this study was to develop a simple, sensitive, cost-effective and rapid method for processing large numbers of environmental and pecan samples for Salmonella detection. This study was also aimed at validation of a new protocol for the detection of Salmonella from in-shell pecans. Different DNA template preparation methods, including direct boiling, prespin, multiple washing and commercial DNA extraction kits, were evaluated with pure cultures of Salmonella Typhimurium and with enriched soil, cattle feces and in-shell pecan each spiked individually with Salmonella Typhimurium. PCR detection of Salmonella was conducted using invA and 16S rRNA gene (internal amplification control) specific primers. The effect of amplification facilitators, including bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and gelatin on PCR sensitivity, was also evaluated. Conducting a prespin of sample matrices in combination with the addition of 0·4% (w/v) BSA and 1% (w/v) PVP in PCR mix was the simplest, most rapid, cost-effective and sensitive method for PCR detection of Salmonella, with up to 40 CFU Salmonella per reaction detectable in the presence of over 10(9 ) CFU ml(-1) of background micro-organisms from enriched feces soil or pecan samples. The developed method is rapid, cost-effective and sensitive for detection of Salmonella from different matrices. This study provides a method with broad applicability for PCR detection of Salmonella in complex sample matrices. This method has a potential for its application in different research arenas and diagnostic laboratories. © 2014 The Society for Applied Microbiology.

  11. A novel method for the rapid detection of microbes in blood using pleurocidin antimicrobial peptide functionalized piezoelectric sensor.

    PubMed

    Shi, Xiaohong; Zhang, Xiaoqing; Yao, Qiongqiong; He, Fengjiao

    2017-02-01

    The rapid detection of microbes is critical in clinical diagnosis and food safety. Culture-dependent assays are the most widely used microbial detection methods, but these assays are time-consuming. In this study, a rapid microbial detection method was proposed using a pleurocidin/single-walled carbon nanotubes/interdigital electrode-multichannel series piezoelectric quartz crystal (pleurocidin/SWCNT/IDE-MSPQC) sensor. The selected pleurocidin antimicrobial peptide served as a recognition probe that exhibits broad-spectrum antimicrobial activity and the SWCNT acted as the electronic transducer and cross-linker for the immobilization of pleurocidin on the IDE. The response mechanism of the sensor was based on the specific interaction between pleurocidin and the microbe causing pleurocidin to detach from the SWCNT modified IDE, resulting in a sensitive frequency shift response of the MSPQC. Microbes that may be clinically present in the bloodstream during an infection were successfully detected by the proposed method within 15min. The developed strategy provides a new universal platform for the rapid detection of microbes.

  12. Application of a bioluminescence method for the beer industry: sensitivity of MicroStar-RMDS for detecting beer-spoilage bacteria. Rapid Microbe Detection System.

    PubMed

    Takahashi, T; Nakakita, Y; Watari, J; Shinotsuka, K

    2000-05-01

    We set up the original operating conditions of the MicroStar-Rapid Microbe Detection System (RMDS) to suppress false positives, which have kept this system from practical. The detection limit of our system was between 6.3 x 10(-16) mol and 3.1 x 10(-16) mol in terms of the amount of ATP, which is approximately equal to the ATP content of one yeast cell or 50 lactic acid bacteria cells. The detection time and the detection count were compared between the RMD method and the conventional plate count method (C.P.C. method) using 23 test samples of beer-spoilage Lactobacillus brevis. Judging from the detection time and detection count, 16-24 hours of cultivation for the RMD method corresponded to 40-96 hours of cultivation for the C.P.C. method. The RMD method reached a useful level for our practical use at the point of sensitivity.

  13. 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.

  14. Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method

    PubMed Central

    Abdullah, J.; Saffie, N.; Sjasri, F.A.R.; Husin, A.; Abdul-Rahman, Z.; Ismail, A.; Aziah, I.; Mohamed, M.

    2014-01-01

    An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method. PMID:25763045

  15. Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method.

    PubMed

    Abdullah, J; Saffie, N; Sjasri, F A R; Husin, A; Abdul-Rahman, Z; Ismail, A; Aziah, I; Mohamed, M

    2014-01-01

    An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

  16. 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

    2016-03-10

    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.

  17. 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

  18. Establishment of a proficiency panel for an external quality assessment programme for the detection of bacterial contamination in platelet concentrates using rapid and cultural detection methods.

    PubMed

    Vollmer, T; Schmidt, M; Hourfar, K; Schottstedt, V; Pichl, L; Gubbe, K; Knabbe, C; Dreier, J

    2016-05-01

    Platelet concentrates (PCs) are the main focus regarding the residual risk of transfusion-transmitted bacterial infections. Rapid screening methods for bacterial detection in platelets have been optimized over the last decade, but their external evaluation represents a complicated process. We developed a new type of proficiency panel for bacterial detection in PCs using currently available screening methods (especially rapid methods) suitable for external quality assessment programmes (EQAP). PC samples were inoculated with different bacteria at two concentrations (10E+03 CFU/ml, 10E+05 CFU/ml) and stored under temperature-controlled conditions (1-5 days). Bacterial growth was further prevented by the addition of 0-20 μg/ml cotrimoxazole. Samples were analysed prior to and after storage using rapid detection methods (Bactiflow (BF), bacteria-generic NAT) and cultural methods to determine the influence of storage and antibiotic treatment on bacterial counts and the result outcome. A pilot EQAP was performed with four participants. Testing under the evaluated conditions demonstrated that bacterial counts remained constant prior to and after storage. The supplementation of 10 μg/ml cotrimoxazole did not influence bacterial detection using the two rapid detection methods BF and NAT. Furthermore, the detection of bacteria using cultural methods is still possible despite of antibiotic supplementation. The pilot EQAP confirmed these results. A storage time of up to 3 days proved practicable, showing no considerable influence on bacterial count and outcome of test results. The established proficiency panel provided PC matrix-conform samples with stabilized bacterial counts which can be analysed in parallel by rapid and cultural detection methods. © 2016 International Society of Blood Transfusion.

  19. Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

    PubMed

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Quyen, Than Linh; Engelsmann, Pia; Wolff, Anders; Bang, Dang Duong

    2017-04-01

    Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. BACs-on-beads: a new robust and rapid detection method for prenatal diagnosis.

    PubMed

    Choy, Richard Kwong Wai; Chen, Ying; Sun, Xiao-Fang; Kwok, Yvonne Ka Yin; Leung, Tak Yeung

    2014-04-01

    Karyotyping, the gold standard used for diagnosis of chromosomal abnormalities, is being progressively replaced by rapid aneuploidy testing (RAT) techniques such as quantitative fluorescence-PCR, FISH and multiplex ligation-dependent probe amplification for diagnosing the common aneuploidies or chromosomal microarray analysis for comprehensive genome-wide testing. However, due to technical limitations, current RATs are confined to the detection of common aneuploidies 13, 18, 21 and sex chromosomes. To overcome the limitations of RATs, a bacterial artificial chromosomes-on-beads (BoBs™) assay technology has been introduced for the detection of the common aneuploidies as well as specific microdeletion syndromes. The BoBs assay is a bead-based multiplex assay using polystyrene beads impregnated with two spectrally distinct infrared fluorochromes to create a liquid array of up to 100 unique spectral signatures that supports the analysis of that scale of simultaneous hybridization assays on a minute DNA sample. This review gives an overview on the collective experiences of BoBs applications in prenatal diagnosis.

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  2. 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

  3. An interactive image segmentation method for lithological boundary detection: A rapid mapping tool for geologists

    NASA Astrophysics Data System (ADS)

    Vasuki, Yathunanthan; Holden, Eun-Jung; Kovesi, Peter; Micklethwaite, Steven

    2017-03-01

    Large volumes of images are collected by geoscientists using remote sensing platforms. Manual analysis of these images is a time consuming task and there is a need for fast and robust image interpretation tools. In particular the reliable mapping of lithological boundaries is a critical step for geological interpretation. In this contribution we developed an interactive image segmentation algorithm that harnesses the geologist's input and exploits automated image analysis to provide a practical tool for lithology boundary detection, using photographic images of rock surfaces. In the proposed method, the user is expected to draw rough markings to indicate the locations of different geological units in the image. Image segmentation is performed by segmenting regions based on their homogeneity in colour. This results in a high density of segmented regions which are then iteratively merged based on the colour of different geological units and the user input. Finally, a post-processing step allows the user to edit the boundaries. An experiment was conducted using photographic rock surface images collected by a UAV and a handheld digital camera. The proposed technique was applied to detect lithology boundaries. It was found that the proposed method reduced the interpretation time by a factor of four relative to manual segmentation, while achieving more than 96% similarity in boundary detection. As a result the proposed method has the potential to provide practical support for interpreting large volume of complex geological images.

  4. Rapid and Efficient Method for the Detection of Microplastic in the Gastrointestinal Tract of Fishes.

    PubMed

    Roch, Samuel; Brinker, Alexander

    2017-04-07

    The rising evidence of microplastic pollution impacts on aquatic organisms in both marine and freshwater ecosystems highlights a pressing need for adequate and comparable detection methods. Available tissue digestion protocols are time-consuming (>10 h) and/or require several procedural steps, during which materials can be lost and contaminants introduced. This novel approach comprises an accelerated digestion step using sodium hydroxide and nitric acid in combination to digest all organic material within 1 h plus an additional separation step using sodium iodide which can be used to reduce mineral residues in samples where necessary. This method yielded a microplastic recovery rate of ≥95%, and all tested polymer types were recovered with only minor changes in weight, size, and color with the exception of polyamide. The method was also shown to be effective on field samples from two benthic freshwater fish species, revealing a microplastic burden comparable to that indicated in the literature. As a consequence, the present method saves time, minimizes the loss of material and the risk of contamination, and facilitates the identification of plastic particles and fibers, thus providing an efficient method to detect and quantify microplastics in the gastrointestinal tract of fishes.

  5. Rapid method utilizing the polymerase chain reaction for detection of canine parvovirus in feces of diarrheic dogs.

    PubMed

    Uwatoko, K; Sunairi, M; Nakajima, M; Yamaura, K

    1995-03-01

    By using primers based on the sequence of the VP2 gene of canine parovirus (CPV), we established a rapid and specific assay for identification of the virus from fecal specimens based on the polymerase chain reaction (PCR). By use of a pair of primers, a specific 226-bp sequence was amplified by the PCR. All strains of CPV tested gave a specific amplification product by the PCR, while neither porcine parovirus nor host cell did so. The PCR assay can detect fewer particles of CPV than the conventional methods, being able to detect CPV from fecal specimens in a rapid manner, provided that gel filtration of the samples through a spun column was done to remove inhibitory substances from the fecal specimens. These results suggest that the PCR assay can detect the presence of CPV in dogs early enough to prevent secondary infection by CPV in veterinary hospitals.

  6. [Development and application of TaqMan MGB probe fluorescence quantitative PCR method for rapid detection of Clostridium piliforme].

    PubMed

    Gao, Zheng-qin; Yue, Bing-fei; He, Zheng-ming

    2012-02-01

    To develop a TaqMan MGB probe-based, sensitive and specific fluorescence quantitative PCR assay method for rapid detection of Clostridium piliforme. Primers and probes specific to 16S rRNA gene of Clostridium piliforme were designed. A TaqMan MGB probe-based, fluorescence quantitative PCR method was established. Specificity, sensitivity and stability of the method were assessed, followed by real-time quantitative PCR assay to detect Clostridium piliforme on 1156 clinical specimens during 2008-2011 and compared with conventional PCR assay. The specificity of TaqMan MGB probe-based fluorescence quantitative PCR was high and did not show cross-reactivity with Helicobacter hepaticus, Helicobacter pylori, Campylobacter jejuni, Pasteurella pneumotropica, Escherichia coli or Pseudomonas aeruginosa. The detection limit was 2.2 copies/µl. The correlation coefficient and slope value of standard curve were 0.999 and -3.204, respectively and the efficiency of TaqMan MGB-based probe fluorescence quantitative PCR assay was 100%. When the TaqMan MGB-based probe fluorescence quantitative PCR assay was preformed to detect Clostridium piliforme on 1156 clinical specimens, a total of 101 specimens showed positive on Clostridium piliforme. However, only 44 specimens showed positive when conventional PCR was used. The real-time quantitative PCR for Clostridium piliforme could be completed within 2 hours. The TaqMan MGB-based probe fluorescence quantitative PCR assay method was a reliable, specific, sensitive and useful tool for rapid detection of Clostridium piliforme.

  7. Rapid Screening Method for Detecting Ethinyl Estradiol in Natural Water Employing Voltammetry

    PubMed Central

    2016-01-01

    17α-Ethinyl estradiol (EE2), which is used worldwide in the treatment of some cancers and as a contraceptive, is often found in aquatic systems and is considered a pharmaceutically active compound (PhACs) in the environment. Current methods for the determination of this compound, such as chromatography, are expensive and lengthy and require large amounts of toxic organic solvents. In this work, a voltammetric procedure is developed and validated as a screening tool for detecting EE2 in water samples without prior extraction, clean-up, or derivatization steps. Application of the method we elaborate here to EE2 analysis is unprecedented. EE2 detection was carried out using differential pulse adsorptive cathodic stripping voltammetry (DP AdCSV) with a hanging mercury drop electrode (HMDE) in pH 7.0 Britton-Robinson buffer. The electrochemical process of EE2 reduction was investigated by cyclic voltammetry at different scan rates. Electroreduction of the hormone on a mercury electrode exhibited a peak at −1.16 ± 0.02 V versus Ag/AgCl. The experimental parameters were as follows: −0.7 V accumulation potential, 150 s accumulation time, and 60 mV s−1 scan rate. The limit of detection was 0.49 μg L−1 for a preconcentration time of 150 s. Relative standard deviations were less than 13%. The method was applied to the detection of EE2 in water samples with recoveries ranging from 93.7 to 102.5%. PMID:27738548

  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.

  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. Rapid method for the detection of storage mites in cereals: feasibility of an ELISA based approach.

    PubMed

    Dunn, J A; Thind, B B; Danks, C; Chambers, J

    2008-04-01

    This paper describes the development of rapid immunodiagnostic tests for the detection of storage mite infestations in cereals and cereal products. The study's first phase (proof of concept) involved the production of a species-specific enzyme-linked immunoassay (ELISA) for the flour mite, Acarus siro (L.), a major pest of stored commodities. The specificity of this new assay was assessed against key stored product contaminants (13 species of mites of which three were predatory, five species of insects and five species of fungi) in the presence and absence of grain. The assay was species-specific (no cross-reactivity to other storage contaminants) and was unaffected by the presence of cereal antigens in the extract. In the study's second phase, species- and genera-specific ELISAs were developed for a range of key storage mite pests: the cosmopolitan food mite (Lepidoglyphus destructor), the grocers' itch mite (Glycyphagus domesticus), the grainstack mite (Tyrophagus longior), mites of the Tyrophagus and Glycyphagus generas, and all storage mites. All tests were demonstrably specific to target species or genera, with no cross-reactions observed to other storage pest contaminants or cereals. The final, validation phase, involved a comparative assessment of the species-specific A. siro and the genus-specific Tyrophagus ELISAs with the flotation technique using laboratory and field samples. Both ELISAs were quantitative (0-30 mites per 10 g wheat) and produced good comparative data with the flotation technique (A. siro r(2)=0.91, Tyrophagus spp. r(2)=0.99).

  11. Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates.

    PubMed

    Vollmer, T; Hinse, D; Schottstedt, V; Bux, J; Tapernon, K; Sibrowski, W; Knabbe, C; Dreier, J

    2012-07-01

      Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT-PCR) was evaluated in an inter-laboratory comparison in three different German blood services.   Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10(3) -4·5 × 10(8)  CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods.   The inter-laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT-PCR-screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non-detected positive samples were below the assay's detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10(5) and 2·10 × 10(7)  CFU/ml, respectively, K. pneumoniae: 4·79 × 10(6)  CFU/ml, S. aureus: 6·03 × 10(5)  CFU/ml). All rapid screening methods revealed no false-positive results.   Both BactiFlow and 23S rRNA RT-PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram-negative strains. © 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.

  12. [A simple method for the rapid detection of bacterial hyaluronidase in K hyaluronate-containing gel].

    PubMed

    Balke, E; Weiss, R

    1984-08-01

    For detection of hyaluronidase activities we investigated several groups of bacteria. The bacteria were inoculated on a 1,5% agarose gel in Petri plates of 4 cm diameter or gel discs of 7 mm diameter, containing 0,1% of K-hyaluronate as well as nutritient medium, and were incubated for 2-20 h at 37 degrees C in a moist chamber. Subsequently some ml of a 10% solution of cetylpyridiniumchloride were poured on the gel to precipitate the polymere hyaluronate. If the hyaluronate was depolymerized by hyaluronidase, a translucent area was visible around the colonies. We found out, that a gel layer of 1 mm was sufficient to detect the small amounts of hyaluronidase, which were produced by bacteria within an incubation time of 2 h. These results were confirmed by incubation for 20 h and in some cases 36 h. The hyaluronidase production by different anaerobic Clostridium strains was always proved after a 20 h growth period. The bacteria were inoculated with the whole loop of a self made platin sowing wire loop. By this method quantitative differences of hyaluronidase activities between different strains of bacteria could be detected.

  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. 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.

  15. A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR.

    PubMed

    Nakahara, K; Hataya, T; Uyeda, I

    1999-01-01

    A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples.

  16. A rapid microtiter plate method for the detection of lysozyme release from human neutrophils.

    PubMed

    Moreira-Ludewig, R; Healy, C T

    1992-04-01

    An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng/ml. Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t-Boc-L-methionyl-L-leucyl-L-phenylalanine (N-t-Boc), LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC50 of 2 microM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC50 of 2 microM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. An immunomagnetic separation/loop-mediated isothermal amplification method for rapid direct detection of thermotolerant Campylobacter spp. during poultry production.

    PubMed

    Romero, M R; D'Agostino, M; Arias, A Pérez; Robles, S; Casado, C Fernández; Iturbe, L Orueta; Lerma, O Gurrutxaga; Andreou, M; Cook, N

    2016-02-01

    To develop a rapid test for thermotolerant Campylobacter in poultry faeces. The reported method is based on immunomagnetic separation and loop-mediated isothermal DNA amplification (IMS/LAMP). This LAMP assay is specific (demonstrated using 10 Campylobacter strains and 13 non-Campylobacter bacterial species) and sensitive (95% probability of detecting 22 genome copies). A competitive internal amplification control (IAC) has been incorporated to give unambiguous determination of negative results. Immunoseparation of Campylobacter allows direct LAMP detection from poultry boot swab samples in 90 min without enrichment or DNA purification (74% probability of detecting 10(4) CFU ml(-1) of a boot swab suspension). The analysis of 17 samples from commercial turkey farms showed 100% correlation with parallel results obtained by standard microbiological methods. A rapid test has been developed for direct detection of thermotolerant Campylobacter spp. in boot swab samples, thus bypassing culture enrichment or DNA extraction. The test has potential to be carried out by farm personnel on site. The method offers an inexpensive approach to monitor poultry infection in near real time, assisting flock management and controls to prevent introduction of Campylobacter into the food chain. © 2015 The Society for Applied Microbiology.

  18. 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.

  19. Investigation of fluorescence methods for rapid detection of municipal wastewater impact on drinking water sources

    NASA Astrophysics Data System (ADS)

    Peleato, Nicolas M.; Legge, Raymond L.; Andrews, Robert C.

    2017-01-01

    Fluorescence spectroscopy as a means to detect low levels of treated wastewater impact on two source waters was investigated using effluents from five wastewater facilities. To identify how best to interpret the fluorescence excitation-emission matrices (EEMs) for detecting the presence of wastewater, several feature selection and classification methods were compared. An expert supervised regional integration approach was used based on previously identified features which distinguish biologically processed organic matter including protein-like fluorescence and the ratio of protein to humic-like fluorescence. Use of nicotinamide adenine dinucleotide-like (NADH) fluorescence was found to result in higher linear correlations for low levels of wastewater presence. Parallel factors analysis (PARAFAC) was also applied to contrast an unsupervised multiway approach to identify underlying fluorescing components. A humic-like component attributed to reduced semiquinone-like structures was found to best correlate with wastewater presence. These fluorescent features were used to classify, by volume, low (0.1-0.5%), medium (1-2%), and high (5-15%) levels by applying support vector machines (SVMs) and logistic regression. The ability of SVMs to utilize high-dimensional input data without prior feature selection was demonstrated through their performance when considering full unprocessed EEMs (66.7% accuracy). The observed high classification accuracies are encouraging when considering implementation of fluorescence spectroscopy as a water quality monitoring tool. Furthermore, the use of SVMs for classification of fluorescence data presents itself as a promising novel approach by directly utilizing the high-dimensional EEMs.

  20. Interlaboratory validation of a real-time PCR 24-hour rapid method for detection of Salmonella in foods.

    PubMed

    Cheng, Chorng-Ming; Van Khanh, T; Lin, Wen; Ruby, Richard M

    2009-05-01

    The efficacy of a 24-h Salmonella real-time, or quantitative, PCR (qPCR) detection method was assessed through a collaborative effort involving eight Federal and state laboratories. Eleven foods including mashed potatoes, soft cheese, chili powder, chocolate, eggs, sprouts, apple juice, fish, shrimp, ground beef, and ground chicken were tested. For each food, seven blind samples were distributed to each participant for testing. These included six samples equivalently inoculated with 1 to 5 CFU/25 g of various serotypes of Salmonella (Gaminara, Weltevreden, Heidelberg, Senftenberg, Enteritidis, Newport, Typhimurium, and Kentucky for each food) and 10 to 50 CFU/25 g of the competitor Enterobacter cloacae. The seventh sample was inoculated with 10 to 50 CFU/25 g of the competitor, E. cloacae, only. These samples were tested for Salmonella by using four methods in parallel: (i) 24-h qPCR method detecting Salmonella from modified buffered peptone water enrichment medium; (ii) 48-h qPCR method detecting Salmonella from a secondary selective enrichment broth; (iii) modified Bacteriological Analytical Manual method; and (iv) VIDAS, an immunoassay system. The results of the statistical analysis showed there was no significant (P > or = 0.05) difference between either of the qPCR methods and the modified Bacteriological Analytical Manual method for 10 of 11 foods. For the one exception, sprouts, detection by qPCR required 48 h. Both qPCR methods showed a detection limit of 0.08 to 0.2 CFU/g. These results provide a solid basis for using this 24-h qPCR rapid screening method to detect Salmonella in foods.

  1. Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods.

    PubMed

    Albillos, S M; Reddy, R; Salter, R

    2011-07-01

    Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method. Copyright ©, International Association for Food Protection

  2. Rapid and specific detection of H3 swine influenza virus using reverse transcription loop-mediated isothermal amplification method.

    PubMed

    Gu, H; Qi, X; Li, X; Jiang, H; Wang, Y; Liu, F; Lu, S; Yang, Y; Liu, F

    2010-04-01

    The main objective of our study is to develop a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based system for rapid and specific detection of H3 swine influenza virus (SIV). The system, H3 RT-LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT-LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10-fold more sensitive than the conventional RT-PCR assay and even comparable to the real-time RT-PCR method, with the detection limit of about one plaque-forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT-LAMP and real-time RT-PCR methods, while only 7 were positive with the conventional RT-PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT-PCR and real-time RT-PCR approaches. Our results provide the first direct evidence that RT-LAMP is highly specific and sensitive for detecting H3 SIV. These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings.

  3. 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.

  4. Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.

    PubMed

    Cui, S; Schroeder, C M; Zhang, D Y; Meng, J

    2003-01-01

    To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.

  5. A facile label-free G-quadruplex based fluorescent aptasensor method for rapid detection of ATP

    NASA Astrophysics Data System (ADS)

    Liu, Haisheng; Ma, Changbei; Ning, Feng; Chen, Hanchun; He, Hailun; Wang, Kemin; Wang, Jun

    2017-03-01

    The present work demonstrates a simple, rapid and label-free ATP detection method using a fluorescent aptasensor that is based on G-quadruplex formation. In the absence of ATP, the Thioflavin T (ThT) dye binds to the G-rich ATP aptamer and forms an ATP aptamer/ThT G-quadruplex complex, which results in high fluorescence intensity. Upon addition of ATP, the ATP aptamer/ThT complex will be replaced by the formation of an ATP aptamer/ATP complex. During this process, separation of the ThT dye from the ATP aptamer/ThT complex decreases the fluorescence intensity of the reaction mixture dramatically. This fluorescence aptasensor is highly sensitive and rapid, with a detection limit of 18 nM and a total reaction time of only 10 min. Furthermore, this method is cost-effective and simple, removing the requirement for labeling the detection reagents with a fluorophore-quencher pair.

  6. Comparison of three rapid methods for detection of antibodies to streptolysin O and DNase B.

    PubMed Central

    Hostetler, C L; Sawyer, K P; Nachamkin, I

    1988-01-01

    Three commercial systems were compared for ability to detect antibodies to streptolysin O (ASO) and DNase B (ADB). Streptozyme (Wampole Laboratories, Cranbury, N.J.) exhibited high sensitivity (100%) for detecting ASO but low sensitivity for ADB (22.2%). The LeapStrep (Organon Teknika, Malvern, Pa.) and Check-Spectra (Diagnostic Technology, Hauppauge, N.Y.) tests had low sensitivities for detecting ASO (35.3 and 21.4%, respectively) and ADB (22.2 and 33.3%, respectively). PMID:3045154

  7. Development of rapid, sensitive and non-radioactive tissue-blot diagnostic method for the detection of citrus greening.

    PubMed

    Nageswara-Rao, Madhugiri; Miyata, Shin-Ichi; Ghosh, Dilip; Irey, Mike; Garnsey, Stephen M; Gowda, Siddarame

    2013-01-01

    Citrus huanglongbing (HLB or citrus greening) is one of the most devastating diseases of citrus worldwide. The disease is caused by Gram-negative, phloem-limited α-proteobacterium, 'Candidatus Liberibacter asiaticus', vectored by the psyllid, Diaphorina citri Kuwayama. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection and non-uniform distribution within the tree makes the detection of the pathogen very difficult. Efficient management of HLB disease requires rapid and sensitive detection early in the infection followed by eradication of the source of pathogen and the vector. The polymerase chain reaction (PCR) based method is most commonly employed for screening the infected/suspected HLB plants and psyllids. This is time consuming, cumbersome and not practical for screening large number of samples in the field. To overcome this, we developed a simple, sensitive, non-radioactive, tissue-blot diagnostic method for early detection and screening of HLB disease. Digoxigenin labeled molecular probes specific to 'Ca. L. asiaticus' nucleotide sequences have been developed and used for the detection of the pathogen of the HLB disease. The copy number of the target genes was also assessed using real-time PCR experiments and the optimized real-time PCR protocol allowed positive 'Ca. L. asiaticus' detection in citrus samples infected with 'Ca. L. asiaticus' bacterium. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Rapid detection of bacteria in urine samples by the "three-plug-injection" method using capillary electrophoresis.

    PubMed

    Song, Lin; Li, Wanchen; Li, Guoxia; Wei, Dianjun; Ge, Peng; Li, Guizhen; Zheng, Fang; Sun, Xuguo

    2013-09-15

    This study explored a method that can rapidly detect bacteria in urine samples for the auxiliary determination of urinary tract infections (UTIs). Urine samples from patients with UTIs (230 cases) were obtained using aseptic technique. The urine biochemical assay was then carried out using an automated urine analyzer for all the urine samples. Bacterial species were identified by a combination of bacterial culture technique, morphological observation and the BACT-IST microbial identification/susceptibility analysis system. The most common seven species of bacteria in the study included Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. Bacterial samples were suspended in sample buffer solutions and separated by the "three-plug-injection" method using capillary electrophoresis (CE). Each species of bacteria appeared as a bacterial peak. The mixture of the seven species also provided only one peak. Further analysis showed that the concentration limit for the "three-plug-injection" method is 10(6) colony forming units (CFU)/mL, and there is a good linear relationship between the peak height and bacterial concentration (R(2)=0.99). The effect of urine composition on CE results was also investigated. The results showed that urine composition, i.e., proteins, white blood cells (WBCs) and red blood cells (RBCs), affected the peak retention time but could not affect the separation of bacteria. The results demonstrated that the bacteria in urine samples can be detected within 10min by the "three-plug-injection" method using CE. The "three-plug-injection" method is therefore suitable for the rapid detection of organisms in clinical urine samples from UTIs.

  9. Microarray Method for the Rapid Detection of Glycosaminoglycan–Protein Interactions

    PubMed Central

    Rogers, Claude J.; Hsieh-Wilson, Linda C.

    2014-01-01

    Glycosaminoglycans (GAGs) perform numerous vital functions within the body. As major components of the extracellular matrix, these polysaccharides participate in a diverse array of cell-signaling events. We have developed a simple microarray assay for the evaluation of protein binding to various GAG subclasses. In a single experiment, the binding to all members of the GAG family can be rapidly determined, giving insight into the relative specificity of the interactions and the importance of specific sulfation motifs. The arrays are facile to prepare from commercially available materials. PMID:22057535

  10. 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.

  11. Fluorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation

    SciTech Connect

    Birnboim, H.C.; Jevcak, J.J.

    1981-05-01

    DNA strand breaks can be detected with great sensitivity by exposing crude cell lysates to alkaline solutions and monitoring the rate of strand unwinding. As little as one strand break per chromosome can be detected. Previous methods for measuring strand unwinding have required physical separation of single- from double-stranded molecules. We now describe conditions under which unwinding can be monitored directly using a fluorescent dye, thus greatly simplifying the analysis. Breaks due to irradiation of blood samples by /sup 60/Co gamma-rays at doses as low as 0.05 to 0.1 gray (5 to 10 rads) were detectable. Rapid rejoining of strand breaks during in vitro incubation at 37 degrees could readily be observed following a dose of one gray. Since the procedure is very rapid and cells can be analyzed directly without the requirement for culturing or radiolabeling, the procedure could be useful in cancer chemotherapy if in vivo damage is to be monitored or for testing the in vitro sensitivity of cells to drugs.

  12. Fluorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation

    SciTech Connect

    Birnboim, H.C.; Jevcak, J.J.

    1981-05-01

    DNA strand breaks can be detected with great sensitivity by exposing crude cell lysates to alkaline solutions and monitoring the rate of strand unwinding. As little as one strand break per chromosome can be detected. Previous methods for measuring strand unwinding have required physical separation of single- from double-stranded molecules. Researchers now describe conditions under which unwinding can be monitored directly using a fluorescent dye, thus greatly simplifying the analysis. Breaks due to irradiation of blood samples by 60Co gamma-rays at doses as low as 0.05 to 0.1 gray were detectable. Rapid rejoining of strand breaks during in vitro incubation at 37 degrees could readily be observed following a dose of one gray. Since the procedure is very rapid and cells can be analyzed directly without the requirement for culturing or radiolabeling, the procedure could be useful in cancer chemotherapy if in vivo damage is to be monitored or for testing the in vitro sensitivity of cells to drugs.

  13. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

    PubMed

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 10(2) CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.

  14. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China

    PubMed Central

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient. PMID:28197157

  15. Development of a rapid detection method to detect tdh gene in Vibrio parahaemolyticus using 2-step ultrarapid real-time polymerase chain reaction.

    PubMed

    Kang, Min-Hee; Kim, Il-Wook; Lee, Dong-Woo; Yoo, Mi-Sun; Han, Sang-Hoon; Yoon, Byoung-Su

    2011-01-01

    Thermostable direct hemolysin encoded by tdh gene has been considered an important virulence factor in pathogenic Vibrio parahaemolyticus. Two-step ultrarapid real-time polymerase chain reaction (URRT PCR) with a microchip was devised to detect V. parahaemolyticus carrying tdh gene. This novel method has a 6-μL reaction volume and extremely reduces running time since one cycle can be completed in 10 s or less. Consequently, 35 cycles of URRT PCR was successfully able to detect up to 100 fg (18 copies) of genomic DNA from pathogenic V. parahaemolyticus carrying tdh gene in 6 min. These results indicate that this method is at present the most rapid detection method for tdh gene and pathogenic V. parahaemolyticus.

  16. Rapid method to detect rubella immunoglobulin M and immunoglobulin A antibodies.

    PubMed Central

    Schmitz, H; Shimizu, H; Kampa, D; Doerr, H W

    1975-01-01

    Immunoglobulin (Ig) G was removed from serum specimens by precipitation with gamma chain-specific anti-human IgG of rabbit origin. The remaining rubella virus-specific IgM (and IgA) antibodies were then detected by the rubella hemagglutination-inhibition test. This procedure has proven to be as reliable as estimations carried out with IgM fractions separated on a sucrose density gradient. PMID:1176596

  17. Facile colorimetric method for simple and rapid detection of endotoxin based on counterion-mediated gold nanorods aggregation.

    PubMed

    Wang, Yashan; Zhang, Daohong; Liu, Wei; Zhang, Xiao; Yu, Shaoxuan; Liu, Tao; Zhang, Wentao; Zhu, Wenxin; Wang, Jianlong

    2014-05-15

    Existence of endotoxin in food and injection products indicates bacterial contaminations and therefore poses threat to human health. Herein, a simple and rapid colorimetric method for the effective detection of endotoxin in food and injections based on counterion-mediated gold nanorods aggregation is first proposed. By taking advantage of the color change of unmodified gold nanorods resulted from endotoxin mediated gold nanorods aggregation, endotoxin could be detected in the concentration range of 0.01-0.6 μM. Further, we studied the performance of gold nanorods with different aspect ratios (2.7 and 3.3) in determination of endotoxin and found that gold nanorods with higher aspect ratio (AR) showed superiority in the sensing sensitivity of endotoxin. A good specificity for endotoxin, a detection limit of 0.0084 μM and recoveries ranging from 84% to 109% in spiked food and injection samples are obtained with the colorimetric method. Results demonstrate that the present method provides a novel and effective approach for on-site screening of endotoxin in common products, which is beneficial for monitoring and reducing the risk of bacterial contaminations in food and injections production.

  18. Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis.

    PubMed Central

    Singh, A; Yu, F P; McFeters, G A

    1990-01-01

    A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h. PMID:2407185

  19. 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

  20. Development of a rapid method for the detection of cocoa butter equivalents in mixtures with cocoa butter.

    PubMed

    Barcarolo, R; Anklam, E

    2001-01-01

    A simple and rapid gas chromatographic (GC) method was developed for the detection of cocoa butter equivalents (CBEs) in cocoa buffer (CB). It is based on the use of a 5 m nonpolar capillary column for the separation of the main triglycerides of CB according to their acyl/carbon numbers. The GC procedure was optimized to avoid thermal degradation of the triglycerides. By computing the ratio C54/C50 and (C54/C50) x C52 and by 2-dimensional plotting of these values, authentic CB samples were clearly distinguished from samples containing various CBEs. The detection of little as 1% CBE in CB (corresponding to about 0.3% CBE in chocolate) in a model system was shown to be possible. Under real conditions, for a wide range of CBs, about 2.5% CBEs in CB were detected. With this method, quantitation was possible at a concentration of 5% CBEs in CB mixtures, which corresponds to around 1% in chocolate; this value is far below the maximum level of 5% CBEs allowed to be added to chocolate.

  1. 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.

  2. A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine.

    PubMed

    Kasana, Ramesh Chand; Salwan, Richa; Dhar, Hena; Dutt, Som; Gulati, Arvind

    2008-11-01

    Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram's iodine instead of the reagents just mentioned. Gram's iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram's iodine for the detection of cellulase production by microorganisms using plate assay.

  3. [Nitrate reductase assay for the rapid detection of Staphylococcus aureus methicillin resistance: a breakpoint susceptibility testing method].

    PubMed

    Coban, Ahmet Yılmaz; Demirpek, Uğur; Ciftçi, Alper; Bozdoğan, Bülent

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of hospital- and community-acquired infections. Therefore rapid and accurate detection of MRSA is essential for infection control and prevention of nosocomial spread. In this study, the efficacy of a nitrate reductase assay (NRA) as a breakpoint susceptibility testing method was evaluated for the rapid detection of methicillin resistance in S.aureus A total of 135 S.aureus clinical isolates from our collection were tested for methicillin susceptibility by NRA breakpoint susceptibility method and by broth microdilution method. For NRA breakpoint susceptibility testing three tubes including growth control tube (without drug), test tube (with 4 mg/L cefoxitin) and lyophilized test tube (with 4 mg/L cefoxitin) were used. 50 µl of 0.5 McFarland bacterial suspension of each isolate was inoculated into the tubes. All tubes were incubated at 35ºC. After five-hour incubation, 500 µl of freshly prepared reagent [2 units of 0.2% sulfanilamide, 2 units of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride and 1 unit of concentrated hydrochloric acid] was added into each tube and a color change was watched for. The color changed to violet/purple, if there was bacterial growth. If the color changed to violet/purple in all three tubes, the isolate was identified as methicillin-resistant. If the color changed in growth control tube but not in the test and lyophilized tube, the isolate was identified as methicillin-susceptible. Among 135 isolates tested, 97 had mecA gene and were methicillin-resistant by both microdilution and NRA breakpoint susceptibility method. The remaining 38 clinical isolates did not have this gene and were susceptible to methicillin by both methods used. All results were concordant to the PCR which was considered as the gold standard method. Specificity, sensitivity, positive and negative predictive values were 100%. NRA breakpoint susceptibility test in tubes is an inexpensive and

  4. Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts.

    PubMed

    Llamas, Nuria M; Stewart, Linda; Fodey, Terry; Higgins, H Cowan; Velasco, María Luisa R; Botana, Luis M; Elliott, Christopher T

    2007-09-01

    The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA-bovine thyroglobulin conjugate and OA-N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 microl min(-1); flow rate, 25 microl min(-1). An assay action limit of 126 ng g(-1) was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g(-1), which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of <10%. The chip surface developed was shown to be highly stable, allowing more than 50 analyses per channel. When the concentrations of OA determined with the biosensor method were compared with the values obtained by LC-MS in contaminated shellfish samples, the correlation between the two analytical methods was found to be highly satisfactory (r(2) = 0.991).

  5. Use of Molecular Methods for the Rapid Mass Detection of Schistosoma mansoni (Platyhelminthes: Trematoda) in Biomphalaria spp. (Gastropoda: Planorbidae)

    PubMed Central

    Jannotti-Passos, Liana Konovaloffi; Dos Santos Carvalho, Omar

    2017-01-01

    The low stringency-polymerase chain reaction (LS-PCR) and loop-mediated isothermal amplification (LAMP) assays were used to detect the presence of S. mansoni DNA in (1) Brazilian intermediate hosts (Biomphalaria glabrata, B. straminea, and B. tenagophila) with patent S. mansoni infections, (2) B. glabrata snails with prepatent S. mansoni infections, (3) various mixtures of infected and noninfected snails; and (4) snails infected with other trematode species. The assays showed high sensitivity and specificity and could detect S. mansoni DNA when one positive snail was included in a pool of 1,000 negative specimens of Biomphalaria. These molecular approaches can provide a low-cost, effective, and rapid method for detecting the presence of S. mansoni in pooled samples of field-collected Biomphalaria. These assays should aid mapping of transmission sites in endemic areas, especially in low prevalence regions and improve schistosomiasis surveillance. It will be a useful tool to monitor low infection rates of snails in areas where control interventions are leading towards the elimination of schistosomiasis. PMID:28246533

  6. Use of Molecular Methods for the Rapid Mass Detection of Schistosoma mansoni (Platyhelminthes: Trematoda) in Biomphalaria spp. (Gastropoda: Planorbidae).

    PubMed

    Caldeira, Roberta Lima; Jannotti-Passos, Liana Konovaloffi; Dos Santos Carvalho, Omar

    2017-01-01

    The low stringency-polymerase chain reaction (LS-PCR) and loop-mediated isothermal amplification (LAMP) assays were used to detect the presence of S. mansoni DNA in (1) Brazilian intermediate hosts (Biomphalaria glabrata, B. straminea, and B. tenagophila) with patent S. mansoni infections, (2) B. glabrata snails with prepatent S. mansoni infections, (3) various mixtures of infected and noninfected snails; and (4) snails infected with other trematode species. The assays showed high sensitivity and specificity and could detect S. mansoni DNA when one positive snail was included in a pool of 1,000 negative specimens of Biomphalaria. These molecular approaches can provide a low-cost, effective, and rapid method for detecting the presence of S. mansoni in pooled samples of field-collected Biomphalaria. These assays should aid mapping of transmission sites in endemic areas, especially in low prevalence regions and improve schistosomiasis surveillance. It will be a useful tool to monitor low infection rates of snails in areas where control interventions are leading towards the elimination of schistosomiasis.

  7. New rapid and simple methods for detection of bacteria and determination of their antibiotic susceptibility by using phage mutants.

    PubMed

    Ulitzur, Nirit; Ulitzur, Shimon

    2006-12-01

    Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria ("infectious centers"). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.

  8. 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.

  9. 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.

  10. A rapid high-performance liquid chromatography with fluorescence detection method developed to analyze ochratoxin A in wine.

    PubMed

    Tafuri, Alessio; Meca, Giuseppe; Ritieni, Alberto

    2008-10-01

    A rapid, sensitive, reproducible, and inexpensive method of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the analysis of ochratoxin A (OTA) in wine was developed. It is characterized by direct injection of the wine into the HPLC apparatus, with no need of extraction or cleanup. The method uses acetonitrile, water, and acetic acid (49:49:2, vol/vol/vol, respectively) as the isocratic mobile phase and a 5-microm monolithic C18 column (100 by 3 mm inside diameter). The relative standard deviation obtained in the OTA determination varied between 0.22 and 1.76%, with a mean value of 0.89%, in samples with concentrations between 0.10 and 100 ng/ml. The recovery of OTA ranged from 102% in samples spiked with 1 ng/ml OTA to 120% in samples with 0.10 ng/ml OTA. The method compared favorably with a published method based on an immunoaffinity column cleanup and a chromatographic assay with a C18 conventional HPLC column.

  11. 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

  12. 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

  13. 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

    2012-01-07

    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. This journal is © The Royal Society of Chemistry 2012

  14. A rapid cation-exchange HPLC method for detection and quantification of pyridinium oximes in plasma and tissue.

    PubMed

    Singh, Harry; Moorad-Doctor, Deborah; Ratcliffe, Ruthie H; Wachtel, Katie; Castillo, Andres; Garcia, Gregory E

    2007-03-01

    A rapid and sensitive assay for pyridinium oximes in plasma and tissue was developed. The method was suitable for the analysis of mono- and di-pyridinium oximes and utilizes ultrafiltration followed by cation-exchange high-performance liquid chromatography with UV detection. The assay was originally developed for the measurement of the oxime MMB-4 in plasma for which the lower limit of detection was 0.0005 pg and the limit of quantitation was 0.001 to 2.5 microg. The assay required as little as 50 microL of whole blood or 30 pL of tissue homogenate, and it was used for a pharmacokinetic study from a single intramuscular injection of MMB-4 (dichloride or dimethylsulfonate salt) in the guinea pig. Both salts were found to have similar pharmacokinetic properties in the plasma with a T1/2 of about 34 to 42 min and the area-under-the-curve values increased dose dependently. MMB-4 tissue concentrations were much lower than the plasma. The tissue levels peaked at 5-20 min depending on the tissue. A rank of concentration was diaphragm > heart > thigh muscle.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. Rapid Shell Vial Culture Technique for Detection of Enteroviruses and Adenoviruses in Fecal Specimens: Comparison with Conventional Virus Isolation Method

    PubMed Central

    Van Doornum, G. J. J.; De Jong, J. C.

    1998-01-01

    Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell

  17. Evaluation of methods for H. pylori detection in PPI consumption using culture, rapid urease test and smear examination

    PubMed Central

    Saniee, Parastoo; Khalili-Samani, Saman; Hosseini, Farideh; Malakutikhah, Fahimeh; Mamivand, Marzieh; Shahreza, Somayeh; Sharifi, Amir Houshang

    2015-01-01

    Background Culture, rapid urease test (RUT) and smear examination have been used as reliable methods for diagnosis of H. pylori infection. Accurate performance of these tests requires good quality biopsies with considerable number of bacterial cells. However, consumption of proton pump inhibitors (PPIs) affects growth and urease activity of H. pylori, leading to false negative results. In this study the efficacy of culture, RUT and smear examination was assessed and the effect of PPI consumption was evaluated. Methods Two antral biopsies from 530 dyspeptic patients with and without PPI consumption were used for RUT, culture and smear examination. Statistical analysis was used to determine the association between results of culture, RUT or smear examination and PPI consumption. Sensitivity and specificity of three tests were calculated by standard methods. Results H. pylori infection was detected in 40% of patients by culture, 48.3% by RUT and 21.1% by smear examination and the overall detection rate was 54%. A strong correlation was found between PPI consumption and negative results of culture and RUT (P<0.05) but not smear examination. The sensitivity of RUT was reduced as a result of PPI consumption. This reduction was more profound in 1-hr RUT (92.2% to 74.4%) compared with 24-hr RUT (93.9% to 81.6%). Conclusions Prevalence of H. pylori was declined, compared with previous studies. This decrement could be due to false negative results of H. pylori diagnostic tests, among which culture and RUT are mostly affected by PPI. Accordingly, PPI consumption should be stopped before performance of endoscopy. PMID:25705643

  18. A rapid method for direct detection of metabolic conversion and magnetization exchange with application to hyperpolarized substrates

    NASA Astrophysics Data System (ADS)

    Larson, Peder E. Z.; Kerr, Adam B.; Leon Swisher, Christine; Pauly, John M.; Vigneron, Daniel B.

    2012-12-01

    In this work, we present a new MR spectroscopy approach for directly observing nuclear spins that undergo exchange, metabolic conversion, or, generally, any frequency shift during a mixing time. Unlike conventional approaches to observe these processes, such as exchange spectroscopy (EXSY), this rapid approach requires only a single encoding step and thus is readily applicable to hyperpolarized MR in which the magnetization is not replenished after T1 decay and RF excitations. This method is based on stimulated-echoes and uses phase-sensitive detection in conjunction with precisely chosen echo times in order to separate spins generated during the mixing time from those present prior to mixing. We are calling the method Metabolic Activity Decomposition Stimulated-echo Acquisition Mode or MAD-STEAM. We have validated this approach as well as applied it in vivo to normal mice and a transgenic prostate cancer mouse model for observing pyruvate-lactate conversion, which has been shown to be elevated in numerous tumor types. In this application, it provides an improved measure of cellular metabolism by separating [1-13C]-lactate produced in tissue by metabolic conversion from [1-13C]-lactate that has flowed into the tissue or is in the blood. Generally, MAD-STEAM can be applied to any system in which spins undergo a frequency shift.

  19. A Rapid Method for Direct Detection of Metabolic Conversion and Magnetization Exchange with Application to Hyperpolarized Substrates

    PubMed Central

    Larson, Peder E. Z.; Kerr, Adam B.; Leon Swisher, Christine; Pauly, John M.; Vigneron, Daniel B.

    2012-01-01

    In this work, we present a new MR spectroscopy approach for directly observing nuclear spins that undergo exchange, metabolic conversion, or, generally, any frequency shift during a mixing time. Unlike conventional approaches to observe these processes, such as exchange spectroscopy (EXSY), this rapid approach requires only a single encoding step and thus is readily applicable to hyperpolarized MR in which the magnetization is not replenished after T1 decay and RF excitations. This method is based on stimulated-echoes and uses phase-sensitive detection in conjunction with precisely chosen echo times in order to separate spins generated during the mixing time from those present prior to mixing. We are calling the method Metabolic Activity Decomposition Stimulated-echo Acquisition Mode or MAD-STEAM. We have validated this approach as well as applied it in vivo to normal mice and a transgenic prostate cancer mouse model for observing pyruvate-lactate conversion, which has been shown to be elevated in numerous tumor types. In this application, it provides an improved measure of cellular metabolism by separating [1-13C]-lactate produced in tissue by metabolic conversion from [1-13C]-lactate that has flowed into the tissue or is in the blood. Generally, MAD-STEAM can be applied to any system in which spins undergo a frequency shift. PMID:23143011

  20. Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods.

    PubMed

    Sowmya, N; Thakur, M S; Manonmani, H K

    2012-07-01

      The study describes the development of simple and rapid DNA extraction method in combination with loop-mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples.   In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X-100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay.   The template DNA extraction method developed in this study for food samples is simple, rapid and cost-effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR.   The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  1. A new loop-mediated isothermal amplification method for rapid, simple, and sensitive detection of Leptospira spp. in urine.

    PubMed

    Koizumi, Nobuo; Nakajima, Chie; Harunari, Tsunehito; Tanikawa, Tsutomu; Tokiwa, Toshihiro; Uchimura, Eriko; Furuya, Tokujiro; Mingala, Claro Niegos; Villanueva, Marvin Ardeza; Ohnishi, Makoto; Suzuki, Yasuhiko

    2012-06-01

    We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.

  2. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

    PubMed Central

    Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-01-01

    Objective To analyse molecular detection of coliforms and shorten the time of PCR. Methods Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Conclusions Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN. PMID:25182727

  3. A rapid method for detecting extracellular proteinase activity in Cryptococcus neoformans and a survey of 63 isolates.

    PubMed

    Ruma-Haynes, P; Brownlee, A G; Sorrell, T C

    2000-08-01

    A rapid method to detect extracellular proteolytic activity around colonies of Cryptococcus neoformans was developed with tannic acid used to complex with residual protein in a solid medium. A survey was conducted with 32 isolates of C. neoformans var. gattii and 31 isolates of C. neoformans var. neoformans which were cultured on medium containing gelatin as the sole nitrogen source. The annulus of clearing around fungal colonies was > 1.2 mm in 24 (77%) isolates of C. neoformans var. neoformans compared with only 7 (22%) isolates of C. neoformans var. gattii. There was no difference in proteolytic activity between environmental and human clinical isolates of C. neoformans. However, there was a difference between the size of the annulus around animal isolates of C. neoformans var. neoformans and isolates of the same variety from other sources. The annuli around the 14 animal isolates were all >1.2 mm, while 7 (70%) of 10 human clinical isolates and only 3 (43%) of 7 environmental isolates were scored in the high proteinase range. A difference between the genetic types (as characterised by RAPD typing) of C. neoformans var. gattii was also evident with 17 (77%) of 22 VG-I isolates having a small annulus compared with only 1 (17%) of 6 VG-II and VG-III isolates with annuli of similar size. Relatively low proteinase production by C. neoformans var. gattii may reduce local and systemic spread of infection in mammalian hosts.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  5. Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h☆

    PubMed Central

    Swift, Benjamin M.C.; Denton, Emily J.; Mahendran, Sophie A.; Huxley, Jonathan N.; Rees, Catherine E.D.

    2013-01-01

    The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease. PMID:23811207

  6. [Rapid diagnosis of influenza infection by PCR method--detection of influenza virus HA gene in throat swab].

    PubMed

    Morishita, T; Kobayashi, S; Miyake, T; Ishihara, Y; Isomura, S; Nakajima, S; Nakajima, K

    1992-07-01

    We studied the detection of the HA gene of human influenza viruses in throat swabs obtained from the outbreaks of influenza in school children utilizing the polymerase chain reaction (PCR) method. Sensitivity and specificity of the PCR method was compared to conventional virus isolation using MDCK cells. Three pairs of primers for PCR in detecting the HA genes of AH1, AH3, and B influenza viruses showed both subtype and type specificity. The dilution experiments showed that influenza viruses, as few as 1.1-3.5 plaque-forming units per 50 microliters, were sufficient for the detection of HA genes by PCR method and the detection rate by PCR method was 2-3 fold higher than that by conventional method. Our results showed that the PCR method was a fast, sensitive and reliable method for the diagnosis of influenza infections.

  7. Rapid detection of in vitro antituberculous drug resistance among smear-positive respiratory samples using microcolony detection-based direct drug susceptibility testing method.

    PubMed

    Iftikhar, Irim; Irfan, Seema; Farooqi, Joveria; Azizullah, Zahida; Hasan, Rumina

    2017-01-01

    With the rise in multidrug-resistant tuberculosis, there is a search for newer techniques that will rapidly detect drug-resistant Mycobacterium tuberculosis. Although molecular techniques can detect resistance, culture is still considered gold standard, especially in resource-limited settings where quick, cheap, and easy techniques are needed. The aim of the study was to evaluate microcolony method thin layer agar (TLA) for quick detection of resistance against the first- and second-line antituberculous drugs in clinical isolates. This was a cross-sectional study performed at Aga Khan University Hospital. A total of 87 Z-N stain smear-positive pulmonary samples were received and indirect drug susceptibility test (ID-DST) was performed using Lowenstein-Jensen and mycobacteria growth indicator tube. Direct DST was performed using TLA on 7H10 agar. TLA was observed twice weekly under microscope for 4 weeks. Sensitivity, specificity, and accuracy were calculated for TLA using indirect susceptibility method as the gold standard. Level of agreement was calculated using Kappa score. TLA showed sensitivity of 89% and 95.2% for isoniazid and rifampicin, while for ethionamide, ofloxacin, and injectable aminoglycosides, it was 96.6%, 92.1%, and 100%, respectively. Specificity for the first-line drugs was >95% while second-line drugs ranged from 70% to 100%. Mean time to positivity was 10.2 days by TLA as compared to 43.1 days by ID-DST. TLA is a quick and reliable method in identifying resistance, especially in resource-limited settings. However, additional liquid culture can be set up as backup, especially in patients on therapy to avoid false negative results.

  8. Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours▿

    PubMed Central

    Stanley, Emma C.; Mole, Richard J.; Smith, Rebecca J.; Glenn, Sarah M.; Barer, Michael R.; McGowan, Michael; Rees, Catherine E. D.

    2007-01-01

    The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h. PMID:17259362

  9. Detection of malaria infection in blood transfusion: a comparative study among real-time PCR, rapid diagnostic test and microscopy: sensitivity of Malaria detection methods in blood transfusion.

    PubMed

    Hassanpour, Gholamreza; Mohebali, Mehdi; Raeisi, Ahmad; Abolghasemi, Hassan; Zeraati, Hojjat; Alipour, Mohsen; Azizi, Ebrahim; Keshavarz, Hossein

    2011-06-01

    The transmission of malaria by blood transfusion was one of the first transfusion-transmitted infections recorded in the world. Transfusion-transmitted malaria may lead to serious problems because infection with Plasmodium falciparum may cause rapidly fatal death. This study aimed to compare real-time polymerase chain reaction (real-time PCR) with rapid diagnostic test (RDT) and light microscopy for the detection of Plasmodium spp. in blood transfusion, both in endemic and non-endemic areas of malaria disease in Iran. Two sets of 50 blood samples were randomly collected. One set was taken from blood samples donated in blood bank of Bandar Abbas, a city located in a malarious-endemic area, and the other set from Tehran, a non-endemic one. Light microscopic examination on both thin and thick smears, RDTs, and real-time PCR were performed on the blood samples and the results were compared. Thin and thick light microscopic examinations of all samples as well as RDT results were negative for Plasmodium spp. Two blood samples from endemic area were positive only with real-time PCR. It seems that real-time PCR as a highly sensitive method can be helpful for the confirmation of malaria infection in different units of blood transfusion organization especially in malaria-endemic areas where the majority of donors may be potentially infected with malaria parasites.

  10. A UHPLC method for the rapid separation and quantification of phytosterols using tandem UV/Charged aerosol detection - A comparison of both detection techniques.

    PubMed

    Fibigr, Jakub; Šatínský, Dalibor; Solich, Petr

    2017-06-05

    The presented work describes the development and validation of a rapid UHPLC-UV/CAD method using a core-shell particle column for the separation and quantitative analysis of seven plant sterols and stanols. The phytosterols (ergosterol, brassicasterol, campesterol, fucosterol, stigmasterol, and β-sitosterol) and the phytostanol stigmastanol were separated and analyzed in 8.5min. The sample pre-treatment procedure was optimized to be less time-consuming than any other published method, especially due to no need of derivatization, evaporation and even reconstitution step. The chromatographic separation was performed on the Kinetex 1.7μ Phenyl-hexyl column (100×2.1mm) with a mobile phase acetonitrile/water according to the gradient program at a flow rate of 0.9mLmin(-1) and a temperature of 60°C. A tandem connection of PDA and CAD (Corona Charged Aerosol Detector) was used and both detection techniques were compared. The method was validated using saponification as a first step in sample pre-treatment and an universal CAD as the detector. Recoveries for all analyzed compounds were between 95.4% and 103.4% and relative standard deviation ranged from 1.0% to 5.8% for within-day and from 1.4% to 6.7% for between-day repeatability. The limits of detection were in the range of 0.4-0.6μgmL(-1) for standard solutions and 0.3-1.2μgmL(-1) for phytosterols in real samples. Although several gradient programs and different stationary phases were tested, two compounds, campesterol and campestanol, were not separated. Their peak was quantified as a sum of both analytes. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  12. Rapid Statistical Methods: Part 1.

    ERIC Educational Resources Information Center

    Lyon, A. J.

    1980-01-01

    Discusses some rapid statistical methods which are intended for use by physics teachers. Part one of this article gives some of the simplest and most commonly useful rapid methods. Part two gives references to the relevant theory together with some alternative and additional methods. (HM)

  13. Easy-to-use rapid gene amplification method for direct detection of RNA and DNA viruses in sera and feces from various animals.

    PubMed

    Segawa, Takao; Kobayashi, Yuki; Sase, Yukina; Itou, Takuya; Suzuki, Miwa; Endoh, Tomoko; Nakanishi, Teruyuki; Sakai, Takeo

    2014-06-01

    The development of rapid and simple gene amplification tests is required for detection of pathogens to prevent transmission of infectious diseases between animals or from animals to humans. An easy-to-use rapid gene amplification method that can directly detect RNA and DNA viruses in clinical samples was developed. This method is based on combining loop-mediated isothermal amplification (LAMP) or reverse transcription-LAMP (RT-LAMP) and RNA GEM Tissue, a thermophilic enzyme that extracts nucleic acid by quickly digesting proteins and ribonucleases. The authors named these methods GEM LAMP and GEM RT-LAMP. These methods were able to detect viral DNA and RNA within 70 min in a single tube using only a water bath. The detection capacities were 10-100-fold more sensitive than those of previously established LAMP and RT-LAMP methods. The GEM LAMP and GEM RT-LAMP methods were used to detect macroscopically the presence of DNA and RNA viruses in sera or fecal samples from cattle, pigs, horses, dolphins, penguins, and sea lions using SYBR green I. The GEM LAMP and GEM RT-LAMP methods thus have considerable versatility as tools for detecting pathogens and are applicable to basic human and veterinary medicine, environmental hygiene, and point-of-care-testing. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Evaluation of environmental sampling methods and rapid detection assays for recovery and identification of Listeria spp. from meat processing facilities.

    PubMed

    Kovacević, Jovana; Bohaychuk, Valerie M; Barrios, Pablo Romero; Gensler, Gary E; Rolheiser, Deana L; McMullen, Lynn M

    2009-04-01

    Studies that isolated Listeria spp. from the environment of two meat processing facilities were conducted. Samples were collected in the processing environment of the facilities with three different sampling methods (cotton swab, sterile sponge, and composite-ply tissues) to evaluate their ability to recover Listeria spp. A total of 240 samples for each sampling method were collected and tested. The cotton swab method of sampling was significantly (P < 0.01) less efficient in recovery of Listeria spp. than the sterile-sponge and composite-ply tissue methods, which were similar (P > 0.05) in their ability to recover Listeria spp. The specificity and sensitivity of four detection methods (conventional culture, Petrifilm Environmental Listeria Plates [ELP], lateral-flow immunoprecipitation [LFI], and automated PCR) were evaluated for identification of Listeria spp. Facilities were visited until a minimum of 100 positive and 100 negative samples per detection method were collected. The LFI and PCR methods were highly sensitive (95.5 and 99.1%, respectively) and specific (100%) relative to the culture method. The ELP method was significantly less efficient (P < 0.01) than LFI and PCR in detection of Listeria spp., with lower sensitivity (50.6%) and specificity (91.5%). Kappa values indicated excellent agreement of the LFI and PCR assays and moderate agreement of the ELP method to the culture method. Overall, ELP was easy to use but less efficient in detection of Listeria spp. from environmental samples, while the LFI and PCR methods were found to be excellent alternatives to culture, considering performance and time and labor inputs.

  15. PCR-single-strand conformational polymorphism method for rapid detection of rifampin-resistant Mycobacterium tuberculosis: systematic review and meta-analysis.

    PubMed

    Xu, Hong-Bin; Jiang, Rui-Hai; Sha, Wei; Li, Ling; Xiao, He-Ping

    2010-10-01

    The reference standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as culture on Lowenstein-Jensen or Middlebrook 7H10/11 medium, are very slow to give results; and due to the emergence of multidrug-resistant M. tuberculosis and extensively drug-resistant M. tuberculosis, there is an urgent demand for new, rapid, and accurate drug susceptibility testing methods. PCR-single-strand conformational polymorphism (PCR-SSCP) analysis has been proposed as a rapid method for the detection of resistance to rifampin, but its accuracy has not been systematically evaluated. We performed a systematic review and meta-analysis to evaluate the accuracy of PCR-SSCP analysis for the detection of rifampin-resistant tuberculosis. We searched the Medline, Embase, Web of Science, BIOSIS, and LILACS databases and contacted authors if additional information was required. Ten studies met our inclusion criteria for rifampin resistance detection. We applied the summary receiver operating characteristic (SROC) curve to perform the meta-analysis and to summarize diagnostic accuracy. The sensitivity of PCR-SSCP analysis for the rapid detection of rifampin-resistant tuberculosis was 0.79 (95% confidence interval [CI], 0.75 to 0.82), the specificity was 0.96 (95% CI, 0.94 to 0.98), the positive likelihood ratio was 16.10 (95% CI, 5.87 to 44.13), the negative likelihood ratio was 0.20 (95% CI, 0.10 to 0.40), and the diagnostic odds ratio was 100.93 (95% CI, 31.95 to 318.83). PCR-SSCP analysis is a sensitive and specific test for the rapid detection of rifampin-resistant M. tuberculosis. Additional studies in countries with a high prevalence of multidrug-resistant M. tuberculosis and also cost-effectiveness analysis are required in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in M. tuberculosis.

  16. 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.

  17. A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

    PubMed Central

    2012-01-01

    Background Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population. Results We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM. Conclusions These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population. PMID:22958243

  18. Rapid Detection Methods for Asphalt Pavement Thicknesses and Defects by a Vehicle-Mounted Ground Penetrating Radar (GPR) System

    PubMed Central

    Dong, Zehua; Ye, Shengbo; Gao, Yunze; Fang, Guangyou; Zhang, Xiaojuan; Xue, Zhongjun; Zhang, Tao

    2016-01-01

    The thickness estimation of the top surface layer and surface layer, as well as the detection of road defects, are of great importance to the quality conditions of asphalt pavement. Although ground penetrating radar (GPR) methods have been widely used in non-destructive detection of pavements, the thickness estimation of the thin top surface layer is still a difficult problem due to the limitations of GPR resolution and the similar permittivity of asphalt sub-layers. Besides, the detection of some road defects, including inadequate compaction and delamination at interfaces, require further practical study. In this paper, a newly-developed vehicle-mounted GPR detection system is introduced. We used a horizontal high-pass filter and a modified layer localization method to extract the underground layers. Besides, according to lab experiments and simulation analysis, we proposed theoretical methods for detecting the degree of compaction and delamination at the interface, respectively. Moreover, a field test was carried out and the estimated results showed a satisfactory accuracy of the system and methods. PMID:27929409

  19. Rapid Detection Methods for Asphalt Pavement Thicknesses and Defects by a Vehicle-Mounted Ground Penetrating Radar (GPR) System.

    PubMed

    Dong, Zehua; Ye, Shengbo; Gao, Yunze; Fang, Guangyou; Zhang, Xiaojuan; Xue, Zhongjun; Zhang, Tao

    2016-12-06

    The thickness estimation of the top surface layer and surface layer, as well as the detection of road defects, are of great importance to the quality conditions of asphalt pavement. Although ground penetrating radar (GPR) methods have been widely used in non-destructive detection of pavements, the thickness estimation of the thin top surface layer is still a difficult problem due to the limitations of GPR resolution and the similar permittivity of asphalt sub-layers. Besides, the detection of some road defects, including inadequate compaction and delamination at interfaces, require further practical study. In this paper, a newly-developed vehicle-mounted GPR detection system is introduced. We used a horizontal high-pass filter and a modified layer localization method to extract the underground layers. Besides, according to lab experiments and simulation analysis, we proposed theoretical methods for detecting the degree of compaction and delamination at the interface, respectively. Moreover, a field test was carried out and the estimated results showed a satisfactory accuracy of the system and methods.

  20. Early detection and rapid response

    USGS Publications Warehouse

    Westbrooks, Randy G.; Eplee, Robert E.; Simberloff, Daniel; Rejmánek, Marcel

    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.

  1. 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.

  2. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Rapid method for detection of influenza a and B virus antigens by use of a two-photon excitation assay technique and dry-chemistry reagents.

    PubMed

    Koskinen, Janne O; Vainionpää, Raija; Meltola, Niko J; Soukka, Jori; Hänninen, Pekka E; Soini, Aleksi E

    2007-11-01

    New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.

  4. Rapid Method for Detection of Influenza A and B Virus Antigens by Use of a Two-Photon Excitation Assay Technique and Dry-Chemistry Reagents▿

    PubMed Central

    Koskinen, Janne O.; Vainionpää, Raija; Meltola, Niko J.; Soukka, Jori; Hänninen, Pekka E.; Soini, Aleksi E.

    2007-01-01

    New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed. PMID:17855571

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

    PubMed

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

    2009-07-01

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

  6. Development of a real-time reverse transcription loop-mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus.

    PubMed

    Yu, Xuewu; Shi, Lin; Lv, Xiaoping; Yao, Wei; Cao, Minghui; Yu, Hanxun; Wang, Xiurong; Zheng, Shimin

    2015-05-14

    Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine. Recently, the outbreak of the epidemic disease has been a serious problem in swine industry. The objective of this study is to develop a rapid, sensitive, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of porcine epidemic diarrhea virus (PEDV) in less equipped laboratories. The optimal reaction condition of the current real-time RT-LAMP for PEDV was 62 °C for 45 min. It was capable of detecting PEDV from clinical samples and differentiating PEDV from several related porcine viruses, while it did not require additional expensive equipment. The minimum detection limit of the real-time RT-LAMP assay was 0.07PFU per reaction for PEDV RNA, making this assay approximately 100-fold more sensitive than that of one-step RT-PCR. By screening a panel of clinical specimens, the results showed that this method presented a similar sensitivity with real-time RT-PCR and was somewhat sensitive than one-step RT-PCR in detection of clinical samples. In this study, we have developed a new real-time RT-LAMP method, which is rapid, sensitive and efficient to detect PEDV.This method holds great promises not only in laboratory detection and discrimination of PEDV but also in large scale field and clinical studies.

  7. Direct real-time PCR with ethidium monoazide: a method for the rapid detection of viable Cronobacter sakazakii in powdered infant formula.

    PubMed

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

    2012-09-01

    The goal of this study was to establish a rapid assay for the specific detection of viable Cronobacter sakazakii in powdered infant formula (PIF). Samples were subjected to treatment multiple times with ethidium monoazide with a concentration gradient (gEMA) prior to PCR to discriminate viable from dead C. sakazakii cells. To improve the current detection limits, we developed a new buffer for direct quantitative real-time PCR (DqPCR) without DNA isolation. Using 17 PIF samples, our rapid assay was compared with the new U.S. Food and Drug Administration (FDA) method published in the Bacteriological Analytical Manual in 2012. Although both the new FDA method and our rapid assay, which consists of DqPCR combined with gEMA (gEMA-DqPCR), produced negative results for all 17 PIF samples, 5 of the 17 PIFs were positive by DqPCR when they were not treated with EMA. Furthermore, for PIF samples artificially contaminated with viable C. sakazakii, gEMA-DqPCR successfully detected between 1 and 9 CFU of viable C. sakazakii in 300 g of PIF within 9 h, including a 6-h preincubation. Our results indicate that multiple EMA treatments are required to avoid false-positive results due to the contamination of commercial PIF with dead or injured C. sakazakii cells. Our rapid assay may also improve the sensitivity of the screening portion required by the new FDA method published in the Bacteriological Analytical Manual in 2012.

  8. Rapid detection of Enterococcus spp. direct from blood culture bottles using Enterococcus QuickFISH method: a multicenter investigation.

    PubMed

    Deck, Melissa K; Anderson, Erica S; Buckner, Rebecca J; Colasante, Georgia; Davis, Thomas E; Coull, James M; Crystal, Benjamin; Latta, Phyllis Della; Fuchs, Martin; Fuller, Deanna; Harris, Will; Hazen, Kevin; Klimas, Lisa L; Lindao, Daniel; Meltzer, Michelle C; Morgan, Margie; Shepard, Janeen; Stevens, Sharon; Wu, Fann; Fiandaca, Mark J

    2014-04-01

    The performance of a diagnostic method for detection and identification of Enterococcus spp. directly from positive blood culture was evaluated in a clinical study. The method, Enterococcus QuickFISH BC, is a second-generation peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) test, which uses a simplified, faster assay procedure. The test uses fluorescently labeled PNA probes targeting 16S rRNA to differentiate Enterococcus faecalis from other Enterococcus spp. by the color of the cellular fluorescence. Three hundred fifty-six routine blood culture samples were tested; only 2 discordant results were recorded. The sensitivities for detection of Enterococcus faecalis and non-faecalis Enterococcus were 100% (106/106) and 97.0% (65/67), respectively, and the combined specificity of the assay was 100%. The combined positive and negative predictive values of the assay were 100% (171/171) and 98.9% (185/187), respectively.

  9. 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

  10. Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of subtype H7N9 avian influenza virus.

    PubMed

    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.

  11. Validation of three rapid screening methods for detection of verotoxin-producing Escherichia coli in foods: interlaboratory study.

    PubMed

    Capps, Katherine L; McLaughlin, Emiline M; Murray, Alistair W A; Aldus, Clare F; Wyatt, Gary M; Peck, Michael W; van Amerongen, Aart; Ariëns, Renata M C; Wichers, Jan H; Baylis, Christopher L; Wareing, David R A; Bolton, Frederick J

    2004-01-01

    An interlaboratory study was conducted for the validation of 3 methods for the detection of all verotoxin-producing Escherichia coli (VTEC) in foods. The methods were a multi-analyte 1-step lateral flow immunoassay (LFIA) for detection of E. coli O157 and verotoxin (VT); an enzyme-linked immunosorbent assay targeted against VT1, VT2, and VT2c (VT-ELISA); and a polymerase chain reaction (PCR) method for detection of VT genes (VT-PCR). Aliquots (25 g or 25 mL) of 4 food types (raw minced [ground] beef, unpasteurized milk, unpasteurized apple juice [cider], and salami) were individually inoculated with low numbers (<9 to 375 cells/25 g) of 6 test strains of E. coli (serogroups O26, O103, O111, O145, and O157) with differing VT-producing capabilities. Five replicates for each test strain and 5 uninoculated samples were prepared for each food type. Fourteen participating laboratories analyzed samples using the LFIA, 9 analyzed the samples by ELISA, and 9 by PCR. The LFIA for O157 and VT had a specificity (correct identification of negative samples) of 92 and 94%, respectively, and a sensitivity (correct identification of positive samples) of 94 and 55%, respectively. The VT-ELISA and VT-PCR had a specificity of 98 and 99%, respectively, and a sensitivity of 89 and 72%, respectively.

  12. Method for the rapid detection and molecular characterization of DNA alkylating agents by MALDI-TOF mass spectrometry.

    PubMed

    Garaguso, Ignazio; Halter, Roman; Krzeminski, Jacek; Amin, Shantu; Borlak, Jürgen

    2010-10-15

    Metabolic activation of polycyclic aromatic hydrocarbons (PAH) may cause DNA adduct formation. While these are commonly detected by the ³²P-postlabeling assay, this method is not informative on the chemical nature of the alkylating agent. Here we report a simple and reliable method that employs MALDI-TOF-MS with 2,5-dihydroxybenzoic acid (DHB) matrix layer (ML) sample preparations for the detection and structural characterization of PAH-DNA adducts. The method involves the enzymatic digestion of DNA to 2'-deoxynucleotides followed by solid phase extraction to remove salt and other contaminants prior to MALDI-MS analysis. By collision induced dissociation (CID) structurally relevant fragments are obtained to permit characterization of the alkylating molecules and the adducted nucleotide. Next to guanosine, adenosine and cytidine adducts formed from reactions with (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) are identified at a sensitivity of <100 fmol and a mass accuracy of <10 ppm. Studies with (±)-anti-benzo[c]-chrysene-9,10-diol-11,12-epoxide (B[c]ChDE) further document the versatility and usefulness of the method. When compared with the ³²P-postlabeling assay MALDIMS only indentified deoxycytidine as well nucleoside and dinucleotides adducts. Therefore, this sensitive method enables molecular specification and characterization of adducted nucleotides and of the alkylating agent, and thus, provides comprehensive information that is beyond the ³²P-postlabeling assay.

  13. A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex.

    PubMed

    Guk, Kyeonghye; Keem, Joo Oak; Hwang, Seul Gee; Kim, Hyeran; Kang, Taejoon; Lim, Eun-Kyung; Jung, Juyeon

    2017-09-15

    Rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe. A dCas9/sgRNA-SG I based detection approach has advantages over monoclonal antibody in conventional immunoassay systems due to its ability to interact with the target gene in a sequence-specific manner. The detection limit of MRSA was as low as 10 cfu/ml and was found to be sufficient to effectively detect MRSA. Unlike conventional gene diagnosis methods in which PCR must be accompanied or genes are isolated and analyzed, the target gene can be detected within 30min with high sensitivity without performing a gene separation step by using cell lysates. We showed that the fluorescence signal of the MRSA cell lysate was more than 10-fold higher than that of methicillin-susceptible S. aureus (MSSA). Importantly, the present approach can be applied to any target other than MRSA by simply changing the single-guide RNA (sgRNA) sequence. Because dCas9/sgRNA-SG I based detection approach has proved to be easy, fast, sensitive, and cost-efficient, it can be applied directly at the point of care to detect various pathogens as well as MRSA in this study. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Validation of the TaqMan Influenza A Detection Kit and a rapid automated total nucleic acid extraction method to detect influenza A virus in nasopharyngeal specimens.

    PubMed

    Bolotin, Shelly; De Lima, Cedric; Choi, Kam-Wing; Lombos, Ernesto; Burton, Laura; Mazzulli, Tony; Drews, Steven J

    2009-01-01

    This study describes the validation of the TaqMan Influenza A Detection Kit v2.0 combined with an automated nucleic acid extraction method. The limit of detection of this assay was determined by probit regression (95% confidence interval) to be 2 influenza A/PR/8/34 (H1N1) virus particles per microlitre. One hundred and eleven specimens previously tested using the Seeplex RV assay and viral culture methods were tested using the TaqMan Influenza A Detection Kit. Compared to the aggregate gold-standard, the sensitivity and specificity of the TaqMan Influenza A Detection Kit were 100% (35/35) and 97% (74/76), respectively. Because of its accuracy, quick turn-around-time and lyophilized bead form, the TaqMan Influenza A Detection Kit, combined with the NucliSense easyMAG automated extraction method, constitutes a reliable protocol for influenza A diagnosis.

  15. Evaluation of a rapid method for the detection of streptococcal group A antigen directly from throat swabs.

    PubMed Central

    Venezia, R A; Ryan, A; Alward, S; Kostun, W A

    1985-01-01

    Throat swabs from 196 pediatric patients were processed by a direct extraction-latex agglutination method (Group A Strep Direct Antigen Identification Test [DAI]) that detects group A streptococci in the specimen. The method requires a 45-min enzymatic extraction period at 37 degrees C and a 4-min reaction period with antibody-linked latex particles. The results were compared with those of the culture and fluorescent antibody methods and the clinical presentation of the patient for pharyngitis. Ninety-three percent of the specimens resulted in agreement by all tests, and 28% were culture positive for group A streptococci. Compared with the culture method, the DAI had a sensitivity and a specificity of 83% and 99%, respectively. The positive predictive values were 98% versus the culture method and 93% versus the fluorescent antibody method, whereas the negative predictive values were 94% versus both other methods. Of the 14 discrepant results when both clinical presentation of an acute pharyngitis and the test results were compared, the culture method provided the best correlation. An additional 64 specimens were processed by the DAI and another direct extraction-latex agglutination method (Culturette Ten-Minute Group A Strep ID Test), and the results were compared with those of the culture method. This group had a 40.6% culture isolation rate for group A streptococci. The sensitivity and specificity of the DAI and Strep ID methods versus the culture method were 81 and 100%, and 77 and 97%, respectively. These results indicate that the DAI is accurate for diagnosing group A streptococcal pharyngitis directly from throat swabs. However, negative results in the presence of a symptomatic patient must be confirmed by standard culture techniques. PMID:3884656

  16. Rapid sample screening method for authenticity controlling vanilla flavors using a CE microchip approach with electrochemical detection.

    PubMed

    Avila, Mónica; González, María Cristina; Zougagh, Mohammed; Escarpa, Alberto; Ríos, Angel

    2007-11-01

    Five vanilla-related flavors of food significance, vanillic alcohol (VOH), ethyl maltol (EMA), maltol (MAL), ethyl vanillin (EVA) and vanillin (VAN), were separated using CE microchips with electrochemical detection (CE-ED microchips). A +2 kV driving voltage for both injection and separation operation steps, using a borate buffer (pH 9.5, 20 mM) and 1 M nitric acid in the detection reservoir allowed the selective and sensitive detection of the target analytes in less than 200 s with reproducible control of EOF (RSD(migration times)<3%). The analysis in selected real vanilla samples was focusing on VAN and EVA because VAN is a basic fragrance compound of the vanilla aroma, whereas EVA is an unequivocal proof of adulteration of vanilla flavors. Fast detection of all relevant flavors (200 s) with an acceptable resolution (R(s) >1.5) and a high accuracy (recoveries higher than 90%) were obtained with independence of the matrices and samples examined. These results showed the reliability of the method and the potential use of CE microchips in the food control field for fraudulent purposes.

  17. Rapid discrimination between buffalo and cow milk and detection of adulteration of buffalo milk with cow milk using synchronous fluorescence spectroscopy in combination with multivariate methods.

    PubMed

    Durakli Velioglu, Serap; Ercioglu, Elif; Boyaci, Ismail Hakki

    2017-03-22

    This research paper describes the potential of synchronous fluorescence (SF) spectroscopy for authentication of buffalo milk, a favourable raw material in the production of some premium dairy products. Buffalo milk is subjected to fraudulent activities like many other high priced foodstuffs. The current methods widely used for the detection of adulteration of buffalo milk have various disadvantages making them unattractive for routine analysis. Thus, the aim of the present study was to assess the potential of SF spectroscopy in combination with multivariate methods for rapid discrimination between buffalo and cow milk and detection of the adulteration of buffalo milk with cow milk. SF spectra of cow and buffalo milk samples were recorded between 400-550 nm excitation range with Δλ of 10-100 nm, in steps of 10 nm. The data obtained for ∆λ = 10 nm were utilised to classify the samples using principal component analysis (PCA), and detect the adulteration level of buffalo milk with cow milk using partial least square (PLS) methods. Successful discrimination of samples and detection of adulteration of buffalo milk with limit of detection value (LOD) of 6% are achieved with the models having root mean square error of calibration (RMSEC) and the root mean square error of cross-validation (RMSECV) and root mean square error of prediction (RMSEP) values of 2, 7, and 4%, respectively. The results reveal the potential of SF spectroscopy for rapid authentication of buffalo milk.

  18. Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7.

    PubMed

    Bao, Hongmei; Wang, Xiurong; Zhao, Yuhui; Sun, Xiaodong; Li, Yanbing; Xiong, Yongzhong; Chen, Hualan

    2012-01-01

    A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method.

    PubMed

    Zhou, Yang; Wu, Jiege; Lin, Feng; Chen, Naifu; Yuan, Shaofei; Ding, Lina; Gao, Li; Hang, Bangxing

    2015-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV.

  20. A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers

    PubMed Central

    Magnin, Sandrine; Viel, Erika; Baraquin, Alice; Valmary-Degano, Severine; Kantelip, Bernadette; Pretet, Jean-Luc; Mougin, Christiane; Bigand, Marthe; Girardo, Benoît; Borg, Christophe; Ferrand, Christophe

    2011-01-01

    The analysis of KRAS mutations has become a prerequisite for anti-epidermal growth factor receptor therapy in patients with metastatic colorectal cancers. KRAS mutations are associated with resistance to treatment by monoclonal antibodies such as cetuximab and panitumumab and thus are correlated with a shorter progression-free survival. BRAF mutations also may play a role in treatment decisions. The widespread use of these targeted therapies has generated the need to develop cost-effective methods for routine KRAS and BRAF analysis. The aim of this study was to compare a multiplex SNaPshot assay with DNA sequencing and high-resolution melting analysis for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations. Thus 110 routinely formalin-fixed and paraffin-embedded tissue blocks were tested by each method. The SNaPshot analysis detected KRAS and BRAF codon 600 mutations in, respectively, 34.5% (n = 38) and 10% (n = 11) of these tissue blocks. These results were confirmed by direct DNA sequencing and by high-resolution melting analysis. The costs and time constraints of each detection method were compared at the same time. In conclusion, our newly designed multiplex SNaPshot assay is a fast, inexpensive, sensitive, and robust technique for molecular diagnostic practices and patient selection. PMID:21742054

  1. Design of a biological method for rapid detection of presence of PCR inhibitors in aged bone DNA.

    PubMed

    Ghasemi, Akram; Mahdieh, Nejat; Tavallaei, Mahmood; Aslani, Mohammad Mehdi; Zafari, Zahra; Shirkavand, Atefeh; Farzad, Maryam Sharafi; Naderi, Mahdi; Azariyan, Sajjad Habibi; Zeinali, Sirous

    2012-01-01

    Molecular human identification is one of the most important tests performed in forensic laboratories. Some of these tests are applied for identification of human remains from natural disasters, wars, etc., but problems may occur as a result of DNA degradation and external DNA contamination. We investigated effects of bacterial DNA on identifying the presence or absence of PCR inhibitors in aged bone DNA. DNA samples were extracted from blood, bone remains and Escherichia coli. These DNA were amplified using human and bacterial specific primers. Using different blood, aged bone, and bacterial DNA dilutions along with PCR based methods; we checked their positive, negative effects, or detecting presence of inhibitors in aged bone DNA by PCR method. Our observation indicated that the addition of bacterial DNA could be a valid biological method for testing the quality of bone DNA to enable us to obtain a usable profile for the identification of human remains. This method will help to test the presence of inhibitors, quantity or even quality of DNA which are of importance in profiling archeological remains. Our method will help to determine if PCR failure is due to presence of inhibitors or lack of amplifiable DNA either because of degradation, minute amount or absence of human DNA.

  2. Rapid and sensitive method for the analysis of carbon monoxide in blood using gas chromatography with flame ionisation detection.

    PubMed

    Sundin, Anna-Maja; Larsson, Jan Erik

    2002-01-05

    In order to measure changes in physiological CO concentrations in blood with good accuracy, a method was developed using gas chromatography with flame ionisation detection (250 degrees C). A nickel catalyst system was fitted to convert CO to methane at 375 degrees C after separation with a molecular sieve column at 35 degrees C. Helium was used as carrier at 30 ml/min. Porcine or human blood (400 microl) was sampled in gastight tubes and treated with sulfuric acid and saponin (800 microl). Accuracy was 1.4% and 1.5% (RSD), respectively. Precision was 2.8% (porcine blood). Limit of detection was 0.01 nmol/ml gas and limit of quantification 12 nmol/ml blood. Calibration was made in the interval 12-514 nmol/ml blood (corresponding to 0.1-6% COHb). Samples were stable for at least a month at +4 degrees C. This paper describes a method with high sensitivity and good accuracy, suitable for analysis of low CO concentrations.

  3. Rapid detection of new and expanding human settlements in the Limpopo province of South Africa using a spatio-temporal change detection method

    NASA Astrophysics Data System (ADS)

    Kleynhans, W.; Salmon, B. P.; Wessels, K. J.; Olivier, J. C.

    2015-08-01

    Recent development has identified the benefits of using hyper-temporal satellite time series data for land cover change detection and classification in South Africa. In particular, the monitoring of human settlement expansion in the Limpopo province is of relevance as it is the one of the most pervasive forms of land-cover change in this province which covers an area of roughly 125 000 km2. In this paper, a spatio-temporal autocorrelation change detection (STACD) method is developed to improve the performance of a pixel based temporal Autocorrelation change detection (TACD) method previously proposed. The objective is to apply the algorithm to large areas to detect the conversion of natural vegetation to settlement which is then validated by an operator using additional data (such as high resolution imagery). Importantly, as the objective of the method is to indicate areas of potential change to operators for further analysis, a low false alarm rate is required while achieving an acceptable probability of detection. Results indicate that detection accuracies of 70% of new settlement instances are achievable at a false alarm rate of less than 1% with the STACD method, an improvement of up to 17% compared to the original TACD formulation.

  4. 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.

  5. 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.

  6. 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

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

    PubMed

    Yeh, Hung-Yueh; Shoemaker, Craig A; Klesius, Phillip H

    2005-10-01

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars 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 amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.

  8. Validation of rapid microbiological methods.

    PubMed

    Peris-Vicente, Juan; Carda-Broch, Samuel; Esteve-Romero, Josep

    2015-06-01

    Classical microbiological methods currently have unacceptably long cycle times. Rapid microbiological methods have been available on the market for decades and have been applied by the clinical and food industries. However, their implementation in the pharmaceutical industry has been hampered by stringent regulations on validation and comparison with classical methods. To encourage the implementation of these methodologies, they must be validated to assess that the results are straightforward. A comparison with traditional methods should be also performed. In this review, information about the validation of rapid microbiological methods reported in the literature is provided as well as an explanation of the difficulty of validation of these methods. A comparison with traditional methods is also discussed. This information is useful for industries and laboratories that can potentially implement these methods. © 2014 Society for Laboratory Automation and Screening.

  9. Diagnostics method for the rapid quantitative detection and identification of low-level contamination of high-purity water with pathogenic bacteria.

    PubMed

    Minogue, Elizabeth; Reddington, Kate; Dorai-Raj, Siobhan; Tuite, Nina; Clancy, Eoin; Barry, Thomas

    2013-09-01

    High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in human infection. Current culture-based techniques for the detection of microorganisms from HPW can be slow and laborious. The aim of this study was to develop a rapid method for the quantitative detection and identification of pathogenic bacteria causing low-level contamination of HPW. A novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimized to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. Sterile HPW, spiked with a bacterial load ranging from 10 to 10(3) cfu/100 ml, was filtered and the bacterial cells were removed from the filters by sonication. Total genomic DNA was then purified from these bacteria and subjected to testing with the developed novel multiplex real-time PCR diagnostics assay. The specific P. aeruginosa and Burkholderia genus assays have an analytical sensitivity of 3.5 genome equivalents (GE) and 3.7 GE, respectively. This analysis demonstrated that it was possible to detect a spiked bacterial load of 1.06 × 10(2) cfu/100 ml for P. aeruginosa and 2.66 × 10(2) cfu/100 ml for B. cepacia from a 200-ml filtered HPW sample. The rapid diagnostics method described can reliably detect, identify, and quantify low-level contamination of HPW with P. aeruginosa and the Burkholderia genus in <4 h. We propose that this rapid diagnostics method could be applied to the pharmaceutical and clinical sectors to assure the safety and quality of HPW, medical devices, and patient-care equipment.

  10. Advances in rapid drug detection technology.

    PubMed

    Zou, Wen-Bo; Yin, Li-Hui; Jin, Shao-Hong

    2017-08-15

    Spurious/Falsely-labeled/Falsified/Counterfeit (SFFC)drugs have become a major threat to public health, especially in rural areas of developing countries.The goal of this review is to provide an overview of rapid detection technologies for counterfeits recently reported, such as Near Infrared Spectroscopy, Near Infrared Chemical Imaging, Raman Spectroscopy, X-Ray Fluorescence, X-RayPowder Diffraction, Ion Mobility Spectrometry, Ion MobilityMass Spectrometry,Isotope Ratio Mass Spectrometry and visual analytical methods The advantages of each of these detection methods are introduced. Examples of characterization of SFFC drugs using the detection technology mentioned are presented. In addition, new characteristics and trends of SFFC drugs are listed and the solution is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

    Rigano, Luciano A; Marano, María R; Castagnaro, Atilio P; Do Amaral, Alexandre Morais; Vojnov, Adrian A

    2010-06-18

    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. 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 bacteria. The

  12. 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

  13. A simple and rapid method for detection of paraquat in human plasma by high-performance liquid chromatography

    PubMed Central

    Sun, Bin; Chen, Yuguo

    2015-01-01

    We have developed an effective analytical method to determine the concentration of paraquat in human plasma by high-performance liquid chromatography (HPLC), which can provide methodological support for diagnosis, therapy, and prognostic evaluation of acute poisoning related to paraquat. The plasma samples were deproteinized with 35% perchloric acid. Then the paraquat was extracted from the samples and separated on Ultimate XB-C18 column with a mobile phase consisting of 0.1 M phosphate buffer (containing 75 mM sodium heptane sulfonate) and methyl cyanides (PH 3.0), flow rate of 1 mL/min, column temperature of 28°C and a detection wave length on 258 nm. A good linearity was obtained within the range of 0.2 to 500 g/ml in human plasma. The average rate of absolute and relative recovery were about 100.6% and 101.31%, accompanied by the variations less than 3% and 6% respectively. The within- and between-day relative standard deviations were all less than 6%. After repeated freeze and thaw of plasma samples, no significant difference was observed for the concentration of paraquat (P>0.05). The method was validated by successfully applying in one patient with acute paraquat poisoning. Due to the celerity, accuracy and no-interference by other components of blood sample, this method was suitable for determination the concentration of paraquat in human plasma. PMID:26770298

  14. Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses.

    PubMed

    Zhang, Zhujun; Liu, Dong; Sun, Wenqiang; Liu, Jing; He, Lihong; Hu, Jiao; Gu, Min; Wang, Xiaoquan; Liu, Xiaowen; Hu, Shunlin; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2017-01-01

    Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.

  15. Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses

    PubMed Central

    Liu, Dong; Sun, Wenqiang; Liu, Jing; He, Lihong; Hu, Jiao; Gu, Min; Wang, Xiaoquan; Liu, Xiaowen; Hu, Shunlin; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2017-01-01

    Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples. PMID:28575115

  16. 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.

  17. Rapid LC-MS method for the detection of common fragrances in personal care products without sample preparation.

    PubMed

    Famiglini, Giorgio; Termopoli, Veronica; Palma, Pierangela; Capriotti, Fabiana; Cappiello, Achille

    2014-05-01

    An LC-MS method for the analysis of personal care and household products without sample preparation is presented. The method takes advantage of the Direct-electron ionization (EI) LC-MS interface for the quantitation of principal components, as well as for the identification of unknown or undeclared ingredients. The technique has proven its inertness toward matrix effects and the electron ionization allows quantitation and library identification. Commercially available products (shower gel, perfume, and hand cream) were diluted with methanol and injected directly into a nano-LC column. Limonene, linalool, and citral were selected as target compounds because of their use as fragrances in toiletry and detergent products. These and all other fragrances are commonly determined with GC-MS analysis, prior to sample cleanup, a procedure that can lead to analytes loss. The selected compounds are not detected with ESI because of their poor or very low response. Figures of merit and validation studies were executed and special attention was devoted to matrix-effects evaluation, because a sample preparation procedure is not involved. No matrix effects were observed, and the repeatability was excellent even after several weeks of operation. Products composition was investigated in full scan mode to determine the presence of unknown or not listed ingredients.

  18. Performance of two commercial rapid methods for sampling and detection of Listeria in small-scale cheese producing and salmon processing environments.

    PubMed

    Schirmer, Bjørn C T; Langsrud, Solveig; Møretrø, Trond; Hagtvedt, Therese; Heir, Even

    2012-11-01

    Two commercially available all-in-one swab rapid detection systems for Listeria spp. (InSite Listeria Test and Path-Chek hygiene Listeria) were tested for performance in cheese production environments and salmon processing facilities. Sampling was conducted both on clean surfaces and during production. A total of 338 samples were taken using the swabs (175 in cheese environments, 163 in salmon environments). Conventional sampling using sterile cloths and standardized qualitative detection of Listeria spp. according to NMKL method no. 136 was performed in parallel from 64 sampling sites in the salmon processing facilities and 40 sampling sites in the cheese production facilities. Results showed that both rapid swab tests detected Listeria spp.; however, they returned significant amounts of false positives. Presence of Listeria spp. was indicated in 47% and 41% of all swabs in the cheese and salmon environments, respectively. Enrichment followed by selective plating and Listeria specific PCR confirmed none of the 82 presumptive positive swabs from the cheese environment and 16 of 67 presumptive positive swabs from the salmon environments, respectively. Further analysis showed that several other bacteria, including Enterococcus spp. and Carnobacterium maltaromaticum, were the source of false positive swab results. From salmon processing facilities, using cloth sampling and standard analyses, 22% Listeria positive sampling sites were confirmed compared to 9% and 11% positives obtained using InSite or Path-Chek detection systems. From the cheese production environments, no Listeria positive sites were confirmed using either swab or cloth sampling. In conclusion, the use of these rapid detection methods was not suited in the selected environments due to large numbers of false positives, caused by the background flora.

  19. Rapid and sensitive method for determining free amino acids in honey by gas chromatography with flame ionization or mass spectrometric detection.

    PubMed

    Nozal, Ma J; Bernal, J L; Toribio, M L; Diego, J C; Ruiz, A

    2004-08-20

    This paper describes a rapid, sensitive and specific method for determination of free amino acids in honey involving a new reaction of derivatization and gas chromatography (GC) with flame ionization (FID) and mass spectrometric (MS) detection. The method allows the determination of 22 free amino acids in honey samples in a short time: 8 and 5 min for GC-FID and GC-MS, respectively. Quantitation was performed using Norvaline as internal standard, with detection limits ranging between 0.112 and 1.795 mg/L by GC-FID and between 0.001 and 0.291 mg/L by GC-MS in the selected-ion monitoring mode. The method was validated and applied to a set of 74 honey samples belonging to four different botanical origins: eucaliptus, rosemary, orange and heather. The statistical treatment of data shows a correct classification of different origins over 90%.

  20. 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.

  1. 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

  2. [Development of a single-tube PCR-pyrosequencing method for simultaneous and rapid detection of the genetic polymorphism of warfarin metabolizing enzymes].

    PubMed

    Shi, Hong; Yu, Run-Liu; Ma, Jin-Fei; Ren, Xu-Yi

    2011-11-01

    The purpose of this article is to develop a new high throughput method for detecting genetic polymorphism of warfarin metabolism-related genes rapidly in a single tube. Genomic DNA from human peripheral blood was extracted, and amplified with biotinylated primer to obtain single-stranded templates for pyrosequencing. Then, the single-stranded tem-plates were subjected to Pyrosequencing analysis using PyroMark ID instrument. Simultaneously, Sanger sequencing was also applied to sequence the products as a control to check the reliability of the pyrosequencing result.. The results dis-played that three variants of the warfarin metabolism-related genetic polymorphism (CYP2C9*2, CYP2C9*3, and VKORC1(-1693)) could be simultaneously detected using three different sequencing primers in a single-tube (one test), and 96 tests could be carried out each time. Repeat test and reliability test indicated that the agreement between the pyrosequencing and the Sanger sequencing methods was 100%. . All of these demonstrated that pyrosequencing could accurately and rapidly detect the genetic polymorphism of the warfarin drug metabolism-related genes with high throughput. Compar-ing with simplex pyrosequencing, the method established in the present study was much more economical and timesaving. It has a great value in personalized medical treatment and could be extended to the other genetic diseases.

  3. A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus.

    PubMed

    Wei, Haiyan; Zeng, Jing; Deng, Congliang; Zheng, Chengzhong; Zhang, Ximeng; Ma, Dan; Yi, Yong

    2013-03-01

    A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ∼100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.

  4. 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.

  5. 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

  6. Rapid detection of tdh and trh mRNAs of Vibrio parahaemolyticus by the transcription-reverse transcription concerted (TRC) method.

    PubMed

    Masuda, Noriyoshi; Yasukawa, Kiyoshi; Isawa, Yuichi; Horie, Ryuichi; Saitoh, Juichi; Ishiguro, Takahiko; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki; Hayashi, Toshinori

    2004-01-01

    We developed a novel method named the transcription-reverse transcription concerted (TRC) method and an instrument that allowed rapid and completely homogeneous real-time monitoring of RNA isothermal sequence amplification without any post-amplification analysis in our previous study [Ishiguro et al., Anal. Biochem., 314, 77-86 (2003)]. In this study, we newly established rapid and sensitive TRC systems for the detection of the mRNAs transcribed from two major virulence genes of Vibrio parahaemolyticus: the tdh gene encoding the thermostable direct hemolysin (tdh) and the trh gene encoding the thermostable direct hemolysin-related hemolysin. Examination of the standard RNAs prepared in vitro showed that a positive result, increase in the fluorescence intensity to the cut-off value within 25 min, was obtained for as few as 100 copies of RNA. The TRC method specific to the trh mRNA detected the mRNAs transcribed from the trh1 and trh2 genes, two representative trh variants sharing 84% sequence identity. The detection time gave a linear relationship to the logarithm of starting RNA copies ranging from 10(3) to 10(7) copies, showing that quantitative analysis is possible. The detection time for 10(3) copies of the standard RNAs ranged from 11 to 15 min. Examination of the total RNAs extracted from the standard strains of V. parahaemolyticus demonstrated that the new TRC systems are sufficiently sensitive to detect as few as 100 CFUs of the strains carrying the target genes. Total RNA preparations extracted from 24 strains of V. parahaemolyticus, 52 strains belonging to 31 other species of the genus Vibrio and 11 strains belonging to 8 species of non-Vibrio genera were examined. The results of the detection of tdh- and trh-specific mRNAs by the two TRC systems and those of the respective genes by the DNA colony hybridization method agreed. We conclude that the new TRC systems are rapid, highly sensitive, easy to manipulate, and are suitable for routine examination of

  7. Rapid Change Detection Algorithm for Disaster Management

    NASA Astrophysics Data System (ADS)

    Michel, U.; Thunig, H.; Ehlers, M.; Reinartz, P.

    2012-07-01

    This paper focuses on change detection applications in areas where catastrophic events took place which resulted in rapid destruction especially of manmade objects. Standard methods for automated change detection prove not to be sufficient; therefore a new method was developed and tested. The presented method allows a fast detection and visualization of change in areas of crisis or catastrophes. While often new methods of remote sensing are developed without user oriented aspects, organizations and authorities are not able to use these methods because of absence of remote sensing know how. Therefore a semi-automated procedure was developed. Within a transferable framework, the developed algorithm can be implemented for a set of remote sensing data among different investigation areas. Several case studies are the base for the retrieved results. Within a coarse dividing into statistical parts and the segmentation in meaningful objects, the framework is able to deal with different types of change. By means of an elaborated Temporal Change Index (TCI) only panchromatic datasets are used to extract areas which are destroyed, areas which were not affected and in addition areas where rebuilding has already started.

  8. Rapid detection method for hepatitis A virus from lettuce by a combination of filtration and integrated cell culture-real-time reverse transcription PCR.

    PubMed

    Hyeon, Ji-Yeon; Chon, Jung-Whan; Park, Chankyu; Lee, Joong-Bok; Choi, In-Soo; Kim, Moo-Sang; Seo, Kun-Ho

    2011-10-01

    We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture-real-time reverse transcription PCR (ICC-real-time RT-PCR) to detect infectious HAV. The most suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC-real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC-real-time RT-PCR detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC-real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC-real-time RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC-real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV in vegetables.

  9. Urinary kidney injury molecule‑1 as an early diagnostic biomarker of obstructive acute kidney injury and development of a rapid detection method.

    PubMed

    Jin, Yingli; Shao, Xiaona; Sun, Bo; Miao, Chunsheng; Li, Zhengqiang; Shi, Yan

    2017-03-01

    The aim of the present study was to investigate whether urinary kidney injury molecule‑1 (KIM‑1) presents a suitable early diagnostic biomarker of obstructive nephropathy‑induced acute kidney injury (AKI), and to develop a rapid detection method for urinary KIM‑1. Obstructive AKI was induced in an experimental rat model by a unilateral ureteral obstruction (UUO) operation. Macro‑ and micromorphological kidney alterations were determined by visual observation and hematoxylin and eosin (HE) staining, respectively. Kidney functions were evaluated by detecting urea nitrogen and creatinine levels in rat urine and blood. Urinary KIM‑1 levels were measured using an enzyme‑linked immunosorbent assay, and the protein expression levels of KIM‑1, α‑smooth muscle actin (α‑SMA) and vimentin in kidney tissues were detected using immunohistochemical assays. In order to measure KIM‑1 levels, colloidal gold immunochromatographic strips were developed based on the colloidal gold immunochromatographic assay. The results indicated that KIM‑1 levels were significantly higher in the UUO group when compared with the Sham group. KIM‑1 levels in the urine and kidney tissues exhibited a time‑dependent increase, together with increasing obstructive AKI in the UUO group. In addition, KIM‑1 levels were demonstrated to be a more sensitive biomarker of early obstructive AKI, when compared with α‑SMA and vimentin. A colloidal gold‑based immunochromatographic strip was developed, whereby the detection of urinary KIM‑1 could be completed within 5‑10 min. In conclusion, results of the present study demonstrated that urinary KIM‑1 may be a valuable biomarker for the early diagnosis of obstructive AKI, and the use of a colloidal gold immunochromatographic strip may be a promising method for the rapid detection of urinary KIM‑1.

  10. Urinary kidney injury molecule-1 as an early diagnostic biomarker of obstructive acute kidney injury and development of a rapid detection method

    PubMed Central

    Jin, Yingli; Shao, Xiaona; Sun, Bo; Miao, Chunsheng; Li, Zhengqiang; Shi, Yan

    2017-01-01

    The aim of the present study was to investigate whether urinary kidney injury molecule-1 (KIM-1) presents a suitable early diagnostic biomarker of obstructive nephropathy-induced acute kidney injury (AKI), and to develop a rapid detection method for urinary KIM-1. Obstructive AKI was induced in an experimental rat model by a unilateral ureteral obstruction (UUO) operation. Macro- and micromorphological kidney alterations were determined by visual observation and hematoxylin and eosin (HE) staining, respectively. Kidney functions were evaluated by detecting urea nitrogen and creatinine levels in rat urine and blood. Urinary KIM-1 levels were measured using an enzyme-linked immunosorbent assay, and the protein expression levels of KIM-1, α-smooth muscle actin (α-SMA) and vimentin in kidney tissues were detected using immunohistochemical assays. In order to measure KIM-1 levels, colloidal gold immunochromatographic strips were developed based on the colloidal gold immunochromatographic assay. The results indicated that KIM-1 levels were significantly higher in the UUO group when compared with the Sham group. KIM-1 levels in the urine and kidney tissues exhibited a time-dependent increase, together with increasing obstructive AKI in the UUO group. In addition, KIM-1 levels were demonstrated to be a more sensitive biomarker of early obstructive AKI, when compared with α-SMA and vimentin. A colloidal gold-based immunochromatographic strip was developed, whereby the detection of urinary KIM-1 could be completed within 5–10 min. In conclusion, results of the present study demonstrated that urinary KIM-1 may be a valuable biomarker for the early diagnosis of obstructive AKI, and the use of a colloidal gold immunochromatographic strip may be a promising method for the rapid detection of urinary KIM-1. PMID:28075469

  11. Development and application of a multiplex PCR method for rapid differential detection of subgroup A, B, and J avian leukosis viruses.

    PubMed

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

    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 × 10(3) 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.

  12. 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. Published by Elsevier Ltd.

  13. Development of a Semi-nested PCR-Based Method for Specific and Rapid Detection of Alternaria solani Causing Potato Early Blight in Soil.

    PubMed

    Gu, Qing; Yang, Zhi-Hui; Zhao, Dong-Mei; Zhang, Dai; Wang, Qian; Ma, Li-Song; Zhu, Jie-Hua

    2017-09-01

    Early blight, caused by Alternaria solani, is one of the most devastating diseases of potato that causes severe yield loss worldwide. The infected potato debris existed in the soil serve as the initial infection sources for the next growing potato. Current identification of A. solani in soil relies primarily on cultural and morphological characteristics, which are time-consuming and inaccurate. In this study, a semi-nested PCR method was developed using primers based on internal transcribed spacer region that is specific to A. solani. 20 isolates including 6 Alternaria species and 10 other species of common potato pathogens were used to examine the specificity of the primers. The primer set ptAsQ-F/ptAs-R was highly specific to A. solani, as a product of 251 bp was amplified only from A. solani isolates and no amplification signal was observed from other tested species. The sensitivity of this method determined using A. solani genomic DNA was 10 fg. This PCR assay was also successfully employed to detect A. solani in soil with the detection sensitivity of one conidia spore in 0.5 g of soil. To the best of our knowledge, this is the first report of molecular detection of A. solani in soil, which provides a useful tool for early and rapid detection of early blight in soil before next growing season.

  14. An Alternative Chemical Redox Method for the Production of Bispecific Antibodies: Implication in Rapid Detection of Food Borne Pathogens

    PubMed Central

    Owais, Mohammad; Kazmi, Shadab; Tufail, Saba; Zubair, Swaleha

    2014-01-01

    Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen Listeria monocytogenes (L. monocytogenes) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches. PMID:24637674

  15. An evaluation of the Charm test--a rapid method for the detection of penicillin in milk.

    PubMed

    Thorogood, S A; Wood, P D; Prentice, G A

    1983-05-01

    Using the Charm radioactive assay technique as a rapid test for the determination of penicillin in milk in experiments with milks containing known concentrations of penicillin, the coefficient of variation was found to be 15 and 11% for raw whole and reconstituted skim-milks respectively. Storage of reagents for up to 4 d increased the variance of the test by more than 20% and the estimated penicillin concentration 0.4%/d of storage up to 25 d. In comparison with the Bromocresol Purple Test, which was capable of 100% accuracy in defining a threshold of 0.02 i.u. penicillin/ml milk, the Charm Test falsely classified 15.6% of the samples.

  16. 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.

  17. 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.

  18. 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.

  19. Rapid method for detection of gram-positive and -negative bacteria in milk from cows with moderate or severe clinical mastitis.

    PubMed

    Yazdankhah, S P; Sørum, H; Larsen, H J; Gogstad, G

    2001-09-01

    A rapid method for demonstration of gram-positive and gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), which causes decolorization of gram-negative bacteria, whereas gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination, or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with moderate or severe clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92 and 100% for gram-positive and gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples. The detection limit was 5 x 10(6) CFU/ml for Staphylococcus aureus and 1 x 10(6) CFU/ml for Escherichia coli in spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.

  20. 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

  1. Coffee ring aptasensor for rapid protein detection.

    PubMed

    Wen, Jessica T; Ho, Chih-Ming; Lillehoj, Peter B

    2013-07-02

    We introduce a new biosensing platform for rapid protein detection that combines one of the simplest methods for biomolecular concentration, coffee ring formation, with a sensitive aptamer-based optical detection scheme. In this approach, aptamer beacons are utilized for signal transduction where a fluorescence signal is emitted in the presence of the target molecule. Signal amplification is achieved by concentrating aptamer-target complexes within liquid droplets, resulting in the formation of coffee ring "spots". Surfaces with various chemical coatings were utilized to investigate the correlation among surface hydrophobicity, concentration efficiency, and signal amplification. On the basis of our results, we found that the increase in the coffee ring diameter with larger droplet volumes is independent of surface hydrophobicity. Furthermore, we show that highly hydrophobic surfaces produce enhanced particle concentration via coffee ring formation, resulting in signal intensities 6-fold greater than those on hydrophilic surfaces. To validate this biosensing platform for the detection of clinical samples, we detected α-thrombin in human serum and 4-fold-diluted whole blood. Coffee ring spots from serum and blood produced detection signals up to 40 times larger than those from samples in liquid droplets. Additionally, this biosensor exhibits a lower limit of detection of 2 ng/mL (54 pM) in serum, and 4 ng/mL (105 pM) in blood. On the basis of its simplicity and high performance, this platform demonstrates immense potential as an inexpensive diagnostic tool for the detection of disease biomarkers, particularly for use in developing countries that lack the resources and facilities required for conventional biodetection practices.

  2. Rapid detection of deletions in hotspot C-terminal segment region of MECP2 by routine PCR method: report of two classical Rett syndrome patients of Indian origin.

    PubMed

    Khajuria, Rajni; Sapra, Savita; Ghosh, Manju; Gupta, Neerja; Gulati, Sheffali; Kalra, Veena; Kabra, Madhulika

    2009-04-01

    Rett syndrome (RS) is an X-linked dominant neurodevelopment disorder with normal prenatal and postnatal development till 6-18 months, followed by stagnation and regression of acquired skills. RS primarily manifests in females, and there are a few reports with males having RS. Sporadic or de novo mutations of the methyl CpG binding protein 2 (MECP2) gene have been reported in 70-90% of affected girls. Conventional methods such as fluorescence in situ hybridization, real-time PCR, southern blotting, multiplex ligation-dependent probe amplification, and DNA sequencing have been previously reported for the detection of insertions or deletions in the MECP2 gene. Here, we report detection of two deletions of 44 bp (c.1157_1200del44 or p.L386fs) and 38 bp (c.1151_1188del38 or p.P384fs) in exon 4 or C-terminal segment (CTS) region of MECP2 using a simple PCR technique that is rapid, accurate, and cost effective as compared to other techniques. The deletions were detected by routine PCR amplification followed by 2% agarose gel electrophoresis. We suggest that a simple PCR can easily detect deletions in the hotspot CTS region of the MECP2 gene and can be used for routine molecular diagnostics of RS.

  3. Development and application of a reverse transcription loop-mediated isothermal amplification method for rapid detection of Duck hepatitis A virus type 1.

    PubMed

    Yang, Limin; Li, Jing; Bi, Yuhai; Xu, Lei; Liu, Wenjun

    2012-12-01

    We developed and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Duck hepatitis A virus type 1 (DHAV-1). The amplification could be finished in 1 h under isothermal conditions at 63 °C by employing a set of four primers targeting the 2C gene of DHAV-1. The RT-LAMP assay showed higher sensitivity than the RT-PCR with a detection limit of 0.1 ELD(50) 0.1 ml(-1) of DHAV-1. The RT-LAMP assay was highly specific; no cross-reactivity was observed from the samples of other related viruses, bacteria, allantoic fluid of normal chicken embryos, or the livers of uninfected ducks. Thirty clinical samples were subjected to detection by RT-LAMP, RT-PCR, and virus isolation, which obtained completely consistent, positive results. As a simple, rapid, and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of DHAV-1.

  4. Rapid PCR-based method for detection and differentiation of Didymiaceae and Physaraceae (myxomycetes) in environmental samples.

    PubMed

    Kamono, Akiko; Fukui, Manabu

    2006-12-01

    Ecological studies of myxomycetes have been limited by the absence of universal cultivation techniques and the lack of life stage independent identification methods. We designed a novel PCR primer pair for the specific amplification of small subunit ribosomal RNA gene of Didymiaceae and Physaraceae. The primers produced amplicons from 192 fruiting body samples belonging to 10 genera. Twenty-four samples yielded longer fragments and sequence analysis revealed the presence of intron(s). As for the exonic regions, while sequence heterogeneities within a single species/varietas/forma were frequently observed, identical sequences were obtained only from identical species/varietas. The effectiveness of this primer pair in the analysis of morphologically unidentifiable samples was confirmed with the applications to samples of environmental plasmodium/sclerotium and soil. Denaturing gradient gel electrophoresis analysis was also tested with the soil samples. The results presented here demonstrate this PCR-based method can facilitate further ecological studies of Physaraceae and Didymiaceae in the environment.

  5. 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.

  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. Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique.

    PubMed

    Kawana, Shuichi; Nakagawa, Katsuhiro; Hasegawa, Yuki; Yamaguchi, Seiji

    2010-11-15

    A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using ²H₈-Val, ²H₃-Leu, ²H₃-Met and ²H₅-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients=1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood.

  8. Performance and Specificity of the Covalently Linked Immunomagnetic Separation-ATP Method for Rapid Detection and Enumeration of Enterococci in Coastal Environments

    PubMed Central

    Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Ferguson, Donna

    2014-01-01

    The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment. PMID:24561583

  9. Rapid quantitative analysis of individual anthocyanin content based on high-performance liquid chromatography with diode array detection with the pH differential method.

    PubMed

    Wang, Huayin

    2014-09-01

    A new quantitative technique for the simultaneous quantification of the individual anthocyanins based on the pH differential method and high-performance liquid chromatography with diode array detection is proposed in this paper. The six individual anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside, petunidin 3-glucoside, petunidin 3-rutinoside, and malvidin 3-rutinoside) from mulberry (Morus rubra) and Liriope platyphylla were used for demonstration and validation. The elution of anthocyanins was performed using a C18 column with stepwise gradient elution and individual anthocyanins were identified by high-performance liquid chromatography with tandem mass spectrometry. Based on the pH differential method, the high-performance liquid chromatography peak areas of maximum and reference absorption wavelengths of anthocyanin extracts were conducted to quantify individual anthocyanins. The calibration curves for these anthocyanins were linear within the range of 10-5500 mg/L. The correlation coefficients (r(2)) all exceeded 0.9972, and the limits of detection were in the range of 1-4 mg/L at a signal-to-noise ratio ≥5 for these anthocyanins. The proposed quantitative analysis was reproducible with good accuracy of all individual anthocyanins ranging from 96.3 to 104.2% and relative recoveries were in the range 98.4-103.2%. The proposed technique is performed without anthocyanin standards and is a simple, rapid, accurate, and economical method to determine individual anthocyanin contents.

  10. Detection sensitivity of influenza rapid diagnostic tests

    PubMed Central

    Sakai-Tagawa, Yuko; Ozawa, Makoto; Yamada, Shinya; Uchida, Yuko; Saito, Takehiko; Takahashi, Kazuo; Sugaya, Norio; Tashiro, Masato; Kawaoka, Yoshihiro

    2014-01-01

    We compared the sensitivity of influenza rapid diagnostic tests (IRDTs) currently available in Japan for various influenza virus strains, including human H7N9 and H5N1 isolates. We found that all of the IRDTs examined detected these viruses, but their detection sensitivities differed. PMID:25079880

  11. 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

  12. A Simple and Rapid Test-card Method to Detect Hepatitis B Surface Antigen and Antibody: Potential Application in Young Children and Infants.

    PubMed

    Wu, Fu-Yu; Liao, Yu-Wun; Wu, Jia-Feng; Chen, Huey-Ling; Hsu, Hong-Yuan; Chang, Mei-Hwei; Ni, Yen-Hsuan

    2016-06-01

    Hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) were conventionally determined by enzyme immunoassays. We aimed to apply a rapid, simple, and accurate method to detect HBsAg and its antibody. We collected 1463 serum samples from healthy volunteers, hepatitis B carriers, and children of HBsAg-positive mothers. The test card that we examined is a chromatographic immunoassay for the qualitative detection of either HBsAg or anti-HBs. We then compared the results of the test card to the results of the conventional enzyme-immunoassay method, which is regarded as a standard. In the use of the test card to check HBsAg, the sensitivity was 88.8% and the specificity was 100%. The median hepatitis B virus viral load was significantly higher in the true-positive group [10(3.71) copies/mL (range, 10(2)-10(9.03) copies/mL)] than in the false-negative group [10(2) copies/mL (range, 10(2)-10(3.26) copies/mL)] (p = 0.005). In those who were younger than 2 years, the diagnostic accuracy of the HBsAg test card was 100%. Then, 1272 samples were tested for anti-HBs rapid test card. The sensitivity was 91.8% and the specificity was 96.5%. The median anti-HBs titer was significantly higher in the true-positive group (295.8 mIU/mL) than in the false-negative group (42.3 mIU/mL; p < 0.001). Because of (1) the limited amount of blood sample required and (2) most of the young hepatitis B virus carriers having high viremia, and no concerns of false negativity, the test card is a good rapid screening tool for the detection of HBsAg and anti-HBs in pediatric group. Copyright © 2015. Published by Elsevier B.V.

  13. [Rapid detection of mutations related to Mycobacterium leprae drug resistance by using Hp-rPCR (hairpin primer- real time PCR) method].

    PubMed

    Kai, Masanori

    2014-03-01

    Rapid and simple detection method of drug resistance bacteria is required. In the present study, Hp-rPCR (hairpin primer-real time PCR) was applied to Mycobacterium leprae genes to detect mutations. Target sites of the method were as follows: first base and second base on 53rd codon and second base on 55th codon infolP1 gene for dapsone resistance, first base on 441st codon and 451st codon and second base on 456th and 458th codon in rpoB gene for rifampicin resistance, and first base on 89th codon and second base on 91st codon in gyrA gene for quinolone resistance which were common mutation sites in clinical reports. The total number of the target sites was 9. Mycobacterium leprae, Thai-53, Zensho-2 and Zensho-4 were used as reference bacteria in the present study and clear, reliable results were obtained. Double-blind study was conducted using 15 samples. The number of target sites was calculated as 135 in total by 9 sites in 15 samples. There was only one misreading in the blind samples and the sensitivity was more than 99%.

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

    USDA-ARS?s Scientific Manuscript database

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

  15. 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.

  16. 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.

  17. 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.

  18. 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 Central

    Aebischer, Andrea; Wernike, Kerstin; Beer, Martin

    2014-01-01

    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

  19. A method for rapid detection and genotype identification of hepatitis C virus 1-6 by one-step reverse transcription loop-mediated isothermal amplification.

    PubMed

    Nyan, Dougbeh-Chris; Swinson, Kevin L

    2016-02-01

    Hepatitis C virus (HCV) is probably the leading cause of liver cirrhosis and hepatocellular carcinoma globally. Diagnostic tools conventionally used for the detection and identification of HCV infection are technically demanding, time-consuming, and costly for resource-limited environments. This study reports the development of the first rapid loop-mediated reverse transcription isothermal amplification assay that rapidly detects and identifies HCV genotypes in blood components. RNA extracted from donor plasma and serum specimens was applied to a one-step reverse transcription loop-mediated isothermal amplification reaction performed with HCV-specific oligonucleotides. Reactions were conducted at 63.5 °C for 30-60 min. The diagnostic characteristics of the assay were investigated and validated with clinical specimens. Electrophoretic analysis of amplification revealed detection and identification of HCV genotypes 1-6. Positive amplification revealed unique ladder-like banding patterns that identified each HCV genotype. The assay demonstrated a sensitivity of 91.5% and specificity of 100%. Rapid naked-eye detection of HCV infection was facilitated by observation of an intense fluorescent glow of amplified targets under UV illumination. These diagnostic characteristics highlight the potential utility of this assay for the rapid detection and genotype identification of HCV infection in field and point-of-care settings in endemic regions and resource-limited environments. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Rapid Radiochemical Methods for Asphalt Paving Material ...

    EPA Pesticide Factsheets

    Technical Brief Validated rapid radiochemical methods for alpha and beta emitters in solid matrices that are commonly encountered in urban environments were previously unavailable for public use by responding laboratories. A lack of tested rapid methods would delay the quick determination of contamination levels and the assessment of acceptable site-specific exposure levels. Of special concern are matrices with rough and porous surfaces, which allow the movement of radioactive material deep into the building material making it difficult to detect. This research focuses on methods that address preparation, radiochemical separation, and analysis of asphalt paving materials and asphalt roofing shingles. These matrices, common to outdoor environments, challenge the capability and capacity of very experienced radiochemistry laboratories. Generally, routine sample preparation and dissolution techniques produce liquid samples (representative of the original sample material) that can be processed using available radiochemical methods. The asphalt materials are especially difficult because they do not readily lend themselves to these routine sample preparation and dissolution techniques. The HSRP and ORIA coordinate radiological reference laboratory priorities and activities in conjunction with HSRP’s Partner Process. As part of the collaboration, the HSRP worked with ORIA to publish rapid radioanalytical methods for selected radionuclides in building material matrice

  1. 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...

  2. 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...

  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 Waterborne Pathogen Detection with Mobile Electronics

    PubMed Central

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

    2017-01-01

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

  5. Multicenter evaluation of the LightCycler methicillin-resistant Staphylococcus aureus (MRSA) advanced test as a rapid method for detection of MRSA in nasal surveillance swabs.

    PubMed

    Peterson, Lance R; Liesenfeld, Oliver; Woods, Christopher W; Allen, Stephen D; Pombo, David; Patel, Parul A; Mehta, Maitry S; Nicholson, Bradly; Fuller, DeAnna; Onderdonk, Andrew

    2010-05-01

    The rate of methicillin-resistant Staphylococcus aureus (MRSA) infection continues to rise in many health care settings. Rapid detection of MRSA colonization followed by appropriate isolation can reduce transmission and infection. We compared the performance of the new Roche LightCycler MRSA advanced test to that of the BD GeneOhm MRSA test and culture. Double-headed swabs were used to collect anterior nasal specimens from each subject. For both tests, DNA was extracted and real-time PCR was performed according to manufacturer's instructions. For culture, one swab of the pair was plated directly to CHROMagar MRSA. The swab paired with the BD GeneOhm MRSA test was also placed into an enrichment broth and then plated to CHROMagar MRSA. Colonies resembling staphylococci were confirmed as S. aureus by standard methods. Discrepant specimens had further testing with additional attempts to grow MRSA as well as sample amplicon sequencing. Agreement between results for the two swabs was 99.3% for those with valid results. A total of 1,402 specimens were tested using direct culture detection of MRSA as the gold standard; 187 were culture positive for MRSA. The LightCycler MRSA advanced test had relative sensitivity and specificity of 95.2% (95% confidence interval [CI]: 91.1% to 97.8%) and 96.4% (95% CI: 95.2% to 97.4%), respectively. The BD GeneOhm assay had relative sensitivity and specificity of 95.7% (95% CI: 91.7% to 98.1%) and 91.7% (95% CI: 90.0% to 93.2%), respectively. Following discrepancy analysis, the relative sensitivities of the LightCycler MRSA advanced test and the BD GeneOhm MRSA assay were 92.2 and 93.2%, respectively; relative specificities were 98.9 and 94.2%, respectively. Specificity was significantly better (P<0.001) with the LightCycler MRSA advanced test. The sensitivity of direct culture was 80.4%. The LightCycler MRSA advanced test is a useful tool for sensitive and rapid detection of MRSA nasal colonization.

  6. A multicenter comparison study between the Endosafe PTS rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products.

    PubMed

    Gee, A P; Sumstad, D; Stanson, J; Watson, P; Proctor, J; Kadidlo, D; Koch, E; Sprague, J; Wood, D; Styers, D; McKenna, D; Gallelli, J; Griffin, D; Read, E J; Parish, B; Lindblad, R

    2008-01-01

    Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

  7. Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens.

    PubMed

    Revathy, Mani; Therese, Kulandai Lily; Bagyalakshmi, Radhakishnan; Chandrasekar, Chokaliingam; Kumar, Suria; Madhavan, Hajib N

    2014-09-01

    As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of P. jirovecii in HIV positive patients. One hundred and fifty sputum specimens from HIV positive (n = 75) and HIV negative (n = 75) patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS), RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis. Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33%) patients by the staining techniques, and in 23 (30.65%) patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank. Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection.

  8. 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.

  9. Comparison of Molecular Detection Method (Nested Polymerase Chain Reaction) with Blood Culture and Paired Widal test for the Rapid Diagnosis of Typhoid Fever.

    PubMed

    Khan, S; Miah, R A; Pal, S; Khatun, S; Fatema, N; Roy, R R; Naheen, C R

    2017-01-01

    Typhoid fever is a major health problem in developing countries in spite of the use of antibiotics and the development of newer antibacterial drugs. Blood culture & serological tests (specially Widal test) which are invariably done in Bangladesh for typhoid fever diagnosis give unacceptable levels of false negative & false positive results respectively. This cross sectional study was done at Bangabandhu Sheikh Mujib Medical University from March 2013 to February 2014. In this study, a polymerase chain reaction-based technique (which has 100% specificity for Salmonella Typhi) was compared with blood culture and widal test among 80 clinically suspected cases of typhoid fever. PCR showed maximum positivity rate (70%) followed by widal test (43.75%) and blood culture (16.25%). PCR showed positive results for 17(48.6%) of 35 typhoid patients with negative results with blood culture and widal test. The results of the study revealed that PCR is rapid and reliable diagnostic technique for detection of S. Typhi in clinically suspected typhoid fever cases, as compared to most commonly done methods such as conventional blood culture, widal test applied.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  11. Development of a Rapid High-Throughput Method for High-Resolution Melting Analysis for Routine Detection and Genotyping of Noroviruses▿

    PubMed Central

    Tajiri-Utagawa, Etsuko; Hara, Masayuki; Takahashi, Kuniaki; Watanabe, Mayumi; Wakita, Takaji

    2009-01-01

    We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major norovirus genotypes were selected. Because we had only small quantities of the patient stool samples used in this study, we constructed plasmids for each genotype and used these to optimize the HRM assay. We selected six stool samples, each positive for one of the six dominant subtypes of noroviruses that have been circulating in Japan, namely, genotypes 4, 8, and 9 from genogroup 1 and genotypes 3, 4, and 10 from genogroup 2. The specific high-resolution derivate plot of the HRM assay for each plasmid was constructed by subtracting the melting-curve shape of the plasmid from the reference or base curve. The RNAs extracted from 14 clinical samples positive for small round structured viruses were then directly analyzed using the HRM assay. The HRM data from the clinical RNA samples corresponded with the genotype results obtained by RT-PCR and sequencing of the clinical samples. In addition, the HRM data from the clinical RNA samples corresponded with the HRM data from the six reference plasmid DNAs, indicating that this assay is useful for the direct detection and genotyping of noroviruses in clinical samples. This assay requires no multiplexing or hybridization probes and provides a new approach to the genetic screening of noroviruses in clinical virology laboratories. PMID:19073870

  12. A rapid and noninvasive method for detecting tissue-limited mosaicism: detection of i(12)(p10) in buccal smear from a child with Pallister-Killian syndrome.

    PubMed

    Velagaleti, Gopalrao V N; Tapper, Jill K; Rampy, Bill A; Zhang, Shuliu; Hawkins, Judy C; Lockhart, Lillian H

    2003-01-01

    Pallister-Killian syndrome (PKS), a rare disorder, is characterized by tissue-limited or tissue-specific mosaicism. The characteristic chromosome abnormality associated with PKS is i(12p), which is seen predominantly in skin fibroblast cultures. Diagnosis of i(12p) has been carried out on buccal smears before and was shown to be an easy and feasible method. All previously published studies used alpha-satellite probes for the diagnosis and as such have several pitfalls. Our approach, using dual-color, locus-specific probes, has high specificity and sensitivity for the diagnosis of i(12p). Using statistical analysis, we have also confirmed that the signal pattern in interphase nuclei is consistent with isochromosome 12p.

  13. Detection of ostreid herpesvirus-1 (OsHV-1) by PCR using a rapid and simple method of DNA extraction from oyster larvae.

    PubMed

    Batista, Frederico M; Taris, Nicolas; Boudry, Pierre; Renault, Tristan

    2005-04-06

    A DNA extraction procedure was developed for the detection of ostreid herpesvirus-1 (OsHV-1) using the polymerase chain reaction (PCR) in oyster larvae. The DNA extraction procedure developed was tested on 8 larval samples. Abnormal nuclei with characteristic features associated with OsHV-1 infections were only observed in samples in which the viral DNA was detected by PCR. A previously described competitive PCR method was applied to detect inhibition during PCR reactions. The results show that the method can be used on small amounts of oyster larvae (3 mg) for the detection of OsHV-1 DNA by PCR.

  14. 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. © 2014 Society for Conservation Biology.

  15. Rapid detection of bacteriuria by laser nephelometry.

    PubMed Central

    Richet, H; Bayardelle, P; Lemarie, J C

    1984-01-01

    A total of 1,002 urine specimens were evaluated by laser nephelometry. This technique was compared with both colony counts, done with a calibrated loop, and serial dilutions. For urine specimens containing between 10(4) and 10(5) bacteria per ml, laser nephelometry detected 75.4% of those detected by colony count and 65.6% of those detected by serial dilution. For specimens where the concentration of bacteria was greater than 10(5) per ml, laser nephelometry detected 95.8 and 92.4% of those detected by colony count and serial dilution, respectively. The mean detection time for bacteriuria varied from 1.57 h for more than 10(5) bacteria per ml to 4.47 h for more than 10(4) bacteria per ml. To determine the number of bacteria according to the voltage growth curve, the passage time at 3 V was used as an index. The mean passage time at 3 V decreased from 5.18 h for fewer than 10(4) bacteria per ml to 1.42 h for more than 10(6) bacteria per ml. The mean passage time at 3 V differed significantly for different concentrations of bacteria. Thus, this index allowed us to predict the number of bacteria in the urine specimens. Laser nephelometry has been used for many years for the immunological determination of proteins; it can now also be considered a tool for rapid screening in bacteriology. PMID:6365961

  16. Rapid Radiochemical Methods for Selected Radionuclides

    EPA Pesticide Factsheets

    The rapid methods documents are supplement guidance in a planned series designed to present radioanalytical laboratory personnel, Incident Commanders (and their designees), and other field response personnel.

  17. Rapid genetic detection of ingested Amanita phalloides.

    PubMed

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

    2014-03-01

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

  18. 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

  19. Colorimetric Integrated PCR Protocol for Rapid Detection of Vibrio parahaemolyticus

    PubMed Central

    Cheng, Kewen; Pan, Daodong; Teng, Jun; Yao, Li; Ye, Yingwang; Xue, Feng; Xia, Fan; Chen, Wei

    2016-01-01

    Rapid detection of pathogens is of great significance for food safety and disease diagnosis. A new colorimetric method for rapid and easy detection of Vibrio parahaemolyticus (V. parahaemolyticus or Vp) has been developed in this research. A specific sequence was designed and integrated with the forward primer for molecular detection of Vp. This specific sequence was tested and treated as the horseradish peroxidase (HRP)-mimicking DNAzyme and could be amplified during the polymerase chain reaction (PCR) process. The products of PCR including the sequence of HRP-mimicking DNAzyme could produce the distinguished color in the presence of catalysis substrates. The optical signal of the catalysis reaction, which is in a linear relationship with the initial template of Vp, could be determined with the naked eye or measured with Ultraviolet-visible (UV-vis) for qualitative and quantitative detections, respectively. Based on the optical signal intensity, rapid and easy detection of Vp was successfully achieved with satisfied sensitivity and specificity. Furthermore, the detection of tdh, trh, tlh and toxR virulence genes of two Vp species (Vp 33847 and Vp 17802) were all performed successfully with this developed colorimetric integrated PCR protocol, which demonstrated potential applicability for the rapid detection of other bacteria. PMID:27690041

  20. Predictions for rapid methods and automation in food microbiology.

    PubMed

    Fung, Daniel Y C

    2002-01-01

    A discussion is presented on the present status of rapid methods and automation in microbiology. Predictions are also presented for development in the following areas: viable cell counts; real-time monitoring of hygiene; polymerase chain reaction, ribotyping, and genetic tests in food laboratories; automated enzyme-linked immunosorbent assay and immunotests; rapid dipstick technology; biosensors for Hazard Analysis Critical Control Point programs; instant detection of target pathogens by computer-generated matrix; effective separation and concentration for rapid identification of target cells; microbiological alert systems in food packages; and rapid alert kits for detecting pathogens at home.

  1. Comparison of antibodies for rapid detection of cytomegalovirus.

    PubMed Central

    Randazzo, D N; Michalski, F J

    1988-01-01

    Antibodies were compared for use in the spin-amplified shell vial method for rapid cytomegalovirus detection. Commercial antibodies by Du Pont Co., Whittaker M.A. Bioproducts, Bartels Immunodiagnostic, Virostat, and Serono Diagnostics were compared at incubation times of 16 to 48 h on 22 patient specimens. Only the Du Pont antibody showed 100% sensitivity and specificity and no nonspecific reactions. PMID:2830312

  2. Comparison of antibodies for rapid detection of cytomegalovirus.

    PubMed

    Randazzo, D N; Michalski, F J

    1988-02-01

    Antibodies were compared for use in the spin-amplified shell vial method for rapid cytomegalovirus detection. Commercial antibodies by Du Pont Co., Whittaker M.A. Bioproducts, Bartels Immunodiagnostic, Virostat, and Serono Diagnostics were compared at incubation times of 16 to 48 h on 22 patient specimens. Only the Du Pont antibody showed 100% sensitivity and specificity and no nonspecific reactions.

  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

    USDA-ARS?s Scientific Manuscript database

    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. Methods of Rapid Evaluation, Assessment, and Appraisal

    ERIC Educational Resources Information Center

    McNall, Miles; Foster-Fishman, Pennie G.

    2007-01-01

    A central issue in the use of rapid evaluation and assessment methods (REAM) is achieving a balance between speed and trustworthiness. In this article, the authors review the key differences and common features of this family of methods and present a case example that illustrates how evaluators can use rapid evaluation techniques in their own…

  5. 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.

  6. FNAS/Rapid Spectral Inversion Methods

    NASA Technical Reports Server (NTRS)

    Poularikas, Alexander

    1997-01-01

    The purpose of this investigation was to study methods and ways for rapid inversion programs involving the correlated k-method, and to study the infrared observations of Saturn from the Cassini orbiter.

  7. Rapid and sensitive immunomagnetic separation-polymerase chain reaction method for the detection of Escherichia coli O157:H7 in raw milk and ice-cream.

    PubMed

    Gooding, C M; Choudary, P V

    1997-02-01

    Escherichia coli O157:H7 in spiked samples of raw milk and ice-cream was enriched in tryptic soy broth for 4 h, captured by immunomagnetic separation, subjected to amplification by polymerase chain reaction of parts of the verotoxin genes (SLT-I and SLT-II), and detected by agarose gel electrophoresis. Using this method, as few as 1 cfu Esch. coli O157:H7/g food could be detected in < 10 h.

  8. Evaluation of rapid and sensitive reverse transcription loop-mediated isothermal amplification method for detecting infectious pancreatic necrosis virus in chum salmon (Oncorhynchus keta).

    PubMed

    Suebsing, Rungkarn; Oh, Myung-Joo; Kim, Jeong-Ho

    2011-07-01

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detecting Infectious pancreatic necrosis virus (IPNV) in chum salmon (Oncorhynchus keta) in Korea. The RT-LAMP is a novel approach of nucleic acid gene amplification with high specificity, sensitivity, and rapidity under isothermal conditions. Based on the VP2/NS gene sequence of VR-299 and Jasper strains, a set of 6 IPNV-specific primers was designed to recognize 8 diverse sequences of the IPNV RNA. The assay was successfully optimized to detect IPNV at 65°C in 30 min. The detection limit was 0.075 tissue culture infectious dose infecting 50% of inoculated cultures per milliliter (TCID(50)/ml) from IPNV-infected rainbow trout gonad (RTG)-2 cells, whereas nested reverse transcription polymerase chain reaction (nRT-PCR) had a sensitivity of 7.5 TCID(50)/ml. Using RT-LAMP assay, field samples were analyzed and the results compared with those of nRT-PCR assay. Two hundred and sixty-six out of 659 (40.4%) samples were IPNV-positive by RT-LAMP, whereas 182 of 659 samples (27.6%) were IPNV-positive by nRT-PCR. The results indicate that RT-LAMP can be a useful tool for early field diagnosis of IPNV.

  9. 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.

  10. [Rapid prototyping: a very promising method].

    PubMed

    Haverman, T M; Karagozoglu, K H; Prins, H-J; Schulten, E A J M; Forouzanfar, T

    2013-03-01

    Rapid prototyping is a method which makes it possible to produce a three-dimensional model based on two-dimensional imaging. Various rapid prototyping methods are available for modelling, such as stereolithography, selective laser sintering, direct laser metal sintering, two-photon polymerization, laminated object manufacturing, three-dimensional printing, three-dimensional plotting, polyjet inkjet technology,fused deposition modelling, vacuum casting and milling. The various methods currently being used in the biomedical sector differ in production, materials and properties of the three-dimensional model which is produced. Rapid prototyping is mainly usedforpreoperative planning, simulation, education, and research into and development of bioengineering possibilities.

  11. 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

  12. Rapid detection methods and prevalence estimation for Borrelia lonestari glpQ in Amblyomma americanum (Acari: Ixodidae) pools of unequal size.

    PubMed

    Bacon, Rendi Murphree; Pilgard, Mark A; Johnson, Barbara J B; Piesman, Joseph; Biggerstaff, Brad J; Quintana, Miguel

    2005-01-01

    DNA was extracted from pools of Amblyomma americanum ticks collected from vegetation at two sites in Fort Leonard Wood, Missouri and tested for the presence of Borrelia spp. Two new methods were developed to detect Borrelia lonestari DNA by targeting the glycerophosphodiester phosphodiesterase (glpQ) gene. The first method detected B. lonestari DNA using a SYBR green I melting curve analysis of the PCR product obtained with glpQ gene primers. The second method, a glpQ TaqMan assay, detected and confirmed the presence of B. lonestari glpQ-specific sequences. Twenty-two of 95 tick pools collected at site A148 contained B. lonestari DNA. None of 19 pools from site A241 contained B. lonestari DNA. No B. burgdorferi sensu lato DNA was detected using a SYBR green I melting curve analysis of the PCR product obtained with outer surface protein A (ospA) primers. The overall B. lonestari infection prevalence (with 95% confidence interval) at site A148 was estimated using two algorithms: minimum infection rate 4.14% (2.45, 5.84) and maximum likelihood with correction 4.82% (3.11, 7.16). The merits of each are discussed. Sequencing of the entire B. lonestari glpQ and partial 16S rRNA genes revealed two genetic variants circulating in this population of A. americanum from Missouri.

  13. Rapid Aminoglycoside NP Test for Rapid Detection of Multiple Aminoglycoside Resistance in Enterobacteriaceae.

    PubMed

    Nordmann, Patrice; Jayol, Aurélie; Dobias, Jan; Poirel, Laurent

    2017-04-01

    The rapid aminoglycoside NP (Nordmann/Poirel) test was developed to rapidly identify multiple aminoglycoside (AG) resistance in Enterobacteriaceae It is based on the detection of the glucose metabolism related to enterobacterial growth in the presence of a defined concentration of amikacin plus gentamicin. Formation of acid metabolites was evidenced by a color change (orange to yellow) of the red phenol pH indicator. The rapid aminoglycoside NP test was evaluated by using bacterial colonies of 18 AG-resistant isolates producing 16S rRNA methylases, 20 AG-resistant isolates expressing AG-modifying enzymes (acetyl-, adenyl-, and phosphotransferases), and 10 isolates susceptible to AG. Its sensitivity and specificity were 100% and 97%, respectively, compared to the broth dilution method, which was taken as the gold standard for determining aminoglycoside resistance. The test is inexpensive, rapid (<2 h), and implementable worldwide. Copyright © 2017 American Society for Microbiology.

  14. 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

  15. Rapid aneuploidy detection or karyotyping? Ethical reflection.

    PubMed

    de Jong, Antina; Dondorp, Wybo J; Timmermans, Daniëlle R M; van Lith, Jan M M; de Wert, Guido M W R

    2011-10-01

    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.

  16. Detection of Coliforms in Drinking Water Using Skin Patches: A Rapid, Reliable Method that Does Not Require an External Energy Source

    PubMed Central

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

    2014-01-01

    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

  17. 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.

  18. A rapid ATR-FTIR spectroscopic method for detection of sibutramine adulteration in tea and coffee based on hierarchical cluster and principal component analyses.

    PubMed

    Cebi, Nur; Yilmaz, Mustafa Tahsin; Sagdic, Osman

    2017-08-15

    Sibutramine may be illicitly included in herbal slimming foods and supplements marketed as "100% natural" to enhance weight loss. Considering public health and legal regulations, there is an urgent need for effective, rapid and reliable techniques to detect sibutramine in dietetic herbal foods, teas and dietary supplements. This research comprehensively explored, for the first time, detection of sibutramine in green tea, green coffee and mixed herbal tea using ATR-FTIR spectroscopic technique combined with chemometrics. Hierarchical cluster analysis and PCA principle component analysis techniques were employed in spectral range (2746-2656cm(-1)) for classification and discrimination through Euclidian distance and Ward's algorithm. Unadulterated and adulterated samples were classified and discriminated with respect to their sibutramine contents with perfect accuracy without any false prediction. The results suggest that existence of the active substance could be successfully determined at the levels in the range of 0.375-12mg in totally 1.75g of green tea, green coffee and mixed herbal tea by using FTIR-ATR technique combined with chemometrics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. A rapid and sensitive LC-ESI-MS/MS method for the detection of YF-49-92.MLS in rat plasma.

    PubMed

    Gao, Qin; Huang, Xin; Jiang, Zhenzhou; Liu, Guanlan; Xiao, Yi; Sun, Lixin; Wang, Tao; Zhang, Luyong; Xie, Shaofei; Huang, Haiyan

    2013-10-01

    YF-49-92.MLS is a novel candidate for TB treatment. An accurate, precise and specific LC-MS/MS method for the quantification of YF-49-92.MLS in rat plasma using verapamil as an IS is reported in this paper. Proper retention time and excellent peak shape were acquired using an Agilent Zorbax(®) SB-C18 column with the mobile phase of 5 mmol/l ammonium acetate, 0.1% formic acid-methanol (30:70, v/v). The LLOQ was 1 ng/ml. The calibration curves encompassed concentrations from 20 to 5000 ng/ml. Intra- and inter-assay precision and accuracy were within 15% by determining low, medium and high concentration samples. Extraction recovery, stability, and matrix effects were also fully validated. This method has been validated to be rapid and sensitive, and successfully applied to the PK study of YF-49-92.MLS in rat plasma.

  20. 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.

  1. 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

    2014-07-07

    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.

  2. 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

  3. 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.

  4. 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.

  5. Staph ID/R: a rapid method for determining staphylococcus species identity and detecting the mecA gene directly from positive blood culture.

    PubMed

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

    2012-03-01

    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.

  6. 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

  7. A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses.

    PubMed

    Yang, Fan; Wang, Guoping; Xu, Wenxing; Hong, Ni

    2017-09-01

    Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus (Citrus aurantifolia), grapevine (Vitis vinifera), peach (Prunus persica), pear (Pyrus spp.), taro (Colocosia esculenta) and tobacco (Nicotiana benthamiana) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Simple and rapid high performance liquid chromatography method for the determination of polidocanol as bulk product and in pharmaceutical polymer matrices using charged aerosol detection.

    PubMed

    Ilko, David; Puhl, Sebastian; Meinel, Lorenz; Germershaus, Oliver; Holzgrabe, Ulrike

    2015-02-01

    Currently, neither the European nor the United States Pharmacopoeia provide a method for the determination of polidocanol (PD) content despite the fact that PD, besides being an excipient, is also used as an active pharmaceutical ingredient. We therefore developed a method where the PD content was determined using a Kinetex C18 column operated at 40°C with water-acetonitrile (15:85, v/v) as mobile phase. A Corona(®) charged aerosol detector was employed for the detection of PD that is lacking a suitable UV chromophore. The method was fully validated. Additionally, the method was applied for the determination of PD release from a pharmaceutical polymer matrix consisting of poly-ɛ-caprolactone and poly(lactic-co-glycolic acid) and PD. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Rapid Modified QuEChERS Method for Pesticides Detection in Honey by High-Performance Liquid Chromatography UV-visible

    PubMed Central

    Ceci, Edmondo; Montemurro, Nicola; Tantillo, Giuseppina; Di Pinto, Angela; Celano, Gaetano Vitale; Bozzo, Giancarlo

    2014-01-01

    The extensive use of pesticides in agriculture plays an important role in bees die-off and allows the presence of residues in hive products, particularly in honey. An accurate and reliable analytical method, based on QuEChERS extractive technique, has been developed for the quantitative determination by high-performance liquid chromatography UV-visible detector of 5 pesticides (Deltamethrin, Dimethoate, Imidacloprid, Acetamiprid, Chlorfenvinphos) in honey. The method, according to Commission Directive 2002/63/EC and Regulation 882/2004/EC, provided excellent results with respect to linearity (correlation coefficient up to 0.993), limits of detection and quantification (0.005 and 0.01 µg/mL for Dimethoate, Deltamethrin and Chlorfenvinphos; 0.02 and 0.05 µg/mL for Acetamiprid and Imidacloprid), recovery values (86.4 to 96.3%), precision and relative expanded uncertainty of a measurement, demonstrating the conformity of the this method with the European directives. The proposed method was applied to 23 samples of Apulian honey. None of the investigated pesticides was detected in these samples. PMID:27800334

  10. 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

  11. Rapid polymerase chain reaction (PCR)-single-stranded conformational polymorphism (SSCP) screening method for the identification of Aspergillus section Nigri species by the detection of calmodulin nucleotide variations.

    PubMed

    Susca, A; Stea, G; Perrone, G

    2007-10-01

    Single-stranded conformational polymorphism (SSCP) analysis for genetic diversity studies has been widely applied to detect indirectly sequence differences up to a single base in amplified DNA fragments of the same length, representing an alternative to gene sequencing. In this study SSCP analysis was used to detect sequence variations contained in an about 180-bp region of the calmodulin gene in order to identify Aspergillus section Nigri species. The method described shows that fluorescence-based SSCP analysis by capillary electrophoresis is cheaper and faster than direct sequencing, and suitable for computer-assisted analyses allowing discrimination between the Aspergillus species belonging to the Nigri section: A. aculeatus, Aspergillus 'atypic uniseriate', A. brasiliensis, A. carbonarius, A. ellipticus, A. foetidus, A. heteromorphus, A. ibericus, A. japonicus, A. niger, and A. tubingensis.

  12. A scoping review of rapid review methods.

    PubMed

    Tricco, Andrea C; Antony, Jesmin; Zarin, Wasifa; Strifler, Lisa; Ghassemi, Marco; Ivory, John; Perrier, Laure; Hutton, Brian; Moher, David; Straus, Sharon E

    2015-09-16

    Rapid reviews are a form of knowledge synthesis in which components of the systematic review process are simplified or omitted to produce information in a timely manner. Although numerous centers are conducting rapid reviews internationally, few studies have examined the methodological characteristics of rapid reviews. We aimed to examine articles, books, and reports that evaluated, compared, used or described rapid reviews or methods through a scoping review. MEDLINE, EMBASE, the Cochrane Library, internet websites of rapid review producers, and reference lists were searched to identify articles for inclusion. Two reviewers independently screened literature search results and abstracted data from included studies. Descriptive analysis was conducted. We included 100 articles plus one companion report that were published between 1997 and 2013. The studies were categorized as 84 application papers, seven development papers, six impact papers, and four comparison papers (one was included in two categories). The rapid reviews were conducted between 1 and 12 months, predominantly in Europe (58 %) and North America (20 %). The included studies failed to report 6 % to 73 % of the specific systematic review steps examined. Fifty unique rapid review methods were identified; 16 methods occurred more than once. Streamlined methods that were used in the 82 rapid reviews included limiting the literature search to published literature (24 %) or one database (2 %), limiting inclusion criteria by date (68 %) or language (49 %), having one person screen and another verify or screen excluded studies (6 %), having one person abstract data and another verify (23 %), not conducting risk of bias/quality appraisal (7 %) or having only one reviewer conduct the quality appraisal (7 %), and presenting results as a narrative summary (78 %). Four case studies were identified that compared the results of rapid reviews to systematic reviews. Three studies found that the conclusions between

  13. 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.

  14. 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.

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

    PubMed

    Bai, Yalong; Song, Minghui; Cui, Yan; Shi, Chunlei; Wang, Dapeng; Paoli, George C; Shi, Xianming

    2013-07-17

    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 pathogens could be extracted directly from complex matrices such as raw milk using ASMNPs. The magnetically separated complexes of genomic DNA and ASMNPs were directly subjected to single PCR (S-PCR) or multiplex PCR (M-PCR) to detect single or multiple pathogens from raw milk samples. Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive) were used as model organisms to artificially contaminate raw milk samples. After magnetic separation and S-PCR, the detection sensitivities were 8 CFU mL(-1) and 13 CFU mL(-1) respectively for these two types of pathogens. Furthermore, this method was successfully used to detect multiple pathogens (S. Enteritidis and L. monocytogenes) from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU mL(-1) and 25 CFU mL(-1), respectively. This method has great potential to rapidly and sensitively detect pathogens in raw milk or other complex food matrices.

  16. 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.

  17. A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters.

    PubMed

    Banerjee, S K; Pandian, S; Todd, E C; Farber, J M

    2002-06-01

    DNA probe-based detection methods were developed and characterized as an alternative to time-consuming and less specific conventional protocols. Digoxigenin-labeled probes were prepared by polymerase chain reaction amplification of the targeted sequences in the specific amplicons generated from genomic DNA. Specific probes with high yields were generated for the detection of the tlh gene of Vibrio parahaemolyticus and the cth gene of V. vulnificus. Colony (Southern) hybridization analyses were carried out using hydrophobic grid membrane filters (HGMFs) to allow biotype-specific differentiation of the two species. Eight strains of V. vulnificus and five strains of V. parahaemolyticus, including one standard (ATCC) strain of each biotype, were examined. Colony lysis, hybridization, and nonradioactive detection parameters were optimized for identification of the target biotypes arranged on the same HGMF and also on a conventional nylon membrane, thereby confirming the specificity of the probes and the comparative usefulness of the HGMFs. The experimental procedure presented here can be completed in 1 day. The protocol was designed specifically to identify the target Vibrio spp. and could potentially be used for the enumeration and differentiation of V. parahaemolyticus and V. vulnificus in foods.

  18. 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.

  19. 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.

  20. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

    PubMed

    McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

    2015-03-01

    Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

  1. 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.

  2. 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.

  3. 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.

  4. Rapid and reliable screening method for detection of 70 pesticides in whole blood by gas chromatography-mass spectrometry using a constructed calibration-locking database.

    PubMed

    Kudo, Keiko; Nagamatsu, Kumi; Umehara, Takahiro; Usumoto, Yosuke; Sameshima, Naomi; Tsuji, Akiko; Ikeda, Noriaki

    2012-03-01

    Pesticide poisoning is one of the most common causes of death by poisoning in Japan, and various kinds of pesticides including organophosphates, carbamates and pyrethroids are listed as causative substances. The purpose of our study was to develop a rapid and reliable screening method for various kinds of pesticides in whole blood by using a unique calibration-locking database and gas chromatography-mass spectrometry. A database of 70 pesticides was constructed using NAGINATA™ software with parameters such as mass spectrum, retention time and qualifier ion/target ion ratio (QT ratio) and calibration curve. Diazepam-d(5) was used as the internal standard for construction of each calibration curve within the range of 0.01-5.0 μg/ml. We examined the applicability of the constructed database by analyzing whole blood samples spiked with 70 pesticides. The pesticides in blood were extracted with hexane under acidic conditions or with an enhanced polymer column (Focus™), subjected to GC-MS, and screened by the pesticides database. Among the 70 pesticides examined, 66 and 62 were successfully identified at the level of 1 and 0.1 μg/ml, respectively, by hexane and 63 and 51 were identified by the Focus column without the use of standard compounds. The time required for data analysis was significantly reduced. Since the established method can produce qualitative and semi-quantitative data without the need for standard substances, this new screening method using NAGINATA™ should be useful for confirming the presence of pesticides in blood in future clinical and forensic cases.

  5. Rapid Detection of Polymyxin-Resistant Enterobacteriaceae from Blood Cultures.

    PubMed

    Jayol, Aurélie; Dubois, Véronique; Poirel, Laurent; Nordmann, Patrice

    2016-09-01

    Enterobacterial strains resistant to polymyxins are being increasingly reported worldwide. The conventional methods for detection of colistin-resistant isolates such as broth microdilution remain time-consuming (24 to 48 h), and methods such as disc diffusion and Etest are not reliable. Recently, the rapid polymyxin NP test was developed for rapid identification of polymyxin-resistant Enterobacteriaceae This test is based on the detection of glucose metabolism related to bacterial growth in the presence of a defined concentration of colistin (or polymyxin B). The formation of acid metabolites is evidenced by a color change of a pH indicator (red phenol) in less than 2 h. In this study, the polymyxin NP test was evaluated for detection of colistin-resistant Enterobacteriaceae directly from blood cultures. The test was performed with 73 blood culture sets (either spiked or clinical blood cultures) with various enterobacterial species. The test exhibited excellent discrimination between polymyxin-resistant and polymyxin-susceptible enterobacterial isolates, and results are obtained from blood cultures within 4 h. It is easy to perform and requires neither subculture nor a centrifugation step. This test is rapid, specific, and sensitive and allows early identification of polymyxin-resistant Enterobacteriaceae directly from blood cultures. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Rapid Detection of Polymyxin-Resistant Enterobacteriaceae from Blood Cultures

    PubMed Central

    Jayol, Aurélie; Dubois, Véronique; Poirel, Laurent

    2016-01-01

    Enterobacterial strains resistant to polymyxins are being increasingly reported worldwide. The conventional methods for detection of colistin-resistant isolates such as broth microdilution remain time-consuming (24 to 48 h), and methods such as disc diffusion and Etest are not reliable. Recently, the rapid polymyxin NP test was developed for rapid identification of polymyxin-resistant Enterobacteriaceae. This test is based on the detection of glucose metabolism related to bacterial growth in the presence of a defined concentration of colistin (or polymyxin B). The formation of acid metabolites is evidenced by a color change of a pH indicator (red phenol) in less than 2 h. In this study, the polymyxin NP test was evaluated for detection of colistin-resistant Enterobacteriaceae directly from blood cultures. The test was performed with 73 blood culture sets (either spiked or clinical blood cultures) with various enterobacterial species. The test exhibited excellent discrimination between polymyxin-resistant and polymyxin-susceptible enterobacterial isolates, and results are obtained from blood cultures within 4 h. It is easy to perform and requires neither subculture nor a centrifugation step. This test is rapid, specific, and sensitive and allows early identification of polymyxin-resistant Enterobacteriaceae directly from blood cultures. PMID:27307457

  7. Rapid extraction methods for organics in soil

    SciTech Connect

    Alsop, G.M.; Conway, R.A.

    1983-03-01

    This article presents rapid methods for the extraction and subsequent analysis of organics in soils potentially contaminated from chemical wastes. A methylene chloride extraction, on both a micro scale in a syringe and a macro scale in a shaken flask, with subsequent gravimetric analysis are described. On wet soils, anhydrous sodium sulfate is added to bind the water before adding methylene chloride and mixing with a vortex touch mixer. A baseline procedure is the direct gravimetric determination of volatile organics corrected with a Karl Fischer determination of water. Estimates of time saved by using the rapid methods, as well as precision and accuracy data, are presented.

  8. 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.).

  9. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations.

    PubMed Central

    Michelmore, R W; Paran, I; Kesseli, R V

    1991-01-01

    We developed bulked segregant analysis as a method for rapidly identifying markers linked to any specific gene or genomic region. Two bulked DNA samples are generated from a segregating population from a single cross. Each pool, or bulk, contains individuals that are identical for a particular trait or genomic region but arbitrary at all unlinked regions. The two bulks are therefore genetically dissimilar in the selected region but seemingly heterozygous at all other regions. The two bulks can be made for any genomic region and from any segregating population. The bulks are screened for differences using restriction fragment length polymorphism probes or random amplified polymorphic DNA primers. We have used bulked segregant analysis to identify three random amplified polymorphic DNA markers in lettuce linked to a gene for resistance to downy mildew. We showed that markers can be reliably identified in a 25-centimorgan window on either side of the targeted locus. Bulked segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome. Genetic walking will be possible by multiple rounds of bulked segregation analysis; each new pair of bulks will differ at a locus identified in the previous round of analysis. This approach will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding. Images PMID:1682921

  10. A unified rapid PCR method for detection of normal and expanded trinucleotide alleles of CAG repeats in huntington chorea and CGG repeats in fragile X syndrome.

    PubMed

    Todorov, Tihomir; Todorova, Albena; Georgieva, Bilyana; Mitev, Vanyo

    2010-06-01

    We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient's genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat's number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.

  11. Evaluation of direct microplate nitrate reductase assay as a rapid method for the detection of multiple and extensively tuberculosis drug resistance.

    PubMed

    Abilleira, Fernanda; Brum, Clarice; von Groll, Andrea; da Silva, Pedro Eduardo

    2015-01-01

    Reports of Mycobacterium tuberculosis resistant to multiple drugs are increasing globally and laboratories are becoming increasingly aware of the need for drug susceptibility testing. In recent years, due to the long time required by conventional drug susceptibility testing, new approaches have been proposed for faster detection of drug resistance, such as the nitrate reductase assay, considered fast and inexpensive, making it a good diagnostic tool for low resource countries. The present study proposed a fast direct colorimetric drug susceptibility testing method in a microplate format using solid medium. The diagnostic accuracy was evaluated by comparing the proportion method with the direct nitrate reductase assay in plates. Frozen sputum samples, known to be positive, were decontaminated and processed by Petroff method. The decontaminated suspension was used to perform direct nitrate reductase assay in 7H11 medium using 1 µ g/ml rifampicin (RIF), 0.2 µg/ml isoniazid (INH), 2 µg/ml ofloxacin (OFX), 6 µg/ml kanamycin (KAN), 2 µg/ml amikacin (AMK) and 10 µg/ml capreomycin (CAP). Eighty-four samples were tested and the results for 69% of them were available within 21 days. The sensitivity and specificity compared to the proportion method, was 98.5% and 100% for INH, 98.3% and 96.2% for RIF, 91.7% and 100% for KAN, 78.8% and 97.3% for OFX, 100% and 100% for AMK and CAP, respectively. The results lead to the conclusion that direct nitrate reductase assay, in this new format, is an accurate, quick and inexpensive method to determine the susceptibility profile of M. tuberculosis and may become an alternative for countries with limited resources.

  12. Simple and rapid simultaneous profiling of minor components of honey by size exclusion chromatography (SEC) coupled to ultraviolet diode array detection (UV-DAD), combined with chemometric methods.

    PubMed

    Beretta, Giangiacomo; Fermo, Paola; Maffei Facino, Roberto

    2012-01-25

    This paper discusses the importance of profiling UV-responsive components, properly integrated with chemometric techniques, in detecting indicative parameters for quality control of honey. The minor components in honeys of different botanical and geographical origins were investigated by size SEC-UV-DAD. We diluted honey with mobile phase before injection into the chromatographic apparatus and a single chromatographic run gave a fast profile of high- (proteins and enzymes), intermediate- (e.g. terpenoid glycosides in lime tree honey) and low-molecular-weight components (secondary metabolites, e.g. kynurenic acid in chestnut honey). The analysis of a total number of 32 honey samples from different regions (Italy, Western Balkan countries, Brazil, Cameroon, Kenya) and of different botanical origins (herbal flower and arboreal flower nectars/honeydews) showed peculiar and characteristic distribution of these markers, which were basically related to their floral origin. Chemometric examination carried out using principal component analysis (PCA) and hierarchical cluster analysis (HCA) of the chromatograms (RT vs. absorption) detected four main clusters in which the groups of (i) chestnut honeys, (ii) honeys from rain forests and (iii) counterfeit/adulterated honeys were clearly separated from the main group of flower nectar honeys. The method is fast, requiring minimal sample handling, and the chromatographic data can be analyzed by multivariate statistical techniques to obtain descriptive information about the honey's quality and composition.

  13. Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.

    PubMed

    Torriani, Sandra; Gatto, Veronica; Sembeni, Silvia; Tofalo, Rosanna; Suzzi, Giovanna; Belletti, Nicoletta; Gardini, Fausto; Bover-Cid, Sara

    2008-01-01

    In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

  14. Rapid detection of antibiotic-resistant organism carriage for infection prevention.

    PubMed

    Diekema, Daniel J; Pfaller, Michael A

    2013-06-01

    Rapid detection of multidrug-resistant organism (MDRO) carriers could help reduce MDRO infections by allowing for faster institution of prevention measures. However, improving the turnaround time (TAT) of a test requires attention to more than the analytic TAT, and will only occur if postanalytic processes (test reporting and care interventions) are also rapid and efficient. Obstacles to rapid MDRO test development include complex evolving resistance mechanisms, performance directly on mixed samples (eg, nares, stool), and adaptation of new methods for routine clinical diagnostic use. Existing data to support the clinical utility of rapid detection (vs standard culture methods) are scant. For these reasons, rapid detection of MDRO carriers remains a work in progress. Future efforts should be on developing rapid tests to detect multidrug-resistant gram-negative rods, particularly those harboring β-lactamases, and on performing clinical trials to determine how best to incorporate rapid detection of MDRO carriage into healthcare-associated infection prevention efforts.

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

    USDA-ARS?s Scientific Manuscript database

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

  16. Method for detecting biomolecules

    SciTech Connect

    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

  17. 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

  18. 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)). © 2011 Taylor & Francis

  19. Short communication: A novel method using immunomagnetic separation with a fluorescent nanobeads lateral flow assay for the rapid detection of low-concentration Escherichia coli O157:H7 in raw milk.

    PubMed

    Huang, Zhen; Cui, Xi; Xie, Quan-Yuan; Liu, Dao-Feng; Lai, Wei-Hua

    2016-12-01

    Escherichia coli O157:H7 is an important serotype of enterohemorrhagic E. coli that was first identified as a human pathogen in 1982. This pathogen causes several serious diseases. In this study, immunomagnetic separation was coupled with a fluorescent nanobeads lateral flow assay to establish a sensitive and rapid detection method for Escherichia coli O157:H7 in raw milk. The pathogen was captured from raw milk by immunomagnetic separation with immunomagnetic nanobeads and then detected using a fluorescent nanobeads lateral flow assay. A fluorescent line was formed in the test line of the test strip and quantitatively detected using a fluorescent reader. Screening times, which included immunomagnetic separation and the fluorescent nanobeads lateral flow assay, were 8, 7, 6, and 5h when 1, 5, 25, and 125 cfu of E. coli O157:H7, respectively, were inoculated into 25mL of raw milk. The established method could be widely applied to the rapid onsite detection of other pathogens to ensure food safety.

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

    PubMed

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

    2017-10-01

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

  1. 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.

  2. 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.

  3. Kit Tests for Rapid Detection of Viable Bacteria and Viruses.

    DTIC Science & Technology

    1980-10-01

    Kit test Bacteria detection Kit bacteria Rapid detection Virus detection Viruses p-Iodonitrotetrazoiiu Viable bacteria Field kit Nucleic acid 21L...AD-A091 796 GEOMET TECHNOLOGIES INC GAITHERSBURG MD F/6 15/2 KIT TESTS FOR RAPID DETECTION OF VIABLE BACTERIA AND VIRUSES . IU) OCT V0 R N MOYER DAAKI...34 ®,,LEYE V TŘ CHEMICAL SYSTEMS LABORATORY CONTRACTOR REPORTo ARCSL-CR-80064 KIT TESTS FOR RAPID DETECTION OF VIABLE BACTERIA AND VIRUSES Final Report by R.H

  4. Rapid field-screening method for PCBs

    NASA Astrophysics Data System (ADS)

    Vo-Dinh, Tuan; Watts, Wendi; Miller, Gordon H.; Pal, A.; Eastwood, DeLyle; Lidberg, Russell L.

    1993-03-01

    The analysis of polychlorinated biphenyls (PCBs) generally requires selectivity and sensitivity. Even after cleanup, PCBs are usually at ultratrace levels in field samples, mixed in with other halocarbons, hydrocarbons, lipids, etc. The levels of PCBs typically found in water, soil, tissue, food, biota, and other matrices of interest are in the parts per billion (ppb) range. Most current measurement techniques for PCBs require chromatographic separations and are not practical for routine analysis. There is a strong need to have rapid and simple techniques to screen for PCBs under field conditions. The use of field screening analysis allows rapid decisions in remedial actions and reduces the need for sample preparations and time- consuming laboratory analyses. Field screening techniques also reduce the cost of clean-up operations. This paper describes a simple screening technique based on room temperature phosphorescence (RTP) and provides an overview of both this analytical procedure to detect trace levels of PCBs in environmental samples.

  5. The rapid detection of cefotaxime-resistant Enterobacteriaceae by HPLC

    PubMed Central

    Robinson, Andrew M; Medlicott, Natalie J; Ussher, James E

    2016-01-01

    Aim: Antibiotic resistance mediated by extended-spectrum β-lactamases (ESBL) and AmpC β-lactamases is widespread and increasingly common, often rendering empiric antibiotic therapy ineffective. In septicemia, delays in initiating effective antibiotic therapy are associated with worse clinical outcomes. With current phenotypic antimicrobial susceptibility testing methods, there is often a delay of 18–24 h before the susceptibility of an isolate is known. Results: Using an HPLC assay, breakdown of the third-generation cephalosporin cefotaxime by ESBL- and AmpC- β-lactamase-producing organisms could be detected within 90 min with 86.4% sensitivity and 100% specificity; sensitivity for ESBL detection was 100%. Conclusion: This assay could be readily established in any clinical laboratory with an HPLC to rapidly detect ESBL-producing Enterobacteriaceae. PMID:28116124

  6. Development and Evaluation of a Molecular Diagnostic Method for Rapid Detection of Histoplasma capsulatum var. farciminosum, the Causative Agent of Epizootic Lymphangitis, in Equine Clinical Samples

    PubMed Central

    Pinchbeck, G. L.; Loughnane, P.; Aklilu, N.; Ashine, T.; Stringer, A. P.; Gordon, L.; Marshall, M.; Christley, R. M.

    2016-01-01

    Histoplasma capsulatum var. farciminosum, the causative agent of epizootic lymphangitis (EZL), is endemic in parts of Africa. Diagnosis based on clinical signs and microscopy lacks specificity and is a barrier to further understanding this neglected disease. Here, a nested PCR method targeting the internal transcribed spacer (ITS) region of the rRNA operon was validated for application to equine clinical samples. Twenty-nine horses with signs of EZL from different climatic regions of Ethiopia were clinically examined. Blood samples and aspirates of pus from cutaneous nodules were taken, along with blood from a further 20 horses with no cutaneous EZL lesions. Among the 29 horses with suspected cases of EZL, H. capsulatum var. farciminosum was confirmed by extraction of DNA from pus and blood samples from 25 and 17 horses, respectively. Positive PCR results were also obtained with heat-inactivated pus (24 horses) and blood (23 horses) spotted onto Whatman FTA cards. Two positive results were obtained among blood samples from 20 horses that did not exhibit clinical signs of EZL. These are the first reports of the direct detection of H. capsulatum var. farciminosum in equine blood and at high frequency among horses exhibiting cutaneous lesions. The nested PCR outperformed conventional microscopic diagnosis, as characteristic yeast cells could be observed only in 14 pus samples. The presence of H. capsulatum var. farciminosum DNA was confirmed by sequencing the cloned PCR products, and while alignment of the ITS amplicons showed very little sequence variation, there was preliminary single nucleotide polymorphism-based evidence for the existence of two subgroups of H. capsulatum var. farciminosum. This molecular diagnostic method now permits investigation of the epidemiology of EZL. PMID:27707938

  7. Development and Evaluation of a Molecular Diagnostic Method for Rapid Detection of Histoplasma capsulatum var. farciminosum, the Causative Agent of Epizootic Lymphangitis, in Equine Clinical Samples.

    PubMed

    Scantlebury, C E; Pinchbeck, G L; Loughnane, P; Aklilu, N; Ashine, T; Stringer, A P; Gordon, L; Marshall, M; Christley, R M; McCarthy, A J

    2016-12-01

    Histoplasma capsulatum var. farciminosum, the causative agent of epizootic lymphangitis (EZL), is endemic in parts of Africa. Diagnosis based on clinical signs and microscopy lacks specificity and is a barrier to further understanding this neglected disease. Here, a nested PCR method targeting the internal transcribed spacer (ITS) region of the rRNA operon was validated for application to equine clinical samples. Twenty-nine horses with signs of EZL from different climatic regions of Ethiopia were clinically examined. Blood samples and aspirates of pus from cutaneous nodules were taken, along with blood from a further 20 horses with no cutaneous EZL lesions. Among the 29 horses with suspected cases of EZL, H. capsulatum var. farciminosum was confirmed by extraction of DNA from pus and blood samples from 25 and 17 horses, respectively. Positive PCR results were also obtained with heat-inactivated pus (24 horses) and blood (23 horses) spotted onto Whatman FTA cards. Two positive results were obtained among blood samples from 20 horses that did not exhibit clinical signs of EZL. These are the first reports of the direct detection of H. capsulatum var. farciminosum in equine blood and at high frequency among horses exhibiting cutaneous lesions. The nested PCR outperformed conventional microscopic diagnosis, as characteristic yeast cells could be observed only in 14 pus samples. The presence of H. capsulatum var. farciminosum DNA was confirmed by sequencing the cloned PCR products, and while alignment of the ITS amplicons showed very little sequence variation, there was preliminary single nucleotide polymorphism-based evidence for the existence of two subgroups of H. capsulatum var. farciminosum This molecular diagnostic method now permits investigation of the epidemiology of EZL.

  8. [Manipulation methods of slow-rapid reinforcing-reducing method].

    PubMed

    Zhao, Xi-Xin; Ran, Peng-Fei; Lü, Xiao-Rui; Wang, Xue-Xia

    2008-06-01

    After consulting literature, following the original meaning of the paper about slow-rapid reinforcing-reducing method in Huangdi's Internal Classic, in combination with explanation of later ages, and comprehension and clinical experience of the authors, it is put forward that the slow-rapid reinforcing-reducing method in Huangdi's Internal Classic is not single manipulation method, but it is a guiding principle for reinforcing-reducing manipulation, it includes many manipulation methods and they were listed, and all of the reinforcing-reducing methods of later ages are developed from these. In the teaching material Acupuncture and Moxibustion Sciences they are included in single reinforcing-reducing method, reducing extension and intension of this definition. The relative description in the teaching material only is slow-rapid reinforcing-reducing method of narrow sense, but the manipulations can be divided into qi-carrying manipulating needle type and three-one pushing-pulling type.

  9. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. 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

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

    PubMed

    Bao, Hongmei; Feng, Xiaoxiao; Ma, Yong; Shi, Jianzhong; Zhao, Yuhui; Gu, Linlin; Wang, Xiurong; Chen, Hualan

    2015-12-01

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Rapid Detection of Convallatoxin Using Five Digoxin Immunoassays

    PubMed Central

    Fink, Susan L.; Robey, Thomas E.; Tarabar, Asim F.; Hodsdon, Michael E.

    2014-01-01

    CONTEXT Cardiac glycosides of plant origin are implicated in toxic ingestions that may result in hospitalization and are potentially lethal. The utility of commonly available digoxin serum assays for detecting foxglove and oleander ingestion has been demonstrated, but no studies have evaluated the structurally similar convallatoxin found in Convallaria majalis (lily of the valley) for rapid laboratory screening, nor has digoxin immune Fab been tested as an antidote for this ingestion. OBJECTIVE We aimed to (1) evaluate multiple digoxin assays for cross-reactivity to convallatoxin, (2) identify whether convallatoxin could be detected in vivo at clinically significant doses and (3) determine whether digoxin immune Fab could be an effective antidote to convallatoxin. MATERIALS AND METHODS Cross-reactivities of purified convallatoxin and oleandrin with five common digoxin immunoassays were determined. Serum from mice challenged with convallatoxin was tested for apparent digoxin levels. Binding of convallatoxin to digoxin immune Fab was determined in vitro. RESULTS Both convallatoxin and oleandrin were detectable by a panel of commonly used digoxin immunoassays, but cross-reactivity was variable between individual assays. We observed measurable apparent digoxin levels in serum of convallatoxin intoxicated mice at sublethal doses. Convallatoxin demonstrated no binding by digoxin immune Fab. CONCLUSION Multiple digoxin immunoassays detect botanical cardiac glycosides including convallatoxin and thus may be useful for rapid determination of severe exposures, but neutralization of convallatoxin by digoxin immune Fab is unlikely to provide therapeutic benefit. PMID:24980812

  13. 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.

  14. Rapid and quantitative detection of hepatitis B virus.

    PubMed

    Liu, Yue-Ping; Yao, Chun-Yan

    2015-11-14

    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.

  15. Rapid Adenovirus typing method for species identification.

    PubMed

    Rayne, Fabienne; Wittkop, Linda; Bader, Clément; Kassab, Somar; Tumiotto, Camille; Berciaud, Sylvie; Wodrich, Harald; Lafon, Marie-Edith

    2017-11-01

    Adenoviruses are characterized by a large variability, reflected by their classification in species A to G. Certain species, eg A and C, could be associated with increased clinical severity, both in immunocompetent and immunocompromised hosts suggesting that in some instances species identification provides clinically relevant information. Here we designed a novel "pVI rapid typing method" to obtain quick, simple and cost effective species assignment for Adenoviruses, thanks to combined fusion temperature (Tm) and amplicon size analysis. Rapid typing results were compared to Sanger sequencing in the hexon gene for 140 Adenovirus-positive clinical samples included in the Typadeno study. Species A and C could be identified with a 100% positive predictive value, thus confirming the value of this simple typing method. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Validation of Rapid Radiochemical Method for Californium ...

    EPA Pesticide Factsheets

    Technical Brief In the event of a radiological/nuclear contamination event, the response community would need tools and methodologies to rapidly assess the nature and the extent of contamination. To characterize a radiologically contaminated outdoor area and to inform risk assessment, large numbers of environmental samples would be collected and analyzed over a short period of time. To address the challenge of quickly providing analytical results to the field, the U.S. EPA developed a robust analytical method. This method allows response officials to characterize contaminated areas and to assess the effectiveness of remediation efforts, both rapidly and accurately, in the intermediate and late phases of environmental cleanup. Improvement in sample processing and analysis leads to increased laboratory capacity to handle the analysis of a large number of samples following the intentional or unintentional release of a radiological/nuclear contaminant.

  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

    USDA-ARS?s Scientific Manuscript database

    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. 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.

  19. Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis

    PubMed Central

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T.

    2012-01-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. PMID:22518861

  20. Recombinase polymerase amplification assay for rapid detection of Francisella tularensis.

    PubMed

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T; Weidmann, Manfred

    2012-07-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

  1. A multiplex real-time PCR-platform integrated into automated extraction method for the rapid detection and measurement of oncogenic HPV type-specific viral DNA load from cervical samples.

    PubMed

    Broccolo, Francesco

    2014-01-01

    The persistent infection with most frequent high-risk (HR)-HPV types (HPV-16, -18, -31, -33, -45, -52, and -58) is considered to be the true precursor of neoplastic progression. HR-HPV detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer, although also the HR-HPV DNA load is considered an ancillary marker for persistent HPV infection. Here, it is described an in-house multiplex quantitative real-time PCR (qPCR)-based typing system for the rapid detection and quantitation of the most common HR-HPV genotypes from cervical cytology screening tests. First, a separate qPCR assay to quantify a single-copy gene is recommended prior to screening (prescreening assay) to verify the adequate cellularity of the sample and the quality of DNA extracted and to normalize the HPV copy number per genomic DNA equivalent in the sample. Subsequently, to minimize the number of reactions, two multiplex qPCR assays (first line screening) are performed to detect and quantify HPV-16, -18, -31, -33, -45, -52, and -58 (HPV-18 and -45 are measured together by single-fluorophore). In addition, a multiplex qPCR assay specific for HPV-18 and HPV-45 is also available to type precisely the samples found to be positive for one of the two strains. Finally, two nucleic acid extraction methods are proposed by using a 96-well plate format: one manual method (supported by centrifuge or by vacuum) and one automated method integrated into a robotic liquid handler workstation to minimize material and hands-on time. In conclusion, this system provides a reliable high-throughput method for the rapid detection and quantitation of HR-HPV DNA load in cervical samples.

  2. Paper-based ELISA to rapidly detect Escherichia coli.

    PubMed

    Shih, Cheng-Min; Chang, Chia-Ling; Hsu, Min-Yen; Lin, Jyun-Yu; Kuan, Chen-Meng; Wang, Hsi-Kai; Huang, Chun-Te; Chung, Mu-Chi; Huang, Kui-Chou; Hsu, Cheng-En; Wang, Chun-Yuan; Shen, Ying-Cheng; Cheng, Chao-Min

    2015-12-01

    Escherichia coli is a generic indicator of fecal contamination, and certain serotypes cause food- and water-borne illness such as O157:H7. In the clinic, detection of bacteriuria, which is often due to E. coli, is critical before certain surgical procedures or in cases of nosocomial infection to prevent further adverse events such as postoperative infection or sepsis. In low- and middle-income countries, where insufficient equipment and facilities preclude modern methods of detection, a simple, low-cost diagnostic device to detect E. coli in water and in the clinic will have significant impact. We have developed a simple paper-based colorimetric platform to detect E. coli contamination in 5h. On this platform, the mean color intensity for samples with 10(5)cells/mL is 0.118±0.002 (n=4), and 0.0145±0.003 (P<0.01⁎⁎) for uncontaminated samples. This technique is less time-consuming, easier to perform, and less expensive than conventional methods. Thus, paper-based ELISA is an innovative point-of-care diagnostic tool to rapidly detect E. coli, and possibly other pathogens when customized as appropriate, especially in areas that lack advanced clinical equipment.

  3. 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.

  4. Rapid fluorophosphate nerve agent detection with lanthanides.

    PubMed

    Menzel, E Roland; Menzel, Laird W; Schwierking, Jake R

    2005-08-15

    We explore the detection of vapors of diisopropylfluorophosphate, a model compound for nerve agents such as Sarin, by means of photoluminescence quenching of filter paper impregnated with sensitized complexes of lanthanides, involving thenoyltrifluoroacetone and 1,10-phenanthroline as sensitizing ligands. We find that the presence of the fluorophosphate vapor is detectable in as little as 2s, by simple visual observation under illumination with a hand-held low intensity ultraviolet lamp.

  5. A rapid method to determine bacterial contamination on hatching eggs. 3. Use of commercial DNA probe kits for detection of specific pathogens after six hours of incubation.

    PubMed

    Pienaar, A C; Coetzee, L; Bragg, R R

    1995-03-01

    The usefulness of commercially available DNA probe kits for the detection of Escherichia coli and Salmonella spp. after only 6 h of incubation, was determined. It was established that the commercially available probe kits used could detect E. coli at initial levels of approximately 4.5 x 10(2) colony-forming units (cfu) per ml after only 6 h of incubation in nutrient broth (NB). Initial bacterial levels as low as 4.5 x 10(1) cfu/ml could be detected when the NB was incubated for 18 h. Salmonella Enteritidis, at initial levels of 2.86 x 10(2) cfu/ml could be detected after 6 h of incubation at 37 degrees C in NB, while initial levels as low as 2.86 x 10(-1) cfu/ml could be detected after 18 h at 37 degrees C in both NB and selected media, as specified by the manufacturers of the probe kits. Commercially available DNA probe kits can therefore be used to detect specific pathogens on the surface of hatching eggs and these probes can be used in conjunction with an egg-washing system, which is used to determine total bacterial contamination, although a longer incubation period greatly improves the sensitivity of these tests.

  6. 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.

  7. 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.

  8. 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.

  9. Waveguide disturbance detection method

    DOEpatents

    Korneev, Valeri A.; Nihei, Kurt T.; Myer, Larry R.

    2000-01-01

    A method for detection of a disturbance in a waveguide comprising transmitting a wavefield having symmetric and antisymmetric components from a horizontally and/or vertically polarized source and/or pressure source disposed symmetrically with respect to the longitudinal central axis of the waveguide at one end of the waveguide, recording the horizontal and/or vertical component or a pressure of the wavefield with a vertical array of receivers disposed at the opposite end of the waveguide, separating the wavenumber transform of the wavefield into the symmetric and antisymmetric components, integrating the symmetric and antisymmetric components over a broad frequency range, and comparing the magnitude of the symmetric components and the antisymmetric components to an expected magnitude for the symmetric components and the antisymmetric components for a waveguide of uniform thickness and properties thereby determining whether or not a disturbance is present inside the waveguide.

  10. Monoclonal antibody technologies and rapid detection assays

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. 16S rRNA Gene Sequence Analysis of Photobacterium damselae and Nested PCR Method for Rapid Detection of the Causative Agent of Fish Pasteurellosis

    PubMed Central

    Osorio, Carlos R.; Collins, Matthew D.; Toranzo, Alicia E.; Barja, Juan L.; Romalde, Jesús L.

    1999-01-01

    The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis. PMID:10388687

  14. A rapid loop-mediated isothermal amplification (LAMP) method for detection of the macrolide-streptogramin type B resistance gene msrA in Staphylococcus aureus.

    PubMed

    Mu, Xiao-Qin; Liu, Bin-Bin; Hui, Ephraim; Huang, William; Yao, Li-Chen; Duo, Li-Bo; Sun, Wen-Ying; Li, Gui-Qiu; Wang, Fu-Xiang; Liu, Shu-Lin

    2016-12-01

    Macrolide-streptogramin type B resistance (the MSB phenotype) is a multidrug resistance phenotype in Staphylococcus aureus conferred by the resistance gene msrA. However, bacteria having the MSB phenotype are susceptible to lincosamides and 16-membered ring macrolides, which makes profiling resistance genes necessary and urgent for timely and appropriate use of antimicrobials. In this study, the loop-mediated isothermal amplification (LAMP) assay was optimized for prompt detection of the msrA gene. msrA gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database and primers were designed using the LAMP primer designing software PrimerExplorer v.4, which together recognize seven distinct regions of the msrA gene. The specific LAMP primer set designed in this study could amplify the msrA gene within 25min at an isothermal temperature of 62°C. More importantly, the msrA gene could be detected at a sensitivity as low as 100pg. Furthermore, this optimized LAMP assay provided swift detection of the msrA gene even directly from human specimens. In conclusion, this assay may have great clinical application potential for detection of the msrA gene.

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

    PubMed

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

    2015-04-01

    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.

  16. A rapid micro-assay method for gelatinolytic activity using tritium-labeled heat-denatured polymeric collagen as a substrate and its application to the detection of enzymes involved in collagen metabolism.

    PubMed

    Sunada, H; Nagai, Y

    1980-06-01

    A rapid micro-assay method for gelatinolytic activitiy has been developed using 3H-labeled heat-denatured polymeric collagen (gelatin) as a substrate to investigate enzymes involved in the post-collagenase catabolism of collagen. The method is based on the incubation of gelatin with enzyme followed by determination of the enzyme digestion products soluble in 67% dioxane. It is sensitive enough to detect microgram levels of gelatin fragments, and can be employed over wide ranges of pH and ionic strength. By applying the method to an embryonic chick skin culture system, three gelatinolytic enzyme fractions which showed high, limited and no caseinolytic activities were demonstrated to be separable by gel chromatography.

  17. 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.

  18. Rapid Column Extraction method for SoilRapid Column Extraction method for Soil

    SciTech Connect

    Maxwell, Sherrod, L. III; Culligan, Brian K.

    2005-11-07

    The analysis of actinides in environmental soil and sediment samples is very important for environmental monitoring as well as for emergency preparedness. A new, rapid actinide separation method has been developed and implemented that provides total dissolution of large soil samples, high chemical recoveries and effective removal of matrix interferences. This method uses stacked TEVA Resin{reg_sign}, TRU Resin{reg_sign} and DGA-Resin{reg_sign} cartridges from Eichrom Technologies (Darien, IL, USA) that allows the rapid separation of plutonium (Pu) neptunium (Np), uranium (U), americium (Am), and curium (Cm) using a single multi-stage column combined with alpha spectrometry. The method combines a rapid fusion step for total dissolution to dissolve refractory analytes and matrix removal using cerium fluoride precipitation to remove the difficult soil matrix. By using vacuum box cartridge technology with rapid flow rates, sample preparation time is minimized.

  19. 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.

  20. 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.

  1. Rapid Detection of Human Pathogenic Orthobunyaviruses

    PubMed Central

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

    2003-01-01

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

  2. A new rapid colorimetric detection method of Al³⁺ with high sensitivity and excellent selectivity based on a new mechanism of aggregation of smaller etched silver nanoparticles.

    PubMed

    Yang, Ningning; Gao, Yuexia; Zhang, Yujie; Shen, Zheyu; Wu, Aiguo

    2014-05-01

    As a pathogenic factor of the Alzheimer׳s disease, aluminum has been associated with the damage of the central nervous system in humans. In this study, we propose a new facile and rapid colorimetric detection method of Al(3+) with excellent selectivity and high sensitivity based on silver nanoparticles (AgNPs) stabilized by reduced glutathione (GSH) in the presence of l-cysteine (Cys). The new mechanism of our Al(3+) detection system based on GSH-AgNPs, i.e., aggregation of smaller etched GSH-AgNPs, are confirmed by TEM, EDS and DLS. The aggregation of smaller etched GSH-AgNPs results in obvious color change of the nanoparticle dispersion from yellow to reddish brown, and red shift and intensity decrease of the surface plasmon resonance (SPR) absorption. The GSH concentration, Cys concentration and pH value of the GSH-AgNPRs-based detection system are respectively optimized to be 10.0 mM, 50.0 mM and 6.0 according to the sensing effect of Al(3+). At the optimized conditions, the selectivity of the GSH-AgNPs detection system for Al(3+) is excellent compared with other ions including K(+), Mg(2+), Fe(3+), Co(2+), Mn(2+), Zn(2+), Cd(2+), Pb(2+), Ca(2+), Ba(2+), Cu(2+), Cr(3+), Hg(2+), Ni(2+), [Formula: see text] , [Formula: see text] , [Formula: see text] , [Formula: see text] and [Formula: see text] . Furthermore, this detection system is very sensitive for Al(3+). The limit of detection (LOD) is 1.2 µM by the naked eyes and 0.16 µM by UV-vis spectra, which are both much lower than the national drinking water standards (7.4 µM). Furthermore, the UV-vis detection offers a good linear detection range from 0.4 to 4.0 µM (R(2)=0.9924), which indicates that our developed detection system can also be used for the quantitative analysis of Al(3+). The detection results of real water samples indicate that this method can be used for real water detection. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. 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

  4. Sex Differences in the Rapid Detection of Emotional Facial Expressions

    PubMed Central

    Sawada, Reiko; Sato, Wataru; Kochiyama, Takanori; Uono, Shota; Kubota, Yasutaka; Yoshimura, Sayaka; Toichi, Motomi

    2014-01-01

    Background Previous studies have shown that females and males differ in the processing of emotional facial expressions including the recognition of emotion, and that emotional facial expressions are detected more rapidly than are neutral expressions. However, whether the sexes differ in the rapid detection of emotional facial expressions remains unclear. Methodology/Principal Findings We measured reaction times (RTs) during a visual search task in which 44 females and 46 males detected normal facial expressions of anger and happiness or their anti-expressions within crowds of neutral expressions. Anti-expressions expressed neutral emotions with visual changes quantitatively comparable to normal expressions. We also obtained subjective emotional ratings in response to the facial expression stimuli. RT results showed that both females and males detected normal expressions more rapidly than anti-expressions and normal-angry expressions more rapidly than normal-happy expressions. However, females and males showed different patterns in their subjective ratings in response to the facial expressions. Furthermore, sex differences were found in the relationships between subjective ratings and RTs. High arousal was more strongly associated with rapid detection of facial expressions in females, whereas negatively valenced feelings were more clearly associated with the rapid detection of facial expressions in males. Conclusion Our data suggest that females and males differ in their subjective emotional reactions to facial expressions and in the emotional processes that modulate the detection of facial expressions. PMID:24728084

  5. A rapid screening method for the detection of mutations in the RET proto-oncogene in multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma families

    SciTech Connect

    Marsh, D.J.; Andrew, S.; Richardson, A.L. |

    1994-09-15

    Multiple endocrine neoplasia type 2A (MEN2A) and familial medullary thyroid carcinoma (FMTC) are autosomal dominant inherited cancer syndromes with incomplete penetrance. Following the identification of mutations in the RET proto-oncogene that segregate with the disease phenotype in MEN2A, MEN2B, and FMTC, genetic screening of individuals with mutations in RET may be performed. The authors have employed restriction endonuclease digestion of polymerase chain reaction products as an alternative to sequence analysis for rapid identification of mutant gene carriers in families in which MEN2A and RMTC are segregating. Twenty-one Australasian MEN2A and FMTC families have been screened for mutations in a cysteine-rich region of the RET proto-oncogene. Seven independent mutations were identified in key individuals in 16 of these families. The authors have identified a mutation in codon 620, 2053 T {r_arrow}C (Cys620Arg), and two mutations in codon 634 of exon 11 of RET, 2095 T {r_arrow} C (Cys634Arg) and 2096 G {r_arrow} A (Cys634Tyr), all three of which were present in both MEN2A and FMTC families. 7 refs., 1 fig., 1 tab.

  6. A new rapid and non-destructive method to detect tephra layers and cryptotephras: applying to the first distal tephrostratigraphic record of the Chaîne des Puys volcanic field (France).

    NASA Astrophysics Data System (ADS)

    Jouannic, Gwénolé; Walter-Simonnet, Anne-Véronique; Bossuet, Gilles; Delabrousse, Eric; Cubizolle, Hervé

    2014-05-01

    Tephrostratigraphy has been considerably developed for 30 years, mainly in palaeo-environmental studies. In such studies, distal tephra layers are important chronological markers, but they are also tools to establish or specify record of past eruptions of a volcanic field. Nowadays, development of effective rapid methods to detect tephra layers in sedimentary records of various compositions is a challenge. Many classic methods for detection of tephra layers, like regular sampling or magnetic susceptibility measurements, have shown their limits. Regular sampling takes a long time, and finding tephra layers remains uncertain. Moreover, magnetic susceptibility maesurements, although it is a non-destructive method, is ineffective when tephra layers are made of volcanic glass shards with differentiated magma composition. X-ray fluorescence (XRF) is also a non-destructive method but it takes a very long time to analyze a core with sufficient high resolution, and measurements only concern the surface of the sediment. We propose a new method allows detection of tephra layers with, for the first time, a 3D resolution: the Computed Tomography Scan (CT- Scan). This method, regularly used in medicine, allows there to obtain pictures of materials density on 3D with inframillimetric measurement ranges. Then, it is possible to detect tephras, cryptotephras (invisible by naked eye), reworked tephra layers even when tephra layers don't outcrop at the surface of the sediment (and are therefore undetectable by usual methods like XRF and magnetic susceptibility). This method has been tried out on tephras sedimented in different types of sediments (silicated, carbonated and organic matter). Our results show that this method is very efficient for peaty environment. Used on coring carried out in Forez Mountains (French Massif Central), CT-Scan allows to detect more tephra layers than usual methods (XRF and magnetic susceptibility). Results presented here allow to build the first

  7. Rapid Detection of Herpes Viruses for Clinical Applications

    NASA Technical Reports Server (NTRS)

    Pierson, Duane; Mehta, Satish

    2013-01-01

    There are eight herpes viruses that infect humans, causing a wide range of diseases resulting in considerable morbidity and associated costs. Varicella zoster virus (VZV) is a human herpes virus that causes chickenpox in children and shingles in adults. Approximately 1,000,000 new cases of shingles occur each year; post-herpetic neuralgia (PHN) follows shingles in 100,000 to 200,000 people annually. PHN is characterized by debilitating, nearly unbearable pain for weeks, months, and even years. The onset of shingles is characterized by pain, followed by the zoster rash, leading to blisters and severe pain. The problem is that in the early stages, shingles can be difficult to diagnose; chickenpox in adults can be equally difficult to diagnose. As a result, both diseases can be misdiagnosed (false positive/negative). A molecular assay has been adapted for use in diagnosing VZV diseases. The polymerase chain reaction (PCR) assay is a non-invasive, rapid, sensitive, and highly specific method for VZV DNA detection. It provides unequivocal results and can effectively end misdiagnoses. This is an approximately two-hour assay that allows unequivocal diagnosis and rapid antiviral drug intervention. It has been demonstrated that rapid intervention can prevent full development of the disease, resulting in reduced likelihood of PHN. The technology was extended to shingles patients and demonstrated that VZV is shed in saliva and blood of all shingles patients. The amount of VZV in saliva parallels the medical outcome.

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

    EPA Science Inventory

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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    EPA Science Inventory

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

  11. Simple rapid method for gene transfer

    SciTech Connect

    Cockburn, A.F.; Meier, H.

    1990-01-30

    The object of the present invention is to provide methods for gene transfer that reduce or eliminate cellular pretreatment steps, e.g., the removal of cell wall by chemical or enzymatic methods, is rapid and can be practiced without the need of additional expensive equipment. Cells, embryos or tissues selected for genetic manipulation are suspended in an Eppendorf tube in an aliquot of the desired genetic material to be transferred to which the resulting mixture is added and is agitated by vortexing from about 30 to about 90 seconds. The cells, embryos or tissue are sedimented and the DNA supernatant removed. After sedimentation, the injected material is resuspended in or on a growth medium to assay for expression.

  12. Rapid Column Extraction Methods for Urine

    SciTech Connect

    Maxwell, S.L. III

    2000-06-09

    A new fecal analysis method that dissolves plutonium oxide was developed at the Westinghouse Savannah River Site. Diphonix Resin (Eichrom Industries), is used to pre-concentrate the actinides from digested fecal samples. A rapid microwave digestion technique is used to remove the actinides from the Diphonix Resin, which effectively extracts plutonium and americium from acidic solutions containing hydrofluoric acid. After resin digestion, the plutonium and americium are recovered in a small volume of nitric acid that is loaded onto small extraction chromatography columns, TEVA Resin and TRU Resin (Eichrom Industries). The method enables complete dissolution of plutonium oxide and provides high recovery of plutonium and americium with good removal of thorium isotopes such as thorium-228.

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  14. Discovering novel interactions at the nuclear pore complex using bead halo: a rapid method for detecting molecular interactions of high and low affinity at equilibrium.

    PubMed

    Patel, Samir S; Rexach, Michael F

    2008-01-01

    A highly sensitive, equilibrium-based binding assay termed "Bead Halo" was used here to identify and characterize interactions involving components of the nucleocytoplasmic transport machinery in eukaryotes. Bead Halo uncovered novel interactions between the importin Kap95 and the nucleoporins (nups) Nic96, Pom34, Gle1, Ndc1, Nup84, and Seh1, which likely occur during nuclear pore complex biogenesis. Bead Halo was also used to characterize the molecular determinants for binding between Kap95 and the family of nups that feature multiple phenylalanine-glycine motifs (FG nups). Binding was sensitive to the number of FG motifs present and to amino acid (AA) residues immediately flanking the FG motifs. Also, binding was reduced but not abolished when phenylalanine residues in all FG motifs were replaced by tyrosine or tryptophan. These results suggest flexibility in the binding pockets of Kap95 and synergism in binding FG motifs. The hypothesis that Nup53 and Nup59 bind directly to membranes through a C-terminal amphipathic alpha helix and to DNA via an RNA recognition motif domain was also tested and validated using Bead Halo. The results support a role for these nups in nuclear pore membrane biogenesis and in gene expression. Finally, Bead Halo detected binding of the nups Gle1, Nup60, and Nsp1 to phospholipid bilayers. This may reflect the known interaction between Gle1 and phosphoinositides and suggests similar interactions for Nup60 and Nsp1. As the Bead Halo assay detected molecular interactions in cell lysates, as well as between purified components, it can be adapted for large-scale proteomic studies using automated robotics and microscopy.

  15. Rapid nested-PCR for tyrosinase gene detection on chip.

    PubMed

    Sciancalepore, Anna Giovanna; Polini, Alessandro; Mele, Elisa; Girardo, Salvatore; Cingolani, Roberto; Pisignano, Dario

    2011-01-15

    The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4°C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16°C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.

  16. Rapid turbidimetric detection of milk powder adulteration with plant proteins.

    PubMed

    Scholl, Peter F; Farris, Samantha M; Mossoba, Magdi M

    2014-02-19

    Development of assays to screen milk for economically motivated adulteration with foreign proteins has been stalled since 2008 due to strong international reactions to the melamine poisoning incident in China and the surveillance emphasis placed on low molecular weight nitrogen-rich adulterants. New screening assays are still needed to detect high molecular weight foreign protein adulterants and characterize this understudied potential risk. A rapid turbidimetric method was developed to screen milk powder for adulteration with insoluble plant proteins. Milk powder samples spiked with 0.03-3% by weight of soy, pea, rice, and wheat protein isolates were extracted in 96-well plates, and resuspended pellet solution absorbance was measured. Limits of detection ranged from 100 to 200 μg, or 0.1-0.2% of the sample weight, and adulterant pellets were visually apparent even at ∼0.1%. Extraction recoveries ranged from 25 to 100%. Assay sensitivity and simplicity indicate that it would be ideally suitable to rapidly screen milk samples in resource poor environments where adulteration with plant protein is suspected.

  17. Rapid Detection of Microorganisms--State of Art and Future Directions

    NASA Astrophysics Data System (ADS)

    Hong, George

    2008-03-01

    For the last several decades, nutrient-based culture growth methods have been accepted as the standard for microorganism detection and identification. However, since the discovery of nucleic acids and molecular breakthrough technologies such as restriction enzymes and polymerase chain reactions, the detection and identification of microorganisms have advanced to culture-independent methods that fall under the category of rapid microbial detections. Here, we present an overview of major rapid microbial detection technologies. These technologies will include both amplification and non-amplification based methods for the detection and identification of target microorganisms. The technologies described can be applied to detecting a wide variety of microorganisms, including bacteria, viruses, mycoplasma, and fungi and have the potential sensitivity to detect a single microorganism. Also in this presentation, we will present examples of real-life applications as well as future challenges for the advancement of the field of rapid microbiology.

  18. Rapid video shot detective based on the dichotomy

    NASA Astrophysics Data System (ADS)

    Zhu, Xing-Hui; Guo, Zong-Ming

    2009-10-01

    Video shot boundary detection is a fundamental step for the organization of large video data. The classical VSB detection is basically a sequential frame to compute frame-by-frame, however this approach is computationally very expensive for large databases .In this work we propose a dichotomy approach for video shot boundary detection. The proposed technique can improve the performance of the algorithm and reduce the calculation. Our experimental results show that the proposed algorithm produces faster detection rapid.

  19. Rapid method for simultaneous determination of flavonoid, saponins and polyacetylenes in folium ginseng and radix ginseng by pressurized liquid extraction and high-performance liquid chromatography coupled with diode array detection and mass spectrometry.

    PubMed

    Qian, Z M; Lu, J; Gao, Q P; Li, S P

    2009-05-01

    A rapid pressurized liquid extraction (PLE) and high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) method for the simultaneous determination of one flavonoid (panasenoside), nine saponins (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) and two polyacetylenes (panaxydol and panaxynol) in folium ginseng and radix ginseng was developed. A Prevail C(18) rocket column (33 mm x 7 mm, 3.0 microm) and gradient elution were used during the analysis. Flavonoid was quantified at 355 nm, and saponins and polyacetylenes were determined at 203 nm. The chromatographic peaks of 12 investigated compounds in samples were unambiguously identified by compared their UV spectra and/or MS data with the related reference compounds. All calibration curves showed good linearity (r>0.999) within the test ranges. The intra- and inter-day variations for 12 analytes were less than 1.17% and 2.17%, respectively. The developed method was successfully applied to determine the investigated compounds in 10 samples of radix ginseng and folium ginseng, respectively. The result showed that PLE combined with rocket column HPLC analysis could provide a rapid method for analysis of compounds in traditional Chinese medicines (TCMs), which is helpful to comprehensive evaluation of quality of radix ginseng and folium ginseng.

  20. Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method.

    PubMed

    DeCory, Thomas R; Durst, Richard A; Zimmerman, Scott J; Garringer, Linda A; Paluca, Gary; DeCory, Heleen H; Montagna, Richard A

    2005-04-01

    The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.

  1. Enhanced Microbial Detection Capabilities by a Rapid Portable Instrument

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

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

  2. Enhanced Microbial Detection Capabilities by a Rapid Portable Instrument

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

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

  3. Rapid and cost-effective method for the detection of the c.533G>A mutation in the HEXA gene.

    PubMed

    Ribeiro, Diogo; Duarte, Ana Joana; Amaral, Olga

    2011-03-01

    Tay-Sachs disease is a rare autosomal recessive neurodegenerative disorder that results from mutations in the HEXA gene, leading to β-hexosaminidase A (HexA) α subunit deficiency. An unusual variant of Tay-Sachs disease is known as the B1 variant. Previous studies indicated that, in northern Portugal, this is not only the most common variant but also one of the most prevalent lysosomal storage diseases. Additionally, this variant might also show a higher prevalence in populations of Portuguese and Spanish ancestry. A single mutation is invariably present in at least one of the alleles of B1 variant patients, HEXA mutation c.533G >A. To implement a method for c.533G >A testing in individuals and populations, we have optimized two distinct mutation analysis techniques, one based on restriction fragment length polymorphism analysis and the other based on allelic discrimination. We present the comparison of both methods and their advantages. Mutation screening by allelic discrimination proved to be particularly useful for the studying of large samples of individuals. It is time saving and highly reproducible, and under the conditions used, its cost is lower than the cost of polymerase chain reaction-based restriction fragment length polymorphism analysis.

  4. Rapid detection of D-Dimers with mLabs® whole blood method for venous thromboembolism exclusion. Comparison with Vidas® D-Dimers assay.

    PubMed

    Gerotziafas, Grigoris T; Ray, Patrick; Gkalea, Vasiliki; Benzarti, Ahlem; Khaterchi, Amir; Cast, Claire; Pernet, Julie; Lefkou, Eleftheria; Elalamy, Ismail

    2016-12-01

    Easy to use point of care assays for D-Dimers measurement in whole blood from patients with clinical suspicion of venous thromboembolism (VTE) will facilitate the diagnostic strategy in the Emergency Department (ED) setting. We prospectively evaluated the diagnostic performance of the point-of-care mLabs® Whole Blood D-Dimers test and we compared it with the Vidas® D-Dimers assay. As part of the diagnostic algorithm applied in patients with clinical suspicion of VTE, the VIDAS® D-Dimers Test was prescribed by the emergency physician in charge. The mLabs® Whole Blood D-Dimers Test was used on the same samples. All patients had undergone exploration with the recommended imaging techniques for VTE diagnosis. Both assays were performed, on 99 emergency patients (mean age was 65 years) with clinical suspicion of VTE. In 3% of patients, VTE was documented with a reference imaging technique. The Bland and Altman test showed significant agreement between the two methods. Both assays showed equal sensitivity and negative predictive value for VTE. The mLabs whole blood assay is a promising point of care method for measurement of D-Dimers and exclusion of VTE diagnosis in the emergency setting which should be validated in a larger prospective study.

  5. Fetal magnetocardiography: Methods for rapid data reduction

    NASA Astrophysics Data System (ADS)

    Mosher, John C.; Flynn, Edward R.; Quinn, A.; Weir, A.; Shahani, U.; Bain, R. J. P.; Maas, P.; Donaldson, G. B.

    1997-03-01

    Fetal magnetocardigraphy (fMCG) provides a unique method for noninvasive observations of the fetal heart. Electrical currents generated by excitable tissues within the fetal heart yield measurable external magnetic fields. Measurements are performed with superconducting quantum interference devices inductively coupled to magnetometer or gradiometer coils, and the resulting signals are converted to digital form in the data acquisition system. The measured fields are usually contaminated by fetal and maternal movements (usually respiration), other physiological fields such as skeletal muscle contraction, the maternal cardiac signal, and environmental electromagnetic fields. Sensitivity to relatively distant sources, both physiological and environmental, is substantially reduced by the use of magnetic gradiometers. Other contaminants may be removed by proper signal conditioning which may be automatically applied using "black box" algorithms that are transparent to the user and highly efficient. These procedures can rapidly reduce the complex signal plus noise waveforms to the desired fMCG with minimal operator interference.

  6. A simple, sensitive, and rapid method for the determination of cotinine in urine by high-performance liquid chromatography with UV detection.

    PubMed

    Rabbaa-Khabbaz, Lydia; Abi Daoud, Rita; Karam-Sarkis, Dolla

    2006-10-01

    Cotinine levels in biological fluids are a reliable indicator of the presence of nicotine. In this paper, a simple and sensitive high-performance liquid chromatography (HPLC) procedure for the determination of cotinine in urine following liquid-liquid extraction with dichloromethane in an alkaline medium is described. Calibration curves show linearity over the 50 to 3000 ng/mL range with low intra- and interday variability as well as good selectivity and specificity. No solid-phase extraction is performed because the liquid dichloromethane extraction step yields excellent results. This method is a good alternative for routine analysis of urinary cotinine in laboratories where gas chromatography or HPLC-mass spectrometry is not available.

  7. A novel strategy for rapid detection of NT-proBNP

    NASA Astrophysics Data System (ADS)

    Cui, Qiyao; Sun, Honghao; Zhu, Hui

    2017-09-01

    In order to establish a simple, rapid, sensitive, and specific quantitative assay to detect the biomarkers of heart failure, in this study, biotin-streptavidin technology was employed with fluorescence immunochromatographic assay to detect the concentration of the biomarkers in serum, and this method was applied to detect NT-proBNP, which is valuable for diagnostic evaluation of heart failure.

  8. Rapid detection of Salmonella in bovine lymph nodes using a commercial real-time PCR system

    USDA-ARS?s Scientific Manuscript database

    Rapid Salmonella detection is needed to help prevent the distribution of contaminated food products. Using traditional culture methods, Salmonella detection can take up to 3-5 days. Using an improved protocol and a commercial real-time PCR system, we have shortened the detection time to under 24 h...

  9. Rapid Detection of Biological and Chemical Threat Agents Using Physical Chemistry, Active Detection, and Computational Analysis

    SciTech Connect

    Chung, Myung; Dong, Li; Fu, Rong; Liotta, Lance; Narayanan, Aarthi; Petricoin, Emanuel; Ross, Mark; Russo, Paul; Zhou, Weidong; Luchini, Alessandra; Manes, Nathan; Chertow, Jessica; Han, Suhua; Kidd, Jessica; Senina, Svetlana; Groves, Stephanie

    2007-01-01

    Basic technologies have been successfully developed within this project: rapid collection of aerosols and a rapid ultra-sensitive immunoassay technique. Water-soluble, humidity-resistant polyacrylamide nano-filters were shown to (1) capture aerosol particles as small as 20 nm, (2) work in humid air and (3) completely liberate their captured particles in an aqueous solution compatible with the immunoassay technique. The immunoassay technology developed within this project combines electrophoretic capture with magnetic bead detection. It allows detection of as few as 150-600 analyte molecules or viruses in only three minutes, something no other known method can duplicate. The technology can be used in a variety of applications where speed of analysis and/or extremely low detection limits are of great importance: in rapid analysis of donor blood for hepatitis, HIV and other blood-borne infections in emergency blood transfusions, in trace analysis of pollutants, or in search of biomarkers in biological fluids. Combined in a single device, the water-soluble filter and ultra-sensitive immunoassay technique may solve the problem of early warning type detection of aerosolized pathogens. These two technologies are protected with five patent applications and are ready for commercialization.

  10. Picture Detection in Rapid Serial Visual Presentation: Features or Identity?

    ERIC Educational Resources Information Center

    Potter, Mary C.; Wyble, Brad; Pandav, Rijuta; Olejarczyk, Jennifer

    2010-01-01

    A pictured object can be readily detected in a rapid serial visual presentation sequence when the target is specified by a superordinate category name such as "animal" or "vehicle". Are category features the initial basis for detection, with identification of the specific object occurring in a second stage (Evans &…

  11. Method for large and rapid terahertz imaging

    DOEpatents

    Williams, Gwyn P.; Neil, George R.

    2013-01-29

    A method of large-scale active THz imaging using a combination of a compact high power THz source (>1 watt), an optional optical system, and a camera for the detection of reflected or transmitted THz radiation, without the need for the burdensome power source or detector cooling systems required by similar prior art such devices. With such a system, one is able to image, for example, a whole person in seconds or less, whereas at present, using low power sources and scanning techniques, it takes several minutes or even hours to image even a 1 cm.times.1 cm area of skin.

  12. Rapid detection of microbial cell abundance in aquatic systems

    DOE PAGES

    Rocha, Andrea M.; Yuan, Quan; Close, Dan M.; ...

    2016-06-01

    The detection and quantification of naturally occurring microbial cellular densities is an essential component of environmental systems monitoring. While there are a number of commonly utilized approaches for monitoring microbial abundance, capacitance-based biosensors represent a promising approach because of their low-cost and label-free detection of microbial cells, but are not as well characterized as more traditional methods. Here, we investigate the applicability of enhanced alternating current electrokinetics (ACEK) capacitive sensing as a new application for rapidly detecting and quantifying microbial cellular densities in cultured and environmentally sourced aquatic samples. ACEK capacitive sensor performance was evaluated using two distinct and dynamicmore » systems the Great Australian Bight and groundwater from the Oak Ridge Reservation in Oak Ridge, TN. Results demonstrate that ACEK capacitance-based sensing can accurately determine microbial cell counts throughout cellular concentrations typically encountered in naturally occurring microbial communities (103 – 106 cells/mL). A linear relationship was observed between cellular density and capacitance change correlations, allowing a simple linear curve fitting equation to be used for determining microbial abundances in unknown samples. As a result, this work provides a foundation for understanding the limits of capacitance-based sensing in natural environmental samples and supports future efforts focusing on evaluating the robustness ACEK capacitance-based within aquatic environments.« less

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

    PubMed

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

    2017-04-15

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

  14. Rapid detection of microbial cell abundance in aquatic systems.

    PubMed

    Rocha, Andrea M; Yuan, Quan; Close, Dan M; O'Dell, Kaela B; Fortney, Julian L; Wu, Jayne; Hazen, Terry C

    2016-11-15

    The detection and quantification of naturally occurring microbial cellular densities is an essential component of environmental systems monitoring. While there are a number of commonly utilized approaches for monitoring microbial abundance, capacitance-based biosensors represent a promising approach because of their low-cost and label-free detection of microbial cells, but are not as well characterized as more traditional methods. Here, we investigate the applicability of enhanced alternating current electrokinetics (ACEK) capacitive sensing as a new application for rapidly detecting and quantifying microbial cellular densities in cultured and environmentally sourced aquatic samples. ACEK capacitive sensor performance was evaluated using two distinct and dynamic systems - the Great Australian Bight and groundwater from the Oak Ridge Reservation in Oak Ridge, TN. Results demonstrate that ACEK capacitance-based sensing can accurately determine microbial cell counts throughout cellular concentrations typically encountered in naturally occurring microbial communities (10(3)-10(6) cells/mL). A linear relationship was observed between cellular density and capacitance change correlations, allowing a simple linear curve fitting equation to be used for determining microbial abundances in unknown samples. This work provides a foundation for understanding the limits of capacitance-based sensing in natural environmental samples and supports future efforts focusing on evaluating the robustness ACEK capacitance-based within aquatic environments. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Rapid quantification method for Legionella pneumophila in surface water.

    PubMed

    Wunderlich, Anika; Torggler, Carmen; Elsässer, Dennis; Lück, Christian; Niessner, Reinhard; Seidel, Michael

    2016-03-01

    World-wide legionellosis outbreaks caused by evaporative cooling systems have shown that there is a need for rapid screening methods for Legionella pneumophila in water. Antibody-based methods for the quantification of L. pneumophila are rapid, non-laborious, and relatively cheap but not sensitive enough for establishment as a screening method for surface and drinking water. Therefore, preconcentration methods have to be applied in advance to reach the needed sensitivity. In a basic test, monolithic adsorption filtration (MAF) was used as primary preconcentration method that adsorbs L. pneumophila with high efficiency. Ten-liter water samples were concentrated in 10 min and further reduced to 1 mL by centrifugal ultrafiltration (CeUF). The quantification of L. pneumophila strains belonging to the monoclonal subtype Bellingham was performed via flow-based chemiluminescence sandwich microarray immunoassays (CL-SMIA) in 36 min. The whole analysis process takes 90 min. A polyclonal antibody (pAb) against L. pneumophila serogroup 1-12 and a monoclonal antibody (mAb) against L. pneumophila SG 1 strain Bellingham were immobilized on a microarray chip. Without preconcentration, the detection limit was 4.0 × 10(3) and 2.8 × 10(3) CFU/mL determined by pAb and mAb 10/6, respectively. For samples processed by MAF-CeUF prior to SMIA detection, the limit of detection (LOD) could be decreased to 8.7 CFU/mL and 0.39 CFU/mL, respectively. A recovery of 99.8 ± 15.9% was achieved for concentrations between 1-1000 CFU/mL. The established combined analytical method is sensitive for rapid screening of surface and drinking water to allow fast hygiene control of L. pneumophila.

  16. Performance Assessment of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of Extensively Drug-resistant Tuberculosis in Clinical Isolates of Mycobacterium tuberculosis

    PubMed Central

    Arjomandzadegan, M; Nazari, R; Zolfaghari, MR; Taherahmadi, M; Sadrnia, M; Titov, LP; Ahmadi, A; Shojapoor, M

    2015-01-01

    ABSTRACT Introduction: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis. Materials and Methods: From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced. Results: Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001). Conclusion: Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance of MDR and XDR forms to anti-tuberculosis drugs using this method. PMID:26624582

  17. A rapid analytical method for cholecalciferol (vitamin D3) in fortified infant formula, milk and milk powder using Diels-Alder derivatisation and liquid chromatography-tandem mass spectrometric detection.

    PubMed

    Abernethy, Grant A

    2012-05-01

    A method for analysing vitamin D(3) (VD3, cholecalciferol) has been established and validated. This method is rapid and cost effective and is intended for use in quality control in the manufacture of fortified infant formulae and milk powders. Milk or reconstituted milk powder was solubilised in methanol and extracted in one step into isooctane, which was separated by centrifugation. A portion of the isooctane layer was then transferred, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione was added to derivatise VD3. The analyte was then re-extracted into a small volume of acetonitrile and analysed by reverse-phase chromatography. Detection was by triple quadrupole mass spectrometer using a selective transition, m/z 560 → 298. An internal standard, deuterium-labelled VD3, was used to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies. The method has been subjected to a single-laboratory validation and has been found to be linear, highly selective and accurate with respect to National Institute of Standards and Technology Standard Reference Material 1849, analyte spiking experiments and comparison with an LC-UV-based method. The repeatability standard deviation was 4.23 %. Significantly for routine laboratories, the method returns results within 2 h, generates minimal waste and minimises health and safety concerns to the analyst.

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

    PubMed

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

    2017-05-15

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

  19. Validation of a Rapid, Robust, Inexpensive Screening Method for Detecting the HLA-B*58:01 Allele in the Prevention of Allopurinol-Induced Severe Cutaneous Adverse Reactions.

    PubMed

    Nguyen, Dinh Van; Vida, Christopher; Chu, Hieu Chi; Fulton, Richard; Li, Jamma; Fernando, Suran L

    2017-01-01

    The HLA B*58:01 allele has been worldwide reported as a pharmacogenetic susceptibility to allopurinol-induced severe cutaneous adverse reactions (SCARs). To prevent these life-threatening conditions, the American College of Rheumatology hingly recommended that the HLA-B*58:01 be screened prior to the initiation of allopurinol therapy. Therefore, we developed a rapid, robust, inexpensive screening method using SYBR® Green real time PCR to detect the HLA-B*58:01 allele. A total of 119 samples were tested. The assay has a sensitivity of 100% (95% CI: 69.15%-100%), a specificity of 100% (95% CI: 96.67%-100%), a positive predictive value of 100% (95% CI: 69.15%-100%) and a negative predictive value of 100% (95% CI: 96.67%-100%). HLA-B*58:01 genotyping results showed 100% agreement with those obtained from Luminex SSO/SBT/SSP. The lowest limit of detection of this method is 0.8 ng/μL of DNA. The unit cost of the test is only $3.8 USD. This novel screening test using SYBR® real time PCR would be appropriate to identify individuals with the HLA-B*58:01 allele for the prevention of allopurinol-induced SCARs.

  20. Validation of a Rapid, Robust, Inexpensive Screening Method for Detecting the HLA-B*58:01 Allele in the Prevention of Allopurinol-Induced Severe Cutaneous Adverse Reactions

    PubMed Central

    Chu, Hieu Chi; Fulton, Richard; Li, Jamma; Fernando, Suran L

    2017-01-01

    The HLA B*58:01 allele has been worldwide reported as a pharmacogenetic susceptibility to allopurinol-induced severe cutaneous adverse reactions (SCARs). To prevent these life-threatening conditions, the American College of Rheumatology hingly recommended that the HLA-B*58:01 be screened prior to the initiation of allopurinol therapy. Therefore, we developed a rapid, robust, inexpensive screening method using SYBR® Green real time PCR to detect the HLA-B*58:01 allele. A total of 119 samples were tested. The assay has a sensitivity of 100% (95% CI: 69.15%-100%), a specificity of 100% (95% CI: 96.67%-100%), a positive predictive value of 100% (95% CI: 69.15%-100%) and a negative predictive value of 100% (95% CI: 96.67%-100%). HLA-B*58:01 genotyping results showed 100% agreement with those obtained from Luminex SSO/SBT/SSP. The lowest limit of detection of this method is 0.8 ng/µL of DNA. The unit cost of the test is only $3.8 USD. This novel screening test using SYBR® real time PCR would be appropriate to identify individuals with the HLA-B*58:01 allele for the prevention of allopurinol-induced SCARs. PMID:27826965

  1. Micromechanical oscillators as rapid biosensor for the detection of active growth of Escherichia coli.

    PubMed

    Gfeller, Karin Y; Nugaeva, Natalia; Hegner, Martin

    2005-09-15

    A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated by common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The sensor is able to detect active growth of Escherichia coli cells within 1 h which is significantly faster than any conventional plating method which requires at least 24 h. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of active bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing by adding antibiotics to the nutritive layer.

  2. Performance Assessment of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of Extensively Drug-resistant Tuberculosis in Clinical Isolates of Mycobacterium tuberculosis.

    PubMed

    Arjomandzadegan, M; Nazari, R; Zolfaghari, M R; Taherahmadi, M; Sadrnia, M; Titov, L P; Ahmadi, A; Shojapoor, M

    2015-09-01

    The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis. From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced. Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001). Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance consisting of MDR and XDR forms to anti-tuberculosis drugs using this method.

  3. Turbo FISH: A Method for Rapid Single Molecule RNA FISH

    PubMed Central

    Shaffer, Sydney M.; Wu, Min-Tzu; Levesque, Marshall J.; Raj, Arjun

    2013-01-01

    Advances in RNA fluorescence in situ hybridization (RNA FISH) have allowed practitioners to detect individual RNA molecules in single cells via fluorescence microscopy, enabling highly accurate and sensitive quantification of gene expression. However, current methods typically employ hybridization times on the order of 2–16 hours, limiting its potential in applications like rapid diagnostics. We present here a set of conditions for RNA FISH (dubbed Turbo RNA FISH) that allow us to make accurate measurements with no more than 5 minutes of hybridization time and 3 minutes of washing, and show that hybridization times can go as low as 30 seconds while still producing quantifiable images. We further show that rapid hybridization is compatible with our recently developed iceFISH and SNP FISH variants of RNA FISH that enable chromosome and single base discrimination, respectively. Our method is simple and cost effective, and has the potential to dramatically increase the throughput and realm of applicability of RNA FISH. PMID:24066168

  4. Assay of olanzapine in human plasma by a rapid and sensitive gas chromatography-nitrogen phosphorus selective detection (GC-NPD) method: validation and comparison with high-performance liquid chromatography-coulometric detection.

    PubMed

    Ulrich, Sven

    2005-08-01

    A gas chromatography-nitrogen phosphorus selective detection (GC-NPD) method with a simple 1-step sample preparation was developed for the assay of the antipsychotic drug olanzapine in plasma. Within a time of analysis of 7 minutes, an HP-5 fused-silica capillary (25 m x 0.2 mm ID, 0.33-microm film thickness, 0.7 mL N2 as carrier gas) provided selectivity with respect to about 30 psychotropic drugs and the internal standard ethylolanzapine. Calibration was linear between 1 and 50 ng/mL and crossed the origin (LOD = 0.3 ng/mL). Intraday precision was 6.7%, 2.7%, and 1.4% at plasma concentrations of 1, 5, and 50 ng/mL, respectively. Interday precision was 4.6% at 20 ng/mL. Accuracy in commercial interlaboratory tests was 108.7% and 88.5%. The method also provided good accuracy in comparison with an HPLC method for patient samples (slope 1.003, r = 0.953) and spiked samples (slope 0.881, r = 0.998). GC-NPD with a simple sample preparation is regarded as an alternative for the assay of olanzapine plasma concentrations in therapeutic drug monitoring (TDM) and in pharmacokinetic studies. Smokers and patients taking concomitant carbamazepine had reduced plasma concentrations of olanzapine. Women and patients older than 60 years had increased plasma olanzapine concentrations.

  5. Optofluidic ring resonator sensors for rapid DNT vapor detection.

    PubMed

    Sun, Yuze; Liu, Jing; Frye-Mason, Greg; Ja, Shiou-jyh; Thompson, Aaron K; Fan, Xudong

    2009-07-01

    We demonstrated rapid 2,4-dinitrotoluene (DNT) vapor detection at room temperature based on an optofluidic ring resonator (OFRR) sensor. With the unique on-column separation and detection features of OFRR vapor sensors, DNT can be identified from other interferences coexisting in the analyte sample mixture, which is especially useful in the detection of explosives from practical complicated vapor samples usually containing more volatile analytes. The DNT detection limit is approximately 200 pg, which corresponds to a solid phase microextraction (SPME) sampling time of only 1 second at room temperature from equilibrium headspace. A theoretical analysis was also performed to account for the experimental results. Our study shows that the OFRR vapor sensor is a promising platform for the development of a rapid, low-cost, and portable analytical device for explosive detection and monitoring.

  6. Rapid detection of small oscillation faults via deterministic learning.

    PubMed

    Wang, Cong; Chen, Tianrui

    2011-08-01

    Detection of small faults is one of the most important and challenging tasks in the area of fault diagnosis. In this paper, we present an approach for the rapid detection of small oscillation faults based on a recently proposed deterministic learning (DL) theory. The approach consists of two phases: the training phase and the test phase. In the training phase, the system dynamics underlying normal and fault oscillations are locally accurately approximated through DL. The obtained knowledge of system dynamics is stored in constant radial basis function (RBF) networks. In the diagnosis phase, rapid detection is implemented. Specially, a bank of estimators are constructed using the constant RBF neural networks to represent the training normal and fault modes. By comparing the set of estimators with the test monitored system, a set of residuals are generated, and the average L(1) norms of the residuals are taken as the measure of the differences between the dynamics of the monitored system and the dynamics of the training normal mode and oscillation faults. The occurrence of a test oscillation fault can be rapidly detected according to the smallest residual principle. A rigorous analysis of the performance of the detection scheme is also given. The novelty of the paper lies in that the modeling uncertainty and nonlinear fault functions are accurately approximated and then the knowledge is utilized to achieve rapid detection of small oscillation faults. Simulation studies are included to demonstrate the effectiveness of the approach.

  7. Fetal magnetocardiography: Methods for rapid data reduction

    SciTech Connect

    Mosher, J.C.; Flynn, E.R.; Quinn, A.; Weir, A.; Shahani, U.; Bain, R.J.; Maas, P.; Donaldson, G.B.

    1997-03-01

    Fetal magnetocardigraphy (fMCG) provides a unique method for noninvasive observations of the fetal heart. Electrical currents generated by excitable tissues within the fetal heart yield measurable external magnetic fields. Measurements are performed with superconducting quantum interference devices inductively coupled to magnetometer or gradiometer coils, and the resulting signals are converted to digital form in the data acquisition system. The measured fields are usually contaminated by fetal and maternal movements (usually respiration), other physiological fields such as skeletal muscle contraction, the maternal cardiac signal, and environmental electromagnetic fields. Sensitivity to relatively distant sources, both physiological and environmental, is substantially reduced by the use of magnetic gradiometers. Other contaminants may be removed by proper signal conditioning which may be automatically applied using {open_quotes}black box{close_quotes} algorithms that are transparent to the user and highly efficient. These procedures can rapidly reduce the complex signal plus noise waveforms to the desired fMCG with minimal operator interference. {copyright} {ital 1997 American Institute of Physics.}

  8. Rapid detection of EBOLA VP40 in microchip immunofiltration assay

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  9. Nanomaterial-enabled Rapid Detection of Water Contaminants.

    PubMed

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

    2015-10-28

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

  10. Rapid and alternative screening methods for microbiological analysis.

    PubMed

    van der Zee, H; Huis in't Veld, J H

    1997-01-01

    Automated analytical instruments for enumerating indicator organisms and diagnostic test kits for pathogens can be used in food microbiology to screen samples and to replace conventional cultural and confirmation steps. Such methods are now available for rapid detection or estimation of groups of (indicator) organisms, pathogenic micro-organisms, bacterial toxins and mycotoxins, and molds. These alternative methods can be classified by the principles on which they are based: modified conventional methods, instrumental measurement of bacterial metabolism, bioluminescence, immunological techniques, DNA techniques, and combinations of these techniques. To meet user expectations, test kits must be accurate, sensitive, specific, rapid (24 h or less), easy to use, and labor-saving. They must also offer the possibility of computerization, a low detection limit, and low investment and running costs. The paper compares the ability of alternative methods to meet these criteria. Variations were found, depending on the techniques used and the target organism of the analysis. Economic reasons can determine whether alternative methods can be used routinely. Adoption of these screening systems also can be hampered by lack of internationally coordinated and accepted validation protocols.

  11. Rapid detection and quantitation of ganciclovir resistance in cytomegalovirus quasispecies.

    PubMed

    Ruiz-Carrascoso, Guillermo; Romero-Gómez, María Pilar; Plaza, Diego; Mingorance, Jesús

    2013-07-01

    Human cytomegalovirus (HCMV) may cause severe or fatal disease among immunocompromised patients. The first line prophylaxis and systemic HCMV disease therapy is ganciclovir (GCV). The presence of GCV-resistant virus has been linked to fatal HCMV disease. The implementation of rapid and sensitive techniques for the early detection and monitoring of GCV-resistance may be helpful to support antiviral therapy management. A pyrosequencing assay for the detection and quantitation of the most frequent mutations conferring moderate- and high-grade GCV resistance was implemented. The pyrosequencing achieved an analytical sensitivity for adequate interpretation of ≥10(3)  copies/ml. The assay was validated with 18 whole blood samples taken over a 6-month period from an umbilical cord blood recipient infected persistently with HCMV and allowed the detection and monitoring of the M460I and A594V GCV-resistant mutations. The percentage of resistant quasispecies ranged from 7.9% to 55.2% for the M460I mutation and from 19.8% to 43% for the A594V mutation. Clearance of the M460I mutation occurred in parallel with a decrease in the HCMV viremia, while the A594V mutation persisted. The pyrosequencing method for detection of GCV is sensitive enough to be used directly on clinical samples for the early identification of resistance mutations and allows the quantitation of resistant and wild type virus quasispecies within hours. The quantitation of minor resistant variants is an important issue to understand their relationship with viral load modification, and potentially anticipate treatment adjustment.

  12. Methods for the detection of marine toxins

    SciTech Connect

    Wekell, M.M.; Manger, R.M.; Hadley, S.W.; Hungerford, J.M.

    1995-12-01

    Toxic materials have been dumped into the seas from waste streams and other pollutant sources such as runoff, etc. For protection of public health, it is essential that rapid, reliable and simple methods exist to detect marine toxins in seafoods. In addition, it is necessary to develop methods requiring a minimum of test material. Pure standards for many of the marine toxins are scarce. Reduced sample requirements extend the utility of detection methods in research and forensic applications as well. In the past, there was much reliance on the animal bioassay; however, this dependence hopefully will be reduced as newer instrumental techniques (chromatographic, mass spectrometric, electrophoretic), biochemical (immunochemical, receptor site assay), and cell bioassay methods are developed with a higher degree of precision and specificity. It is beneficial that a multiplicity of methods be available to detect marine toxins in seafoods. Each method has unique advantages and disadvantages.

  13. MPT 64 Antigen detection for Rapid confirmation of M.tuberculosis isolates

    PubMed Central

    2011-01-01

    Background A new rapid Immunochromatographic test kit(SD MPT64TB Ag Kit) for detection of MPT 64 Antigen in M. tuberculosis isolates using mouse monoclonal MPT 64 Antibody developed by SD Bioline, South Korea was evaluated for rapid identification of M. tuberculosis isolates. We also assessed the sensitivity, specificity and predictive values of this kit. The test kit has an excellent sensitivity, specificity, negative predictive value & positive predictive value. This rapid method is found to be a reliable, rapid and cheaper method for confirming MTB culture isolates in resource poor laboratories. Material/methods: 54 culture isolates of M. tuberculosis in broth & on LJ medium, 12 Non mycobacterial isolates, 10 Non tubercular (NTM) rapidly growing Mycobacteria isolated from pus & 5 smear positive sputum samples were tested for detection of MPT64 antigen using the SD Bioline immunochromatography (ICT)test kit. H37 RV strain was employed as the positive reference control. Findings H37 RV strain showed the presence of MPT64 antigen band. Similar band was formed in all the 54 MTB isolates tested proving 100% sensitivity. MPT64 band formation was not detected in any of the other test isolates which proved the 100% specificity of the test kit. Both PPV & NPV were 100%. Conclusion Tuberculosis is a global pandemic. Rapid identification of MTB culture isolate is very important for drug susceptibility testing. MPT 64 TB Ag detection ICT kit is a rapid, reliable method; it can be a substitute for the molecular identification methods. PMID:21429231

  14. Rapid visual detection of eight meat species using optical thin-film biosensor chips.

    PubMed

    Wang, Wei; Zhu, Yepei; Chen, Ying; Xu, Xinglian; Zhou, Guanghong

    2015-01-01

    Adulteration of meat products has become a very serious issue nowadays. To protect consumer rights, food labeling is required in many countries, and efficient and accurate detection methods are essential as well. This paper reports an innovative method for the rapid detection and identification of meat species based on a silicon-based optical thin-film biosensor chip with which color change results can be perceived by the naked eye without any expensive instruments. This biosensor system can simultaneously and specifically detect eight meat species, including deer, rabbit, duck, chicken, beef, horse, sheep, and pork. The absolute detection limit of this method was 0.5 pg of deer/beef DNA, and the practical detection limit was 0.001%. The biosensor detection can be completed within 30 min after PCR amplification. Therefore, this assay permits specific, sensitive, rapid, and simple detection of meat species in raw or cooked meat products.

  15. Methods of DNA methylation detection

    NASA Technical Reports Server (NTRS)

    Maki, Wusi Chen (Inventor); Filanoski, Brian John (Inventor); Mishra, Nirankar (Inventor); Rastogi, Shiva (Inventor)

    2010-01-01

    The present invention provides for methods of DNA methylation detection. The present invention provides for methods of generating and detecting specific electronic signals that report the methylation status of targeted DNA molecules in biological samples.Two methods are described, direct and indirect detection of methylated DNA molecules in a nano transistor based device. In the direct detection, methylated target DNA molecules are captured on the sensing surface resulting in changes in the electrical properties of a nano transistor. These changes generate detectable electronic signals. In the indirect detection, antibody-DNA conjugates are used to identify methylated DNA molecules. RNA signal molecules are generated through an in vitro transcription process. These RNA molecules are captured on the sensing surface change the electrical properties of nano transistor thereby generating detectable electronic signals.

  16. A rapid method for simultaneous determination of 14 phenolic compounds in Radix Puerariae using microwave-assisted extraction and ultra high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry.

    PubMed

    Du, G; Zhao, H Y; Zhang, Q W; Li, G H; Yang, F Q; Wang, Y; Li, Y C; Wang, Y T

    2010-01-29

    A microwave-assisted extraction (MAE) and ultra high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry (UHPLC-DAD-TOF-MS) method was developed for simultaneous determination of 14 phenolic compounds in the root of Pueraria lobata (Wild.) Ohwi and Pueraria thomsonii Benth. Operational conditions of MAE were optimized by central composite design (CCD). The optimized result was 65% ethanol as extraction solvent, 17mL of extraction volume, 100 degrees C of extraction temperature and 2min of hold time. A Zorbax SB C(18) (50mmx4.6mm I.D., 1.8microm) and gradient elution were used during the analysis. The chromatographic peaks of 14 investigated compounds in samples were successfully identified by comparing their retention time, UV spectra and TOF mass data with the reference substances. All calibration curves showed good linearity (r>0.9997) within the test ranges. The intra-day and inter-day variations were less than 1.77% and 2.88%, respectively. The developed method was successfully applied to determine the investigated compounds in 10 samples of Radix Puerariae Lobatae and Radix Puerariae Thomsonii, respectively. The result indicated that MAE and UHPLC-DAD-TOF-MS system might provide a rapid method for the quality control of Radix Puerariae.

  17. Novel method for the rapid and specific extraction of multiple β2 -agonist residues in food by tailor-made Monolith-MIPs extraction disks and detection by gas chromatography with mass spectrometry.

    PubMed

    Liu, Haibo; Gan, Ning; Chen, Yinji; Ding, Qingqing; Huang, Jie; Lin, Saichai; Cao, Yuting; Li, Tianhua

    2016-09-01

    A quick and specific pretreatment method based on a series of extraction clean-up disks, consisting of molecularly imprinted polymer monoliths and C18 adsorbent, was developed for the specific enrichment of salbutamol and clenbuterol residues in food. The molecularly imprinted monolithic polymer disk was synthesized using salbutamol as a template through a one-step synthesis process. It can simultaneously and specifically recognize salbutamol and clenbuterol. The monolithic polymer disk and series of C18 disks were assembled with a syringe to form a set of tailor-made devices for the extraction of target molecules. In a single run, salbutamol and clenbuterol can be specifically extracted, cleaned, and eluted by methanol/acetic acid/H2 O. The target molecules, after a silylation derivatization reaction were detected by gas chromatography-mass spectrometry. The parameters including solvent desorption, sample pH, and the cycles of reloading were investigated and discussed. Under the optimized extraction and clean-up conditions, the limits of detection and quantitation were determined as 0.018-0.022 and 0.042-0.049 ng/g for salbutamol and clenbuterol, respectively. The assay described was convenient, rapid, and specific; thereby potentially efficient in the high-throughput analysis of β2 -agonists residues in real food samples.

  18. Rapid detection of bacterial pathogens using flourescence spectroscopy and chemometrics

    USDA-ARS?s Scientific Manuscript database

    This work presents the development of a method for rapid bacterial identification based on the fluorescence spectroscopy combined with multivariate analysis. Fluorescence spectra of pure three different genera of bacteria (Escherichia coli, Salmonella, and Campylobacter) were collected from 200...

  19. Nanofluidic devices for rapid detection of virus particles.

    SciTech Connect

    Gourley, Paul Lee; McDonald, Anthony Eugene; Hendricks, Judy K.

    2005-01-01

    Technologies that could quickly detect and identify virus particles would play a critical role in fighting bioterrorism and help to contain the rapid spread of disease. Of special interest is the ability to detect the presence and movement of virions without chemically modifying them by attaching molecular probes. This would be useful for rapid detection of pathogens in food or water supplies without the use of expensive chemical reagents. Such detection requires new devices to quickly screen for the presence of tiny pathogens. To develop such a device, we fabricated nanochannels to transport virus particles through ultrashort laser cavities and measured the lasing output as a sensor for virions. To understand this transduction mechanism, we also investigated light scattering from virions, both to determine the magnitude of the scattered signal and to use it to investigate the motion of virions.

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

    DTIC Science & Technology

    2015-10-01

    Our project work is focused on using a new dielectrophoresis (DEP) microarray technology for rapid isolation and detection of brain -specific RNA and... brain -specific mRNAs, miRNAs and other biomarkers. We have been successful in demonstrating DEP isolation of glioblastoma exosomes from 50 L of un...for detection and accurate diagnosis of Traumatic Brain Injury (TBI) in point-of-care (POC) settings pose an ongoing capability gap. For soldiers in

  1. Rapid detection of Ophiostoma piceae and O. quercus in stained wood by PCR.

    PubMed

    Kim, S H; Uzunovic, A; Breuil, C

    1999-01-01

    A rapid, sensitive, and simple method was developed to detect the sapstain fungi Ophiostoma piceae and O. quercus in stained wood. By using microwave heating for DNA extraction and PCR with internal transcribed spacer-derived-specific primers, detection was feasible within 4 h, even with DNA obtained from a single synnema. This method can easily be extended for the detection of other wood-inhabiting fungi.

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

    PubMed

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

    2017-01-01

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

  3. A new method for the rapid detection of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Gadus chalcogrammus) and ling (Molva molva) using a lateral flow dipstick assay.

    PubMed

    Taboada, Ledicia; Sánchez, Ana; Pérez-Martín, Ricardo I; Sotelo, Carmen G

    2017-10-15

    Species-specific lateral flow dipstick (LFD) assays for the identification of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Gadus chalcogrammus) and ling (Molva molva) in food products were developed. The method comprises a PCR system with four sets of specific primers, for each target species. This step was also devised to dual-labeling of PCR products with biotin and 6-FAM, which are then easily read on a lateral flow dipstick, upon which these products are immobilized by a fixed biotin-ligand and visualized with anti-FAM antibody-coated gold nanoparticles. Sensitivity and selectivity were determined for each of the developed assays. Validation of the assays was performed with DNA extracted from commercial fish products, the identification of all samples by PCR-LFD was coherent with the results found with DNA sequencing. Target species were successfully detected in analyzed commercial samples, demonstrating the applicability of this method to the rapid analysis of food products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Rapid microbead-based DNA detection using dielectrophoresis and impedance measurement

    NASA Astrophysics Data System (ADS)

    Nakano, Michihiko; Ding, Zhenhao; Kasahara, Hiromichi; Suehiro, Junya

    2014-10-01

    Polymerase chain reaction (PCR) is a powerful tool for diagnostic procedures in bacterial and viral infections. The authors propose a new electrical technique for rapid detection of DNA amplified by PCR using dielectrophoresis (DEP) of microbeads. The method is based on dramatic alteration of DEP characteristics of microbeads caused by DNA labeling. DNA-labeled microbeads are trapped on a microelectrode under the action of positive DEP, whereas pristine ones are not. DEP-trapped microbeads are measured impedimetrically to realize rapid and quantitative detection of the amplified DNA. The validity of the proposed method was demonstrated by detection of PCR-amplified DNA of viruses.

  5. A novel method for rapid in vitro radiobioassay

    NASA Astrophysics Data System (ADS)

    Crawford, Evan Bogert

    minutes of sample preparation and counting time. Radiation detector spectra -- e.g. those from high-purity germanium (HPGe) gamma detectors and liquid scintillation detectors -- which contain decay signals from multiple isotopes often have overlapping signals: the counts from one isotope's decay can appear in energy channels associated with another isotope's decay, complicating the calculation of each isotope's activity. The uncertainties associated with analyzing these spectra have been traced in order to determine the effects of one isotope's count rate on the sensitivity and uncertainty associated with each other isotope. The method that was developed takes advantage of activated carbon filtration to eliminate quenching effects and to make the liquid scintillation spectra from different urine samples comparable. The method uses pulse-shape analysis to reduce the interference from beta emitters in the liquid scintillation spectrum and improve the minimum detectable activity (MDA) and minimum quantifiable activity (MQA) for alpha emitters. The method uses an HPGe detector to quantify the activity of gamma emitters, and subtract their isotopes' contributions to the liquid scintillation spectra via a calibration factor, such that the pure beta and pure alpha emitters can be identified and quantified from the resulting liquid scintillation spectra. Finally, the method optionally uses extractive scintillators to rapidly separate the alpha emitters from the beta emitters when the activity from the beta emitters is too great to detect or quantify the activity from the alpha emitters without such a separation. The method is able to detect and quantify all five isotopes, with uncertainties and biases usually in the 10-40% range, depending upon the isotopic mixtures and the activity ratios between each of the isotopes.

  6. An FPGA-Based Rapid Wheezing Detection System

    PubMed Central

    Lin, Bor-Shing; Yen, Tian-Shiue

    2014-01-01

    Wheezing is often treated as a crucial indicator in the diagnosis of obstructive pulmonary diseases. A rapid wheezing detection system may help physicians to monitor patients over the long-term. In this study, a portable wheezing detection system based on a field-programmable gate array (FPGA) is proposed. This system accelerates wheezing detection, and can be used as either a single-process system, or as an integrated part of another biomedical signal detection system. The system segments sound signals into 2-second units. A short-time Fourier transform was used to determine the relationship between the time and frequency components of wheezing sound data. A spectrogram was processed using 2D bilateral filtering, edge detection, multithreshold image segmentation, morphological image processing, and image labeling, to extract wheezing features according to computerized respiratory sound analysis (CORSA) standards. These features were then used to train the support vector machine (SVM) and build the classification models. The trained model was used to analyze sound data to detect wheezing. The system runs on a Xilinx Virtex-6 FPGA ML605 platform. The experimental results revealed that the system offered excellent wheezing recognition performance (0.912). The detection process can be used with a clock frequency of 51.97 MHz, and is able to perform rapid wheezing classification. PMID:24481034

  7. An FPGA-based rapid wheezing detection system.

    PubMed

    Lin, Bor-Shing; Yen, Tian-Shiue

    2014-01-29

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

  8. [Methods of rapid diagnosis in clinical microbiology: Clinical needs].

    PubMed

    Vila, Jordi; Gómez, María Dolores; Salavert, Miguel; Bosch, Jordi

    2017-01-01

    The diagnostic methods of infectious diseases should be fast, accurate, simple and affordable. The speed of diagnosis can play a crucial role in healing the patient, allowing the administration of appropriate antibiotic treatment. One aspect that increasingly determines the need for rapid diagnostic techniques is the increased rates of serious infections caused by multidrug resistant bacteria, which cause a high probability of error in the empirical treatment. Some of the conventional methods such as Gram staining or antigen detection can generate results in less than 1 hour but lack sensitivity. Today we are witnessing a major change in clinical microbiology laboratories with the technological advances such as molecular diagnostics, digital microbiology and mass spectrometry. There are several studies showing that these changes in the microbiological diagnosis reduce the generation time of the test results, which has an obvious clinical impact. However, if we look into the future, other new technologies which will cover the needs required for a rapid microbiological diagnosis are on the horizon. This review provides an in depth analysis of the clinical impact that the implementation of rapid diagnostic techniques will have on unmet clinical needs.

  9. Rapid identification of salmonella serotypes with stereo and hyperspectral microscope imaging Methods

    USDA-ARS?s Scientific Manuscript database

    The hyperspectral microscope imaging (HMI) method can reduce detection time within 8 hours including incubation process. The early and rapid detection with this method in conjunction with the high throughput capabilities makes HMI method a prime candidate for implementation for the food industry. Th...

  10. Rapid Identification of Salmonella Serotypes with Stereo and Hyperspectral Microscope Imaging Methods

    USDA-ARS?s Scientific Manuscript database

    The hyperspectral microscope imaging (HMI) method can reduce detection time within 8 hours including incubation process. The early and rapid detection with this method in conjunction with the high throughput capabilities makes HMI method a prime candidate for implementation for the food industry. Th...

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

    PubMed

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

    2017-05-01

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

  12. [Clinical value of a rapid respiratory syncytial virus antigen detection in point-of-care testing].

    PubMed

    Ding, Y X; Tian, R; Qian, Y; Sun, Y; Deng, J; Wang, F; Zhu, R N; Zhao, L Q

    2017-02-02

    Objective: To evaluate the clinical value of a rapid respiratory syncytial virus (RSV) antigen detection in point-of-care testing (POCT). Method: A total of 209 specimens, including 78 throat swabs (TS) and 131 nasopharyngeal aspirates (NPAs), were collected from inpatients who visited the Children's Hospital Affiliated to the Capital Institute of Pediatrics and were diagnosed as acute respiratory infection from 5 January to 7 February, 2015. These specimens were tested for RSV by a rapid antigen detection kit which was compared with reverse transcription polymerase chain reaction (RT-PCR) and direct immunofluorescence assay (DFA) for RSV detection. Result: Compared with DFA for NPAs, the sensitivity and specificity of rapid antigen detection were 83.9% and 97.3%, respectively, with Kappa value of 0.86; Compared with RT-PCR, the sensitivity (NPAs, 74.2%; TS, 77.8%) and specificity (NPAs, 100.0%; TS, 92.0%) of rapid antigen detection were high, too, with Kappa value of 0.74 in NPAs and 0.62 in TS. However, the RSV positive rate of rapid antigen detection in TS (21.7%) from pediatric patients with acute lower respiratory tract infection was lower than that in NPAs (78.3%), as well as that of RT-PCR (7.3% in TS verse 78% in NPAs). The RSV rapid antigen detection kit can be finished in about 10 minutes. Conclusion: With characteristics of high specificity, high sensitivity, being rapid, efficient and easy to operate in comparison with DFA and RT-PCR, RSV rapid antigen detection in this study is suitable for POCT. For pediatric patients with acute respiratory tract infection, NPA was better than TS for RSV detection.

  13. Rapid Radiochemical Method for Plutonium-238 and ...

    EPA Pesticide Factsheets

    Technical Fact Sheet Technique: Alpha spectrometry Method Developed for: Plutonium-238 and plutonium-239 in building materials Method Selected for: SAM lists this method for qualitative analysis of plutonium-238 and -239 in concrete or brick building materials. Summary of subject analytical method which will be posted to the SAM website to allow access to the method.

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

    SciTech Connect

    Lane, Todd W.; Gantt, Richard W.

    2004-12-01

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

  15. Exoplanet Detection by Astrometric Method

    NASA Astrophysics Data System (ADS)

    Xu, Wei-wei; Liao, Xin-hao; Zhou, Yong-hong; Xu, Xue-qing

    2017-07-01

    As we know, the exoplanets are mostly detected by the methods of radial velocity and transit, up to now only one is found by the astrometric method. As the data of the gaia will be soon released, astrometry will gradually become one of the most important methods for detecting exoplanets. Based on the sequence of star positions obtained by the astrometric method, the solution of the equations of dynamical conditions involving the calculations of planet's mass and orbital parameters is discussed in this paper. Due to the deficiency of the available theory (orbital element method), a new method (coordinate velocity method) is put forward. The differential correction formulae of the two methods, as well as the necessary simulation calculations are presented. In addition, the method established in this paper can be applied to the multi-planet system easily.

  16. Rapid Detection of Small Movements with GNSS Doppler Observables

    NASA Astrophysics Data System (ADS)

    Hohensinn, Roland; Geiger, Alain

    2017-04-01

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

  17. Method for detecting an element

    DOEpatents

    Blackwood, Larry G.; Reber, Edward L.; Rohde, Kenneth W.

    2007-02-06

    A method for detecting an element is disclosed and which includes the steps of providing a gamma-ray spectrum which depicts, at least in part, a test region having boundaries, and which has a small amount of the element to be detected; providing a calculation which detects the small amount of the element to be detected; and providing a moving window and performing the calculation within the moving window, and over a range of possible window boundaries within the test region to determine the location of the optimal test region within the gamma-ray spectrum.

  18. Sensory detection of food rapidly modulates arcuate feeding circuits.

    PubMed

    Chen, Yiming; Lin, Yen-Chu; Kuo, Tzu-Wei; Knight, Zachary A

    2015-02-26

    Hunger is controlled by specialized neural circuits that translate homeostatic needs into motivated behaviors. These circuits are under chronic control by circulating signals of nutritional state, but their rapid dynamics on the timescale of behavior remain unknown. Here, we report optical recording of the natural activity of two key cell types that control food intake, AgRP and POMC neurons, in awake behaving mice. We find unexpectedly that the sensory detection of food is sufficient to rapidly reverse the activation state of these neurons induced by energy deficit. This rapid regulation is cell-type specific, modulated by food palatability and nutritional state, and occurs before any food is consumed. These data reveal that AgRP and POMC neurons receive real-time information about the availability of food in the external world, suggesting a primary role for these neurons in controlling appetitive behaviors such as foraging that promote the discovery of food.

  19. Sensory detection of food rapidly modulates arcuate feeding circuits

    PubMed Central

    Chen, Yiming; Lin, Yen-Chu; Kuo, Tzu-Wei

    2015-01-01

    Summary Hunger is controlled by specialized neural circuits that translate homeostatic needs into motivated behaviors. These circuits are under chronic control by circulating signals of nutritional state, but their rapid dynamics on the timescale of behavior remain unknown. Here we report optical recording of the natural activity of two key cell types that control food intake, AgRP and POMC neurons, in awake behaving mice. We find unexpectedly that the sensory detection of food is sufficient to rapidly reverse the activation state of these neurons induced by energy deficit. This rapid regulation is cell-type-specific, modulated by food palatability and nutritional state, and occurs before any food is consumed. These data reveal that AgRP and POMC neurons receive real-time information about the availability of food in the external world, suggesting a primary role for these neurons in controlling appetitive behaviors such as foraging that promote the discovery of food. PMID:25703096

  20. Individual differences in detecting rapidly presented fearful faces.

    PubMed

    Zhang, Dandan; Wang, Lili; Luo, Yi; Luo, Yuejia

    2012-01-01

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

  1. The rapid eye test to detect drug abuse.

    PubMed

    Tennant, F

    1988-07-01

    Because the current epidemic of drug abuse has touched all levels of society, the primary care physician is increasingly called on to identify, monitor, and treat persons with drug abuse problems. All of the major drugs of abuse, including cocaine, marijuana, amphetamine, phencyclidine, heroin, and alcohol, may produce typical eye signs that can be easily detected by a rapid eye test. These signs include ptosis, abnormal pupil size, nonreactivity of the pupil to a light challenge, nystagmus, and non-convergence. When eye signs are detected, drug use should be confirmed by analysis of body fluids. The rapid eye test is suitable for routine use when screening adolescents, athletes, and employees with jobs where safety is essential.

  2. The design of rapid MicroRNA detection system

    NASA Astrophysics Data System (ADS)

    Wang, Yanfei; Yu, Dongsheng; Chen, Haiyan; Zhang, Zhanying; Fang, Weikai; Lu, Zeyuan; Li, Yanlei; Ji, Yufeng; Guan, Yifu; Xu, Chidong; Jiang, Haihe

    2016-01-01

    In order to detect miRNA quickly, we designed a new portable device for the rapid detection of miRNA, using Opto-electronic detection technology, marking miRNA and isothermal rolling circle amplification and detecting markers which excite fluorescence intensity, the recognition system of characteristic fluorescence analysis was established. By changing the excitation light intensity, miRNA reagent concentration and other parameters, we arrive at the conclusion that there is the linear relationship (R2=0.9947) between miRNA concentration and fluorescence intensity when the miRNA concentration range the instrument can measure is in the range of 0.01-0.1mol and the lowest values measured by the instrument in theory is 7 copies.

  3. Rapid Detection and Quantitative Estimation of Type A Botulinum Toxin by Electroimmunodiffusion

    PubMed Central

    Miller, Carol A.; Anderson, Arthur W.

    1971-01-01

    An experimental system is described for the detection and quantitative estimation of type A botulinum toxin by electroimmunodiffusion. The method is shown to be rapid, specific, and quantitative. As little as 14 mouse LD50 per 0.1 ml of type A toxin was detected within 2 hr. When applied to experimentally contaminated foods such as canned tuna, pumpkin, spinach, green beans, and sausage, the technique detected botulinum toxin rapidly and identified it as to type and quantity. A specific rabbit type A antitoxin was produced for this in vitro system since the equine antitoxin (Center for Disease Control) tested in this experiment was found to be unsuitable. Images PMID:5005291

  4. Rapid detection of hemagglutination using restrictive microfluidic channels equipped with waveguide-mode sensors

    NASA Astrophysics Data System (ADS)

    Ashiba, Hiroki; Fujimaki, Makoto; Awazu, Koichi; Fu, Mengying; Ohki, Yoshimichi; Tanaka, Torahiko; Makishima, Makoto

    2016-02-01

    Hemagglutination is utilized for various immunological assays, including blood typing and virus detection. Herein, we describe a method of rapid hemagglutination detection based on a microfluidic channel installed on an optical waveguide-mode sensor. Human blood samples mixed with hemagglutinating antibodies associated with different blood groups were injected into the microfluidic channel, and reflectance spectra of the samples were measured after stopping the flow. The agglutinated and nonagglutinated samples were distinguishable by the alterations in their reflectance spectra with time; the microfluidic channels worked as spatial restraints for agglutinated red blood cells. The demonstrated system allowed rapid hemagglutination detection within 1 min. The suitable height of the channels was also discussed.

  5. Rapid detection and quantification of impact damage in composite structures

    NASA Technical Reports Server (NTRS)

    Smith, Barry T.

    1992-01-01

    It is shown that a multidisciplinary nondestructive evaluation approach for impact damage detection in composite structures can be used to produce a more efficient inspection. The multidisciplinary NDE approach relies on fast large area thermographic inspections along with detailed ultrasonic volumetric imaging. The thermal inspection technique rapidly identifies the impact damage. The ultrasonic volumetric imaging quantifies the impact generated delaminations through the volume of the structure.

  6. Rapid Chemical Vapor Detection Using Optofluidic Ring Resonators

    NASA Astrophysics Data System (ADS)

    Sun, Yuze; Shopova, Siyka I.; White, Ian M.; Frye-Mason, Greg; Fan, Xudong

    The optofluidic ring resonator (OFRR) is a novel gas sensing technology platform. In an OFRR gas sensor, the OFRR interior surface is coated with a layer of vapor-sensitive polymer. The interaction between the polymer and the gas molecules flowing through the OFRR results in a change in polymer refractive index and thickness, which can be detected by the circulating waveguide modes supported by the circular cross section of the OFRR. Due to the excellent fluidics of a capillary, the OFRR is capable of detecting chemical vapors rapidly with very low sample volume. In addition, the OFRR is highly compatible with gas chromatography (GC) and is a promising platform for development of micro-GC (μGC) with unique multipoint, on-column detection capability. In this chapter, we will discuss the fundamental operational principles of the OFRR gas sensor, followed by examples of rapid detection of several representative vapor analytes. The development of an OFRR-based μGC system and its applications in explosive separation and detection will also be presented.

  7. Rapid detection of sacbrood virus in honeybee using ultra-rapid real-time polymerase chain reaction.

    PubMed

    Yoo, Mi-Sun; Thi, Kim Cuc Nguyen; Van Nguyen, Phu; Han, Sang-Hoon; Kwon, Soon-Hwan; Yoon, Byoung-Su

    2012-01-01

    A real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for the fast and highly sensitive detection of the sacbrood virus (SBV) genome and applied to honeybee samples. Using plasmid DNA containing a partial SBV genome and diluted serially, as few as 1×10(2)copies/μl (correlation co-efficiency >0.99) were detected by the qRT-PCR assay, whereas 1×10(3)copies/μl were detected by the conventional RT-PCR assay. As a rapid detection method, ultra-rapid real-time PCR (URRT-PCR) was carried out with a GenSpector TMC-1000 silicon-glass chip-based thermal cycler, which has a 6μl micro-chamber volume and a fast outstandingly heating/cooling rate. Using this method, 10(3)copies of pBX-SBV3.8 clone were detected within 17 min after 40 PCR cycles, including melting point analysis. To reduce the detection time for SBV, synthesis of the cDNA of the SBV genome from a honeybee sample was attempted for different reaction times and the cDNA was used as the template for URRT-PCR assays. The results indicated that a 5 min reaction time was sufficient to synthesize cDNA as the template for the SBV URRT-PCR assay. This study described a novel PCR-based method that is able to detect an RNA virus in environmental samples within 22 min, including reverse transcription, PCR detection and melting point analysis in real-time.

  8. Outlier detection method in GEEs.

    PubMed

    Pardo, María Del Carmen; Hobza, Tomáš

    2014-09-01

    The generalized estimating equations (GEEs) method has become quite useful in modeling correlated data. However, diagnostic tools to check that the selected final model fits the data as accurately as possible have not been explored intensively. In this paper, an outlier detection tech