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Sample records for rapid detection method

  1. 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.

  2. FACS technology used in a new rapid bacterial detection method.

    PubMed

    Schmidt, M; Hourfar, M K; Nicol, S-B; Spengler, H-P; Montag, T; Seifried, Erhard

    2006-10-01

    Culture methods for bacterial detection (BacT/ALERT or Pall eBDS) are currently implemented in blood donor screening procedures in many countries. Experience in the first years after implementation of these detection assays showed that although the analytical sensitivity was extremely high (about 1 CFU mL(-1)), the majority of these platelets were still transfused before a positive screening result was attained. Rapid technologies were developed to more effectively prevent transfusion-transmitted bacterial infection. In this study, a new rapid bacterial detection method based on fluorescence-activated cell sorting (FACS) technology was developed. Bacteria were stained with thiazole orange dye for 5 min and measurement was taken immediately after staining. The entire process took only 30 min. Six transfusion-relevant bacteria strains were tested in a spiking study. Without pre-incubation in a special bacteria growth medium, analytical sensitivity ranged between 10(5) CFU mL(-1) (Klebsiella oxytoca and Serratia marcesens) and 10(3) CFU mL(-1) (Escherichia coli). Sensitivity could be improved to 10(1) CFU mL(-1) for all tested bacteria by adding a pre-incubation step (6 h at 37 degrees C). Although preliminary in nature, results of our study suggest that bacterial detection by FACS technology in conjunction with a pre-incubation step offers a sensitive alternative technology to culture methods. Additionally, it provides the benefit of a rapid test time and the opportunity of preventing bacterial transmitted infections more effectively.

  3. 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

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

    PubMed

    Ji, Wei; Yao, Weirong

    2015-06-01

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

  5. 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.

  6. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Rapid Methods for the Detection of General Fecal Indicators

    EPA Science Inventory

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

  9. Comparison of rapid cultural methods for the detection of Salmonella species.

    PubMed

    Margot, H; Stephan, R; O'Mahony, E; Iversen, C

    2013-05-15

    Three newly developed rapid cultural methods (Rapid Salmonella, Precis™ Salmonella, IBISA Salmonella) for the detection of Salmonella spp. were compared to a reference method. All methods performed comparably on inclusivity/exclusivity testing. Similar limits of detection were observed for all methods with milk, cocoa and bouillon matrices. Some tea varieties appeared to disturb the normal color formation of all selective agars tested.

  10. [Development of sample pretreatment techniques-rapid detection coupling methods for food security analysis].

    PubMed

    Huang, Yichun; Ding, Weiwei; Zhang, Zhuomin; Li, Gongke

    2013-07-01

    This paper summarizes the recent developments of the rapid detection methods for food security, such as sensors, optical techniques, portable spectral analysis, enzyme-linked immunosorbent assay, portable gas chromatograph, etc. Additionally, the applications of these rapid detection methods coupled with sample pretreatment techniques in real food security analysis are reviewed. The coupling technique has the potential to provide references to establish the selective, precise and quantitative rapid detection methods in food security analysis.

  11. Rapid and Sensitive Plate Method for Detection of Aspergillus fumigatus

    PubMed Central

    Bauters, T. G. M.; Nelis, H. J.

    2000-01-01

    The routine identification of Aspergillus fumigatus in clinical samples involves, apart from direct examination, the isolation of the organism on a plate followed by its microscopic characterization. This approach lacks sensitivity, specificity, and speed. A new procedure has been developed combining microcolony formation on a nylon membrane filter at 45°C with the detection of a specific 4-methylumbelliferyl-α-l-arabinopyranoside cleaving enzyme activity in digitonin permeabilized cells. The test takes approximately 14 h and has an efficiency of 98.2% and false-positive and -negative rates of 0 and 3.1%, respectively. When applied to 188 clinical samples taken from patients with proven or nonproven presence of Aspergillus species, a good agreement with the conventional plate-microscopy method was obtained. PMID:11015405

  12. 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.

  13. 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

  14. The Most Probable Limit of Detection (MPL) for rapid microbiological methods.

    PubMed

    Verdonk, G P H T; Willemse, M J; Hoefs, S G G; Cremers, G; van den Heuvel, E R

    2010-09-01

    Classical microbiological methods have nowadays unacceptably long cycle times. Rapid methods, available on the market for decades, are already applied within the clinical and food industry, but the implementation in pharmaceutical industry is hampered by for instance stringent regulations on validation and comparability with classical methods. Equivalence studies become less relevant when rapid methods are able to detect only one single microorganism. Directly testing this capability is currently impossible due to problems associated with preparing a spiked sample with low microbial counts. To be able to precisely estimate the limit of detection of rapid absence/presence tests, the method of the most probable limit is presented. It is based on three important elements; a relatively precise quantity of microorganisms, a non-serial dilution experiment and a statistical approach. For a set of microorganisms, a limit of detection of one was demonstrated using two different rapid methods.

  15. 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.

  16. A rapid and simple method for the detection and enumeration of Escherichia coli in cleansed shellfish.

    PubMed Central

    Humphrey, T. J.; Gawler, A. H.

    1986-01-01

    A multiple-tube technique based on peptone water incubated at 44 degrees C for 24 h followed by detection of indole was found to be sensitive and specific for the detection of Escherichia coli in oysters and mussels. The method has the advantage of providing rapid results and is both less expensive and less time-consuming than other MPN techniques. PMID:3537117

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2014-05-01

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

  19. 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.

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

    SciTech Connect

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

    2013-01-22

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

  1. 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. PMID:19619859

  2. Method for rapid detection of viable Escherichia coli in water using real-time NASBA.

    PubMed

    Heijnen, Leo; Medema, Gertjan

    2009-07-01

    A rapid real-time NASBA method was developed for detection of Escherichia coli in water samples. In this method, a fragment of the clpB-mRNA is amplified and a specific molecular beacon probe is used to detect the amplified mRNA fragment during the NASBA reaction. The method was shown to be specific and sensitive (1 viable E. coli in 100ml) and can be performed within 3-4h. Different inactivation processes (starvation, heat, UV-irradiation and chlorine) were employed to study the relationship between culturability and the ability to detect E. coli using NASBA. Detection of clpB-mRNA correlated with culturability after starvation or chlorine treatment. After UV-irradiation or heat-inactivation, detection of the increase in production of clpB-mRNA in viable E. coli cells after heat-shock induction correlated with culturability. Application of the NASBA method on tap water, treated sewage and surface water samples showed that culture and NASBA yielded comparable results in these different matrices. This study demonstrates that the NASBA method has high potential as a rapid test for microbiological water quality monitoring.

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

    NASA Astrophysics Data System (ADS)

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

    1995-10-01

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

  4. 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.

  5. 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

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

    PubMed

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

    2014-09-20

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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Development of a rapid and simple method for detection of protein contaminants in carmine.

    PubMed

    Nakayama, Norihisa; Ohtsu, Yutaka; Maezawa-Kase, Daisuke; Sano, Ken-Ichi

    2015-01-01

    Protein contaminants in carmine can cause dyspnea and anaphylactic reactions in users and consumers of products containing this pigment. The method generally used for detection of proteins in carmine has low reproducibility and is time-consuming. In this study, a rapid, simple, and highly reproducible method was developed for the detection of protein contaminants in carmine. This method incorporates acidic protein denaturation conditions and ultrafiltration. To prevent protein aggregation, sodium dodecyl sulfate containing gel electrophoresis running buffer was used for dispersing the carmine before filtration. An ultrafiltration device was used to separate the protein contaminants from carminic acid in the carmine solution. Two ultrafiltration devices were compared, and a cylindrical device containing a modified polyethersulfone membrane gave the best results. The method had high reproducibility. PMID:25892994

  12. Development of a Rapid and Simple Method for Detection of Protein Contaminants in Carmine

    PubMed Central

    Nakayama, Norihisa; Ohtsu, Yutaka; Maezawa-Kase, Daisuke; Sano, Ken-Ichi

    2015-01-01

    Protein contaminants in carmine can cause dyspnea and anaphylactic reactions in users and consumers of products containing this pigment. The method generally used for detection of proteins in carmine has low reproducibility and is time-consuming. In this study, a rapid, simple, and highly reproducible method was developed for the detection of protein contaminants in carmine. This method incorporates acidic protein denaturation conditions and ultrafiltration. To prevent protein aggregation, sodium dodecyl sulfate containing gel electrophoresis running buffer was used for dispersing the carmine before filtration. An ultrafiltration device was used to separate the protein contaminants from carminic acid in the carmine solution. Two ultrafiltration devices were compared, and a cylindrical device containing a modified polyethersulfone membrane gave the best results. The method had high reproducibility. PMID:25892994

  13. 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

  14. Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

    PubMed Central

    Xing, Jian-Ming; Zhang, Su; Du, Ying; Bi, Dan; Yao, Li-Hui

    2009-01-01

    AIM: To develop a new, rapid and accurate reverse dot blot (RDB) method for the detection of intestinal pathogens in fecal samples. METHODS: The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7, Clostridium botulinum, Bacillus cereus, Clostridium perfringens, Vibrio parahaemolyticus, Shigella spp., Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes and Staphylococcus aureus. The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested. Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay. RESULTS: The methods could identify the 12 intestinal pathogens specifically, and the detection limit was as low as 103 CFUs. The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%. CONCLUSION: The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples. PMID:19469006

  15. Comparison of rapid methods of detection of cytomegalovirus in saliva with virus isolation in tissue culture.

    PubMed

    Warren, W P; Balcarek, K; Smith, R; Pass, R F

    1992-04-01

    Two rapid methods for the detection of cytomegalovirus (CMV) in saliva from congenitally and perinatally infected children were assessed by comparison with traditional virus isolation in tissue culture (TC). The polymerase chain reaction (PCR) was used to amplify a 300-bp segment of the CMV gB gene which was detected in ethidium bromide-stained agarose gels. A centrifugation-enhanced microtiter culture method with a monoclonal antibody for the detection of early-antigen fluorescent foci (DEAFF) was also used. Saliva specimens were collected with mouth swabs from children who were between the ages of 1 month and 14 years and who had either prenatal or perinatal CMV infection. One hundred sixty samples were tested by PCR and TC; 65 (40.6%) were found positive by TC, and 58 (36.8%) were found positive by PCR. Although four samples were found positive by PCR and negative by TC, saliva from seronegative and seropositive TC-negative adults were never found positive by PCR. One hundred fifty-two samples were tested by DEAFF and TC; 64 (42.1%) were found positive by TC, and 58 (38.2%) were found positive by DEAFF. With TC results as a standard, the sensitivity and specificity of DEAFF were, respectively, 90.6 and 97.7%. Because of the greater ease of collecting saliva than urine from newborns, both of these rapid methods merit evaluation in screening for congenital infection.

  16. 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

  17. 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

  18. [Research on the method for rapid detection of soil moisture content using spectral data].

    PubMed

    Song, Tao; Bao, Yi-Dan; He, Yong

    2009-03-01

    Spectroscopy technique is one of the qualitative and quantitative analytical techniques developed quickly in recent years. The spectral analysis is a fast and non-destructive method and has been used in many fields such as oil industry, food industry and so on. In the present paper, the spectral band sensitive to soil moisture content was found from the visible/near infrared spectra and a monadic linear regression model based on the data of sensitive spectral band was applied to develop a method for rapid detection of soil moisture content. The spectral data of 52 soil samples were collected by using FieldSpec HandHeld spectroradiometer made by ASD (Analytical Spectral Device) company in the US, and the data of soil moisture content were obtained by experiment. The spectral band sensitive to soil moisture content was achieved by correlation coefficient method. Then, the data of sensitive spectral band were used to build monadic linear regression model of soil moisture content. Finally, the model was employed for the prediction of soil moisture content. Correlation coefficient (r) of prediction and root mean square error of prediction (RMSEP) were used as the evaluation standards. The results indicated that the r and RMSEP for the prediction of soil moisture content were 0.966 5 and 0.012 1 respectively. Thus, it is concluded that the method used in this paper is an available method for the rapid detection of soil moisture content based on the visible/near-infrared spectra.

  19. Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese.

    PubMed

    Botsaris, George; Slana, Iva; Liapi, Maria; Dodd, Christine; Economides, Constantinos; Rees, Catherine; Pavlik, Ivo

    2010-07-31

    Mycobacterium avium subsp. paratuberculosis (MAP) may have a role in the development of Crohn's disease in humans via the consumption of contaminated milk and milk products. Detection of MAP from milk and dairy products has been reported from countries on the European continent, Argentina, the UK and Australia. In this study three different methods (quantitative real time PCR, combined phage IS900 PCR and conventional cultivation) were used to detect the presence of MAP in bulk tank milk (BTM) and cheese originating from sheep, goat and mixed milks from farms and products in Cyprus. During the first survey the presence of MAP was detected in 63 (28.6%) of cows' BTM samples by quantitative real time PCR. A second survey of BTM used a new combined phage IS900 PCR assay, and in this case MAP was detected in 50 (22.2%) samples showing a good level of agreement by both methods. None of the herds tested were known to be affected by Johne's disease and the presence of viable MAP was confirmed by conventional culture in only two cases of cows BTM. This suggests that either rapid method used is more sensitive than the conventional culture when testing raw milk samples for MAP. The two isolates recovered from BTM were identified by IS1311 PCR REA as cattle and sheep strains, respectively. In contrast when cheese samples were tested, MAP DNA was detected by quantitative real time PCR in seven (25.0%) samples (n=28). However no viable MAP was detected when either the combined phage IS900 PCR or conventional culture methods were used.

  20. 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

  1. [Rapid detection of extended-spectrum beta-lactamases by flow cytometry method].

    PubMed

    Duyan, Serhat; Kılıç, Abdullah; Yılmaz, Soner; Ardıç, Nurittin

    2015-10-01

    Extended-spectrum beta-lactamases (ESBL), produced by Enterobacteriaceae members are enzymes that especially cause a resistance to cephalosporin group antibiotics commonly used in clinics. Early and rapid detection of ESBL production is crucial for antimicrobial treatment and infection control; however the methods used for this purpose are time consuming (24 to 48 hours). The aim of this study was to determine a flow cytometry based-test which provides to detect ESBL producing bacteria in a short time. A total of 38 ESBL-producing (29 Escherichia coli, 9 Klebsiella pneumoniae) and 10 non-producing (5 E.coli, 5 K.pneumoniae) Enterobacteriaceae strains isolated between 2012 and 2013 were included in this study. The identification and antibiotic susceptibility tests of the isolates were performed by using Phoenix(TM) 100 automated system (Becton Dickinson, USA). The presence of bla(TEM), bla(SHV), bla(CTX-M1), bla(CTX-M2) and bla(CTX-M9) genes were investigated in ESBL positive isolates via polymerase chain reaction method. At least one of the ESBL genes were detected in 36 out of 38 isolates and no genes were detected in two E.coli isolates. In flow cytometric method, the percentages of death cells exposed to cephalosporin [(ceftazidime (CAZ) or cefotaxime (CTX)] and clavulanic acid (CLA) combination, were compared with death cells exposed only to cephalosporin (CAZ or CTX). CLA index values (CAZ-CLA and CTX-CLA indices) were obtained for CTX and CAZ. Index values which was higher than 1.5 just for one cephalosporin were accepted as GSBL positive. The mean index values for CTX-CLA in ESBL positive strains according to their genotypic characteristics were between 1.14 and 7.22, while those values for CAZ-CLA were between 0.85 and 5.6. When the two groups of 38 ESBL positive and 10 ESBL negative strains were evaluated, statistically significant difference was detected for both CAZ-CLA and CTX-CLA indices (p< 0.005). CTX-CLA indices (p= 0.001) shown a better

  2. Evaluation of Colorimetric Methods Using Nicotinamide for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis▿

    PubMed Central

    Mirabal, Niuris C.; Yzquierdo, Sergio L.; Lemus, Dihadenys; Madruga, Mariela; Milián, Yoslaine; Echemendía, Miguel; Takiff, Howard; Martin, Anandi; Van der Stuyf, Patrick; Palomino, Juan Carlos; Montoro, Ernesto

    2010-01-01

    The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is sufficiently difficult that many laboratories do not attempt it. Most pyrazinamide resistance is caused by mutations that inactivate the pyrazinamidase enzyme needed to convert the prodrug pyrazinamide to its active form. We evaluated two newer and simpler methods to assess pyrazinamidase activity, the nitrate reductase and malachite green microtube assays, using nicotinamide in place of pyrazinamide. A total of 102 strains were tested by these methods and the results compared with those obtained by the classic Wayne assay. Mutations in the pncA gene were identified by sequencing the pncA genes from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the pncA gene were found in 14 of 16 strains that were pyrazinamide resistant and in 1 of 4 strains that were sensitive by the Wayne assay. Both of these simple methods, used with nicotinamide, are promising and inexpensive alternatives for the rapid detection of pyrazinamide resistance in limited-resource countries. PMID:20554826

  3. 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

  4. 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

  5. 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.

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

    PubMed Central

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

    2014-01-01

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

  7. Fluorescein: a rapid, sensitive, nonlethal method for detecting skin ulceration in fish.

    PubMed

    Noga, E J; Udomkusonsri, P

    2002-11-01

    There is a need to develop simple, rapid, and accurate methods for assessing health in fish populations. In this study we demonstrate that use of fluorescein, a nontoxic fluorescent dye, can rapidly and easily detect the presence of skin ulcers in all fish tested, including rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus punctatus), goldfish (Carassius auratus), and hybrid striped bass (Morone saxatilis male X M. chrysops female). Exposure of fish to as little as 0.10 mg fluorescein per milliliter of water for 3 minutes was sufficient to identify experimentally induced lesions, even pinpoint ulcerations. Such lesions were not visible to the naked eye but were clearly demarcated with fluorescein treatment. Examination of fish that appeared clinically normal often revealed the presence of focal ulcerations, which might have been a consequence of damage during capture, but it also might suggest that skin ulceration may be common even in "clinically normal" fish. Exposure of either nonulcerated or experimentally ulcerated hybrid striped bass to an excessively high concentration of fluorescein had no apparent effect on health or survival. Our studies suggest that fluorescein may be a highly useful tool for rapid health screening in fish populations.

  8. [Rapid biochemical method for the detection of paralytical shellfish poisoning toxins in shellfish from seafood market].

    PubMed

    Liu, Yuan-yuan; Cheng, Jin-ping; Gao, Li-li; Wang, Jin-hui; Song, Yu-ling; Chen, Xue; Wang, Wen-hua

    2010-07-01

    A rapid biochemical method was discussed in order to detect low concentration of paralytic shellfish poisoning (PSP) in sea food. The mice were injected with PSP extract of bivalves (1 and 0.2 microg/kg respectively, as STX equivalents) purchased from seafood market. ACh, AChE, NO and NOS in blood were studied at 15, 60, 120 min respectively. The results showed that at low dose (0.2 microg/kg) and 15 min, only the contents of ACh changed significantly compared with control group (p < 0.05), which was (141.2 +/- 14.8) microg/mg, while the contents of NO and the activities of NOS changed until 120 min, compared to control group (p < 0.05) ,which were (68.7 +/- 3.8) micromol/g and (40.1 +/- 4.9) U/mg respectively. At high dose the contents of ACh changed at all three time point. It can be suggested that the contents of ACh is the only one of four indexes which can response to PSP at low dose in an early stage (15 min) and may be selected as a biochemical index for rapid detection of PSP.

  9. 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.

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

    PubMed Central

    Chappell, Cynthia; Gonzales, Christopher; Okhuysen, Pablo

    2015-01-01

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

  11. 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.

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

    PubMed

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

    2015-08-01

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

  13. Simple, Rapid and Inexpensive Quantitative Fluorescent PCR Method for Detection of Microdeletion and Microduplication Syndromes

    PubMed Central

    Stofanko, Martin; Gonçalves-Dornelas, Higgor; Cunha, Pricila Silva; Pena, Heloísa B.; Vianna-Morgante, Angela M.; Pena, Sérgio Danilo Junho

    2013-01-01

    Because of economic limitations, the cost-effective diagnosis of patients affected with rare microdeletion or microduplication syndromes is a challenge in developing countries. Here we report a sensitive, rapid, and affordable detection method that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR). Our procedure is based on the finding of genomic regions with high homology to segments of the critical microdeletion/microduplication region. PCR amplification of both using the same primer pair, establishes competitive kinetics and relative quantification of amplicons, as happens in microsatellite-based Quantitative Fluorescence PCR. We used patients with two common microdeletion syndromes, the Williams-Beuren syndrome (7q11.23 microdeletion) and the 22q11.2 microdeletion syndromes and discovered that MQF-PCR could detect both with 100% sensitivity and 100% specificity. Additionally, we demonstrated that the same principle could be reliably used for detection of microduplication syndromes, by using patients with the Lubs (MECP2 duplication) syndrome and the 17q11.2 microduplication involving the NF1 gene. We propose that MQF-PCR is a useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes, ideally suited for use in developing countries, but having general applicability as well. PMID:23620743

  14. 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

  15. 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.

  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. 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.

  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. A simple, rapid, sensitive method detecting homoserine lactone (HSL)-related compounds in microbial extracts.

    PubMed

    Singh, Maya Prakash; Greenstein, Michael

    2006-04-01

    A simple, rapid, sensitive microtiter plate method detecting N-acyl homoserine lactone (HSL)-related compounds was established using an Agrobacterium tumefaciens strain harboring a traG::lacZ/traR reporter gene responsive to HSLs. This strain did not produce its own HSL, but the traG::lacZ reporter gene was induced only when its transcription activator TraR detected a cognate exogenous HSL. Therefore, the assay was expected to be highly specific for HSL-related compounds. Induction of the reporter gene, leading to production of beta-galactosidase enzyme, was measured by using two different beta-galactosidase substrates, X-gal and Galacton-Star, for colorimetric and chemiluminometric detection, respectively. The screen was validated in both the 96-well and 384-well plate formats, and extracts derived from 696 different microbial isolates, mostly unidentified actinomycetes isolated from diverse locations, were tested. Crude extracts of 81 (11.64%) cultures tested positive for HSL-related compounds, and an additional 34 (4.8%) crude extracts showed a moderate to weak signal for HSLs. Data from the fractionated samples, however, suggested a much higher prevalence of HSL signals in these extracts. Of 144 crude extracts fractionated into 10 individual samples at a 10x concentration, 72 (50%) cultures tested positive for HSLs. Six cultures were active only in the crude extract, 18 were active both in crude and one or more of their fractions, and an additional 48 were active in just one or more of their fractions. This finding may be the first to suggest such a high prevalence of HSL-signals found in nature, and a large number of actinomycetes in our collection appeared to produce HSL-related compounds.

  20. A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water.

    PubMed

    Taguri, Toshitsugu; Oda, Yasunori; Sugiyama, Kanji; Nishikawa, Toru; Endo, Takuro; Izumiyama, Shinji; Yamazaki, Masayuki; Kura, Fumiaki

    2011-07-01

    Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0×10(3)(3.48log)counts mL(-1). We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥3.48 or <3.48log total bacterial counts mL(-1) were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for

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

    PubMed

    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

  3. 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

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

    PubMed

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

    2014-12-29

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

  5. A rapid method for detection of five known mutations associated with aminoglycoside-induced deafness

    PubMed Central

    Bardien, Soraya; Human, Hannique; Harris, Tashneem; Hefke, Gwynneth; Veikondis, Rene; Schaaf, H Simon; van der Merwe, Lize; Greinwald, John H; Fagan, Johan; de Jong, Greetje

    2009-01-01

    Background South Africa has one of the highest incidences of multidrug-resistant tuberculosis (MDR-TB) in the world. Concomitantly, aminoglycosides are commonly used in this country as a treatment against MDR-TB. To date, at least five mutations are known to confer susceptibility to aminoglycoside-induced hearing loss. The aim of the present study was to develop a rapid screening method to determine whether these mutations are present in the South African population. Methods A multiplex method using the SNaPshot technique was used to screen for five mutations in the MT-RNR1 gene: A1555G, C1494T, T1095C, 961delT+C(n) and A827G. A total of 204 South African control samples, comprising 98 Mixed ancestry and 106 Black individuals were screened for the presence of the five mutations. Results A robust, cost-effective method was developed that detected the presence of all five sequence variants simultaneously. In this pilot study, the A1555G mutation was identified at a frequency of 0.9% in the Black control samples. The 961delT+C(n) variant was present in 6.6% of the Black controls and 2% of the Mixed ancestry controls. The T1095C, C1494T and A827G variants were not identified in any of the study participants. Conclusion The frequency of 0.9% for the A1555G mutation in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. Future larger studies are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. The high frequencies of the 961delT+C(n) variant observed in the controls suggest that this change is a common non-pathogenic polymorphism. This genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is important in a low-resource country like South Africa where, despite their adverse side-effects, aminoglycosides will continue to be used

  6. Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water.

    PubMed Central

    Arias, C R; Garay, E; Aznar, R

    1995-01-01

    A nested PCR for the detection of Vibrio vulnificus in fish farms was developed as an alternative to cultural methods by using universal primers flanking the V. vulnificus-specific sequences directed against 23S rRNA genes. This specific assay detected 10 fg of DNA or 12 to 120 cells in artificially inoculated samples without enrichment and within 24 h. PMID:7574657

  7. Detection of indoor PCB contamination by thermal desorption of dust. A rapid screening method?

    PubMed

    Wilkins, Ken; Bøwadt, Søren; Larsen, Kjeld; Sporring, Sune

    2002-01-01

    Although PCB in caulking materials has been forbidden for many years in most of Europe, including Denmark, there has been continued interest to measure PCB levels in the air of contaminated buildings and blood of the occupants (Mengon and Schlatter 1993, Fromme et al. 1996, Ewers et al. 1998, Currado and Harrad 1998, Gabrio et al. 2000). The relatively low priority for investigations of this contamination is probably due to the small quantities inhaled compared to exposure via food, and the rapid metabolism of the most volatile congeners demonstrated by low concentrations of all congeners in the blood of exposed persons (Ewers et al. 1998, Gabrio et al. 2000). There is, however, evidence that PCB containing caulking materials have been used even during the '90s (Fromme et al. 1996). In Denmark, it is estimated that 75 t PCB is still in buildings (Organization of Sealant Branch's Manufacturers and Distributors 2000). During an investigation of dust from buildings with excessive microbial growth (including 35 rooms from 9 buildings), the analysis of semivolatile compounds by thermal desorption-GC/MS of samples from a single building surprisingly revealed large amounts of PCBs containing 3, 4 and 5 chlorine atoms, 10-20 times the amounts found in samples from other buildings. Extraction of the dust by SFE followed by GC/ECD analysis for 12 PCB congeners showed that there was approximately 20 times the total PCB concentrations in dust from the polluted building compared to the levels in the other buildings. Subsequent headspace analysis of caulking material from the polluted building revealed this to be the source. Shelf dust functions as a passive sampling medium and, thus, can be used as a screening method to detect PCB and other semivolatile pollution indoors.

  8. 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

  9. 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.

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

    PubMed

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

    2015-12-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene.

    PubMed

    Wei, Xiuying; Shi, Xingming; Zhao, Yan; Zhang, Jing; Wang, Mei; Liu, Changjun; Cui, Hongyu; Hu, Shunlei; Quan, Yanming; Chen, Hongyan; Wang, Yunfeng

    2012-08-01

    A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

  13. Validation and operational application of a rapid method for rabies antigen detection.

    PubMed

    Saturday, Greg A; King, Robin; Fuhrmann, Leslie

    2009-01-01

    The Veterinary Laboratory Europe (VLE) deployed a direct, rapid immunohistochemical test (DRIT), developed by the Centers for Disease Control and Prevention (CDC), to the US military veterinary units in Iraq and Afghanistan. The test detects rabies virus antigen in fresh and frozen brainstem samples from various animal species. The goal was to conduct surveillance in 2 countries without current rabies diagnostic capabilities and little historical data on rabies prevalence. During the deployment the DRIT was evaluated, and Veterinary Corps officers and technicians were trained to operate the test. Civilian veterinarians from both Iraq and Afghanistan were organized for a lecture forum on rabies, a sample collection lab, and familiarization with the DRIT. Samples collected in Iraq and Afghanistan were tested in the field with the DRIT, and compared to the traditional laboratory standard utilizing direct fluorescent antibody testing at VLE and the CDC with 100% agreement.

  14. A rapid method for the detection of tryptophanase in anaerobic bacteria.

    PubMed

    Karim, M R; Qadri, S M; Flournoy, D J

    1981-01-01

    A total of 633 anaerobic bacteria were examined for tryptophanase production using a rapid method which distinguishes within 5 to 180 minutes between anaerobes that contain tryptophanase and those that do not. Of the 196 tryptophanase-positive isolates tested, 99% showed tryptophanase activity within 2 hours as compared with 94.4% in 24 hours by a conventional method. A total of 299 tryptophanase-negative organisms were tested. Ninety three percent of these remained negative after 24 hours as compared with 95.3% when tested within a 24-h conventional method. Additional information was obtained on the sensitivity of this test and the time-dependent production of indole by tryptophanase.

  15. 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.

  16. The development of an efficient and rapid enzyme linked fluorescent assay method for the detection of Listeria spp. from foods.

    PubMed

    Sewell, A M; Warburton, D W; Boville, A; Daley, E F; Mullen, K

    2003-03-15

    Conventional isolation methods, including the Health Products and Food Branch (HPFB), Health Canada method used for the isolation and identification of Listeria species and Listeria monocytogenes from foods and environmental samples, can take a week or more to complete and are usually labor-intensive. This has led to the development of various rapid methods which attempt to generate results comparable to standard methods but in a reduced time-frame with less hands-on operation. Our previous work with rapid detection systems indicated that the recommended enrichment protocols failed to grow the Listeria to detectable levels in a reliable and consistent manner. In the present study, a novel enrichment protocol is described and consists of samples being pre-enriched in Palcam broth (incubated at 35 degrees C for 26 h), enriched in UVM 2 (30 degrees C for 26 h) and then plated and analysed by a rapid detection kit, with results being generated after 52 h of incubation. In total, 200 naturally contaminated samples were analysed by both the HPFB standard method and the Palcam method. The results showed that the Palcam method is comparable to the HPFB method. Further analysis involved a rapid detection system, which applies ELISA techniques and automation in an enzyme-linked fluorescent assay (ELFA) system. This system, referred to as the Vitek Immuno Diagnostic Assay System or VIDAS, can identify Listeria to the genus or species (L. monocytogenes) level. In this comparison, an additional 324 naturally contaminated samples were analysed by both the Palcam and ELFA methods. The sensitivity and specificity of the ELFA method were 98.1% and 97.0%, respectively, while the efficiency was 97.5%. False-negative and false-positive rates were 1.9% and 3.0%, respectively. These results show that the ELFA method (when using the Palcam method for pre- and secondary enrichment) was efficient and gave reliable results after 52 h of incubation, and met Health Canada's criteria for

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

    PubMed Central

    Ortiz, A; Ritter, E

    1996-01-01

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

  18. 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

  19. A reverse transcription loop-mediated isothermal amplification method for rapid detection of bovine viral diarrhea virus.

    PubMed

    Fan, Qing; Xie, Zhixun; Xie, Liji; Liu, Jiabo; Pang, Yaoshan; Deng, Xianwen; Xie, Zhiqin; Peng, Yi; Wang, Xiuqing

    2012-12-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimized to detect bovine viral diarrhea viral (BVDV) RNA. The RT-LAMP assay is highly sensitive and able to detect 4.67×10(0)copies of BVDV RNA. Additionally, the RT-LAMP method is capable of detecting both genotypes of BVDV. No cross-reaction with other bovine viruses was observed. The ability of RT-LAMP to detect BVDV RNA from bovine fecal swabs was also evaluated. Of the 88 fecal swabs, 38 were found to be positive by RT-LAMP assay, whereas 39 were positive by real-time RT-PCR. Taken together, the BVDV specific RT-LAMP method is highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples.

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

    PubMed Central

    Tang, Qing; Tian, Shuguang; Yu, Nong; Zhang, Xi; Jia, Xiaodong; Zhai, Hongyan

    2016-01-01

    Aspergillus fumigatus is a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection of A. fumigatus infection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection of A. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatus strains, including 5 species of the Aspergillus genus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 102 copies for the same target. Clinical samples from a total of 69 patients with probable IA (n = 14) and possible IA (n = 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection of A. fumigatus in clinical testing has been developed. PMID:26791368

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

    PubMed

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

    2016-04-01

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

  2. 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.

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

    PubMed

    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

  4. 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

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

    PubMed

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

    2016-08-01

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

  6. 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.

  7. 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.

  8. Veterinary drug residues in meat: Concerns and rapid methods for detection.

    PubMed

    Reig, Milagro; Toldrá, Fidel

    2008-01-01

    The use of substances having hormonal or thyreostatic action as well as β-agonists is banned in the European Union. However, sometimes forbidden drugs may be added to feeds for illegal administration to farm animals for promoting increased muscle development or increased water retention and thus obtain an economical benefit. The result is a fraudulent overweight of meat but, what is worse, residues of these substances may remain in meat and may pose a real threat to the consumer either through exposure to the residues, transfer of antibiotic resistance or allergy risk. This has exerted a great concern among European consumers. The control of the absence of these forbidden substances in animal foods and feeds is regulated in the European Union by Directive96/23/EC on measures to monitor certain substances and residues in live animals and animal products. Analytical methodology, including criteria for identification and confirmation, for the monitoring of compliance was also given in Decisions 93/256/EEC and 93/257/EEC. More recently, Decision 2002/657/EC provided rules for the analytical methods to be used in testing of official samples. A crucial step is the screening of veterinary drug residues in live animals, feeds and animal products in view of the remarkable number of samples and large variety of residues to be analysed. In recent years, different rapid methods having easy performance, high sensitivity and high throughput have been proposed and are being extensively used. These methods as well as other new methods are reviewed in this manuscript.

  9. Development of a single aptamer-based surface enhanced Raman scattering method for rapid detection of multiple pesticides.

    PubMed

    Pang, Shintaro; Labuza, Theodore P; He, Lili

    2014-04-21

    The objective of this study was to develop a simple and rapid method that could detect and discriminate four specific pesticides (isocarbophos, omethoate, phorate, and profenofos) using a single aptamer-based capture procedure followed by Surface Enhanced Raman Spectroscopy (SERS). The aptamer is a single stranded DNA sequence that is specific to capture these four pesticides. The thiolated aptamer was conjugated onto silver (Ag) dendrites, a nanostructure that can enhance the Raman fingerprint of pesticides, through Ag-thiol bonds. It was then backfilled with 6-mercaptohexanol (MH) to prevent nonspecific binding. The modified SERS platform [Ag-(Ap + MH)] was then mixed with each pesticide solution (P) for 20 min. After capturing the pesticides, the Ag-(Ap + MH)-P complex was analyzed under a DXR Raman microscope and TQ Analyst software. The results show that the four pesticides can be captured and detected using principal component analysis based on their distinct fingerprint Raman peaks. The limits of detection (LODs) of isocarbophos, omethoate, phorate, and profenofos were 3.4 μM (1 ppm), 24 μM (5 ppm), 0.4 μM (0.1 ppm), and 14 μM (5 ppm) respectively. This method was also validated successfully in apple juice. These results demonstrated the super capacity of aptamer-based SERS in rapid detection and discrimination of multi-pesticides. This technique can be extended to detect a wide range of pesticides using specific aptamers. PMID:24551875

  10. Rapid detection and quantification of Cryptosporidium baileyi oocysts in feces and organs of chickens using a microscopic slide flotation method.

    PubMed

    Abbassi, H; Wyers, M; Cabaret, J; Naciri, M

    2000-03-01

    A simple semiquantitative microscopic slide flotation (MSF) method using modified Sheather's sugar solution (MSSS) is presented for the rapid detection and quantification of Cryptosporidium baileyi oocysts in the feces and mucosal and/or organ scrapings of chickens. Oocyst shedding was evaluated by examination of the surface of coverslips, and the average quantitative score (0-5) recorded for 10 microscopic fields (magnification x250) is reported. The equivalence between these scores and the actual number of oocysts counted per gram of feces was assessed (rs = 0.89; P < 0.001). The applicability of this method was tested by comparison of the kinetics of oocyst shedding in feces of inoculated chickens with those reported by other authors working under similar conditions. In organs the MSF method was compared to histology. Fewer false-negative results were obtained using MSF versus the histology method. The MSF method was particularly more efficient in tracheae with low levels of infection and in the lungs, regardless of the level of infection. The MSF method was also very efficient in detecting oocysts in air sacs from chickens with aerosacculitis. It provides a specific and sufficiently sensitive, simple, rapid, reliable, and low-cost means of diagnosing C. baileyi in the feces and organs of chickens. This method can be used in the routine diagnosis of cryptosporidia in chickens, and it could be extended to other avian species and used in epidemiology studies to evaluate the prevalence of cryptosporidiosis in fowl.

  11. A rapid RT-PCR based method for the detection of BCR-ABL translocation.

    PubMed Central

    Sidorova JYu; Saltykova, L B; Lyschov, A A; Zaritskey AYu; Abdulkadyrov, K M; Blinov, M N

    1997-01-01

    AIMS: To optimise a one step reverse transcriptase polymerase chain reaction (RT-PCR) protocol for BCR-ABL chimaera detection. METHODS: Compared with published RT-PCR procedures, this novel approach has at least two advantages. First, the same enzyme is used for both reverse transcription and PCR. Second, amplification of the target (BCR-ABL chimaera) and control gene (ABL) is performed simultaneously in the same tube. RESULTS: On testing 40 chronic myelogenous leukaemia patients and 10 healthy donors there was a specificity for the newly developed technique. In addition, dilution experiments demonstrated that the protocol was highly sensitive. CONCLUSIONS: The suggested one step PCR strategy is a simple and reliable way to reveal BCR-ABL chimaeras. Images PMID:9497918

  12. 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.

  13. 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.

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-08-16

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

  16. Detection of infective poliovirus by a simple, rapid, and sensitive flow cytometry method based on fluorescence resonance energy transfer technology.

    PubMed

    Cantera, Jason L; Chen, Wilfred; Yates, Marylynn V

    2010-01-01

    The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2A(pro)) as an indication of infection. Quantification of the infectious-virus titers was resolved by using flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2A(pro) were infected with different titers of PV1. After incubation at various time points, cells were harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.

  17. 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.

  18. Rapid deployment intrusion detection system

    SciTech Connect

    Graham, R.H.

    1997-08-01

    A rapidly deployable security system is one that provides intrusion detection, assessment, communications, and annunciation capabilities; is easy to install and configure; can be rapidly deployed, and is reusable. A rapidly deployable intrusion detection system (RADIDS) has many potential applications within the DOE Complex: back-up protection for failed zones in a perimeter intrusion detection and assessment system, intrusion detection and assessment capabilities in temporary locations, protection of assets during Complex reconfiguration, and protection in hazardous locations, protection of assets during Complex reconfiguration, and protection in hazardous locations. Many DOE user-need documents have indicated an interest in a rapidly deployable intrusion detection system. The purpose of the RADIDS project is to design, develop, and implement such a system. 2 figs.

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

    PubMed Central

    Hughes, J H

    1993-01-01

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

  20. 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.

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

    PubMed

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

    2013-11-27

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

  2. 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.

  3. Rapid method for detection of Coxiella burnetii antibodies using high-density particle agglutination.

    PubMed Central

    Nguyen, S V; Otsuka, H; Zhang, G Q; To, H; Yamaguchi, T; Fukushi, H; Noma, A; Hirai, K

    1996-01-01

    A high-density particle agglutination test, using erythrocyte-sensitizing substance from phase II Coxiella burnetii adsorbed to high-density composite particles, was developed for rapid serodiagnosis of Q fever. The test was compared with the microimmunofluorescence test for sensitivity and specificity by using 3,036 human serum samples collected in Gifu Prefecture, Japan. An excellent agreement was found between the two tests for the acute-phase group and paired serum samples, but some discordant results were observed in the single-sample group. The sensitivity and specificity of the high-density particle agglutination test were both 100% in the former group and 81.6 and 99.9%, respectively, in the latter group. The test is a very promising tool for routine serodiagnosis of Q fever because of its simplicity, sensitivity, and specificity. PMID:8940428

  4. 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.

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

    NASA Astrophysics Data System (ADS)

    Tudor, M.

    2015-08-01

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

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

    SciTech Connect

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

    2004-01-01

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

  7. A new spectroscopy method for in situ rapid detection and classification of micro-organisms

    NASA Astrophysics Data System (ADS)

    Garcia-Rubio, Luis H.; Alupoaei, Catalina E.; Olivares, Jose A.; Stark, Peter; Garcia-Lopez, Alicia; Stephans, Christie; Berg, Tracy; Klungness, Greta; Gennaccaro, Angela; Huffman, Debra E.

    2004-12-01

    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.

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

    McGarvey, Michael

    2010-01-01

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

  10. Testing and application of a refined rapid detection method for paralytic shellfish poisoning toxins in UK shellfish.

    PubMed

    Turner, Andrew D; Tarnovius, Sophie; Johnson, Sarah; Higman, Wendy A; Algoet, Myriam

    2015-06-15

    The Scotia Rapid Test for PSP is designed for qualitative identification of saxitoxins at levels in shellfish equivalent to the limit of detection of the biological reference method. However, issues with the method have been reported, including the low assay cross reactivity for some toxins, high numbers of false positive results and the subjective test interpretation. This study focussed on approaches to improve each of these issues. A refined test was found to improve GTX1&4 test sensitivity in samples containing high proportions of GTX1&4. The subjectivity of the test was successfully eliminated through use of an automated scanner, which enabled both the reliable identification of test results as well as the provision of a numerical result which could be utilised for more refined results interpretation. Finally the high proportion of false positive results in comparison with the LC-FLD was investigated, with a modified approach incorporating an additional extract dilution applied to a range of shellfish samples with different toxicities. Results showed highly variable limits of detection of the method and no significant reduction in false positive results when applying the additional dilution, which may be of concern to laboratories in receipt of high numbers of samples containing low concentrations of toxins.

  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

    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.

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

    PubMed

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

    2016-01-01

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

  14. 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

  15. Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture - nested PCR combination assay.

    PubMed

    Saroj, Sunil D; Shashidhar, R; Karani, Manisha; Bandekar, Jayant R

    2008-06-01

    A rapid nested PCR assay for detection of Salmonella from food was developed. The sensitivity of the assay developed was comparable to the traditional culture based methods with an advantage in reduction of assay time. The assay procedure with artificially contaminated samples was able to detect as low as 4CFU Salmonella/25g of food samples (sprout, carrot, cucumber and poultry meat). With two synthetic primers of 26 mer TS11 and 25 mer TS4, a 1.2kb fragment was amplified which served as a template for amplification of final 375bp product using TS11 and TS5 primers. No non-specific amplification from the native microbial flora of food samples was observed. The reaction generates a single band specific to Salmonella which allows the analyst to interpret data at ease and without any confusion. Enriched broth serves as template for the reaction which removes labour intensive DNA isolation procedures. In case of artificially contaminated samples, 6h enriched lactose broth can serve as template. However, for market samples where the organisms are under environmental stress, it is desirable to use template from Rappaport Vasiliadis medium. The assay also employes internal amplification control, which is amplified into a 300bp fragment and thus serves as positive control for the reaction and any possibility of false negative due to inhibitory action of food components on PCR reaction can be ruled out. PMID:18406104

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

    PubMed

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

    2012-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Novel real-time simultaneous amplification and testing method to accurately and rapidly detect Mycobacterium tuberculosis complex.

    PubMed

    Cui, Zhenling; Wang, Yongzhong; Fang, Liang; Zheng, Ruijuan; Huang, Xiaochen; Liu, Xiaoqin; Zhang, Gang; Rui, Dongmei; Ju, Jinliang; Hu, Zhongyi

    2012-03-01

    The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.

  19. 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.

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

    PubMed

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

    2016-05-10

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours.

    PubMed

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

    2007-03-01

    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.

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

    PubMed Central

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

    2014-01-01

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

  4. Comparison of Four Polymerase Chain Reaction Methods for the Rapid Detection of Human Fecal Pollution in Marine and Inland Waters

    PubMed Central

    Bachoon, Dave S.; Miller, Cortney M.; Green, Christen P.; Otero, Ernesto

    2010-01-01

    We compared the effectiveness of three PCR protocols for the detection of Bifidobacterium adolescentis and one PCR protocol for detecting Bacteroidales as indicators of human fecal pollution in environmental samples. Quantitative PCR indicated that a higher concentration of B. adolescentis DNA was recovered from sewage samples on the 0.2 μm filters compared to the 0.45 μm filters, and there was no evidence of qPCR inhibitors in the DNA extracts. With the Matsuki method (1999), B. adolescentis was detected only in undiluted sewage samples. The King method (2007) performed well and detected B. adolescentis in all of the sewage dilutions (from undiluted to 10−4). In contrast, the Bonjoch approach (2004) was effective at detecting B. adolescentis at lower dilutions (10−3) of sewage samples and it gave false positive results with some (3/8) pig fecal samples. Human-specific Bacteroidales (HuBacs) were detected in the lower diluents of sewage samples but was positive in pig (6/8) and cattle fecal samples. PCR detection of B. adolescentis in marine samples from Puerto Rico and freshwater samples from Georgia indicated that the PCR method of King et al. (2007) and the modified Layton method for HuBac were in agreement in detecting human fecal pollution in most sites. PMID:20811614

  5. 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.

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

    PubMed

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

    2016-07-01

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

  7. 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.

  8. A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

    PubMed Central

    Aradaib, Imadeldin E; Majid, Ali A

    2006-01-01

    A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels. Amplification products were not detected when the PCR-based assay was applied to DNA from other blood parasites including Thieleria annulata, Babesia bigemina or nucleic acid free samples. Application of this nPCR-based assay to clinical samples resulted in direct detection of T. evansi from a variety of tissue samples collected from experimentally infected mice and blood from naturally infected camels. The described nPCR-based assay provides a valuable tool to study the epidemiology of T. evansi infection in camels and other susceptible animal populations. PMID:16712737

  9. Simple and rapid detection of Campylobacter spp. in naturally contaminated chicken-meat samples by combination of a two-step enrichment method with an immunochromatographic assay.

    PubMed

    Kawatsu, K; Taguchi, M; Yonekita, T; Matsumoto, T; Morimatsu, F; Kumeda, Y

    2010-08-15

    A simple and rapid method to detect Campylobacter spp. in chicken-meat samples was established. This method consisted of a combination of a two-step enrichment method with a commercially available immunochromatographic assay, named NH Immunochromato Campylobacter (NH IC Campy, Nippon Meat Packers, Ibaraki, Japan), which is able to detect Campylobacter antigen in an enrichment culture within 15 min. The enrichment method did not require much blood or a particular system of generating a microaerobic atmosphere, in contrast to the standard method of enriching Campylobacter spp. in chicken-meat samples. The sensitivity of a combination of the two-step enrichment method with NH IC Campy for detection of non- and freeze-stressed Campylobacter spp. in spiked chicken meat was determined using bacterial cells of Campylobacter jejuni and Campylobacter coli. The detection sensitivities for non-stressed C. jejuni and C. coli were found to range from 5.5 to 1.3x10(1) CFU per 25 g of chicken meat, and those for freeze-stressed C. jejuni and C. coli were found to range from 9.2x10(1) to 1.5x10(2) CFU per 25 g of chicken meat. When a total of 68 chicken-meat samples were tested, the combination method determined that 61 samples were positive for Campylobacter spp. This method was more sensitive than a bacterial culture test, which consists of standard enrichment culturing and plating onto selective agars. Because the combination could be conducted in approximately 48 h, from the beginning of the enrichment culture to final determination, it was more rapid than the bacterial culture test, which requires four to five days. Moreover, the combination was simple to perform. These results suggest that combining the two-step enrichment method with NH IC Campy is useful as a simple and rapid alternative to the conventional bacterial culture test for detecting Campylobacter spp. in naturally contaminated chicken meat samples.

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

    PubMed

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

    2014-08-01

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

  11. 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.

  12. 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.

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

    PubMed Central

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

    1996-01-01

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

  14. 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.

  15. 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.

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

    PubMed

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

    2015-08-01

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

  17. 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 Central

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

    2014-01-01

    Background & objectives: 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. Methods: 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. Results: 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. Interpretation & conclusions: 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. PMID:25366209

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

    PubMed

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

    2016-03-01

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

  19. 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.

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

    PubMed

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

    2015-02-01

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

  1. Sensitive, Rapid Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

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

  2. 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

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

    PubMed

    Liu, Zhongyuan; Dong, Zhijun; Liu, Dongyan

    2016-07-01

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

  4. A rapid simple in situ hybridization method for herpes simplex virus employing a synthetic biotin-labeled oligonucleotide probe: a comparison with immunohistochemical methods for HSV detection.

    PubMed

    Wang, J Y; Montone, K T

    1994-01-01

    We examined 28 paraffin-embedded tissue specimens with histologic evidence of herpes virus infection by in situ hybridization (ISH) utilizing manual capillary action technology (MicroProbe Staining System) and a 21 base synthetic multibiotinylated oligonucleotide probe from the HSV glycoprotein C region. The results were compared to a rapid simple immunohistochemical (IHC) protocol for detection of HSV proteins. HSV was detected by ISH and IHC in all but one specimen which was shown to be positive for varicella zoster virus by direct fluorescent antibody studies. Hybridization signal was confined to the nucleus in all cases. Staining was identified in cells with early as well as late cytopathic effect. IHC produced intense nuclear and/or cytoplasmic signal in infected cells and stained in areas of necrosis which were otherwise spared by ISH. HSV was detected by IHC and/or ISH in 3/5 specimens with histology suggestive of, but not diagnostic for, HSV infection. Both techniques were sensitive and specific for HSV, resulted in rapid detection of the pathogen in routinely processed tissues, and may be useful in cases where the histologic impression is equivocal for HSV infection. ISH for HSV may be preferred because it can identify early HSV infection, which in turn can be treated with antiviral agents.

  5. 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.

  6. Establishment and Application of a Loop-Mediated Isothermal Amplification Method for Simple, Specific, Sensitive and Rapid Detection of Toxoplasma gondii

    PubMed Central

    CAO, Lili; CHENG, Ronghua; YAO, Lin; YUAN, Shuxian; YAO, Xinhua

    2013-01-01

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

  7. 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.

  8. Validation of end-point and real-time PCR methods for the rapid detection of soy allergen in processed products.

    PubMed

    Espineira, Montserrat; Herrero, Beatriz; Vieites, Juan M; Santaclara, Francisco J

    2010-04-01

    This work describes the development and validation of two PCR methods, end-point and real-time PCR, for the detection of soy protein in a wide rage of foodstuffs. These techniques are reliable and sensitive, allowing detection of trace amounts of soybean in processed products. TaqMan real-time PCR was the simpler and more rapid process, with a higher potential for automation and, therefore, currently the most suitable screening method. To verify correct operation of the proposed methodology, ELISA was used for quantitative determination of soy protein. In addition, 35 meat, fish and bakery processed products, which could potentially contain soy but was not declared on the label, were tested for the presence of soy DNA using the proposed methods. The methodologies will be valuable in issues regarding the presence of soy protein in processed products, especially in verifying labelling and security regulations to protect consumer's rights.

  9. 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

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

    PubMed

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

    2016-07-01

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

  11. 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.

  12. A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5' RACE and a molecular beacon probe.

    PubMed

    Lasham, Annette; Herbert, Mike; Coppieters 't Wallant, Natacha; Patel, Rachna; Feng, Sheryl; Eszes, Marika; Cao, Helen; Reid, Glen

    2010-01-01

    Specific detection of mRNA cleavage by 5'RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5'-RNA-linker-mediated RACE (5'-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5'-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. With its sensitivity and specificity, this variation on the 5'RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo.

  13. Rod Photoreceptors Detect Rapid Flicker

    ERIC Educational Resources Information Center

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

    1977-01-01

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

  14. 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.

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

    PubMed

    Janczarek, Monika; Palusińska-Szysz, Marta

    2016-05-01

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

  16. 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

  17. Development of a rapid DNA extraction method and one-step nested PCR for the detection of Naegleria fowleri from the environment.

    PubMed

    Ahmad, Arine Fadzlun; Lonnen, James; Andrew, Peter W; Kilvington, Simon

    2011-10-15

    Naegleria fowleri is a small free-living amoebo-flagellate found in natural and manmade thermal aquatic habitats worldwide. The organism is pathogenic to man causing fatal primary amoebic meningoencephalitis (PAM). Infection typically results from bathing in contaminated water and is usually fatal. It is, therefore, important to identify sites containing N. fowleri in the interests of preventive public health microbiology. Culture of environmental material is the conventional method for the isolation of N. fowleri but requires several days incubation and subsequent biochemical or molecular tests to confirm identification. Here, a nested one-step PCR test, in conjunction with a direct DNA extraction from water or sediment material, was developed for the rapid and reliable detection of N. fowleri from the environment. Here, the assay detected N, fowleri in 18/109 river water samples associated with a nuclear power plant in South West France and 0/10 from a similar site in the UK. Although culture of samples yielded numerous thermophilic free-living amoebae, none were N. fowleri or other thermophilic Naegleria spp. The availability of a rapid, reliable and sensitive one-step nested PCR method for the direct detection of N. fowleri from the environment may aid ecological studies and enable intervention to prevent PAM cases.

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

    PubMed

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

    1993-10-22

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

  19. Methods for rapid and effective PCR-based detection of 'Candidatus Liberibacter solanacearum' from the insect vector Bactericera cockerelli: streamlining the DNA extraction/purification process.

    PubMed

    Lévy, Julien; Hancock, Joseph; Ravindran, Aravind; Gross, Dennis; Tamborindeguy, Cecilia; Pierson, Elizabeth

    2013-06-01

    This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of 'Candidatus (Ca.) Liberibacter solanacearum,' the bacterial causal agent of potato zebra chip disease and eventually for 'Ca. Liberibacter asiaticus' the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (> 97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected 'Ca. Liberibacter' in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average approximately 5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of 'Ca. Liberibacter' pathogens in psyllids. PMID:23865212

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

    PubMed Central

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

    2014-01-01

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

  1. A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis.

    PubMed

    Yamaguchi, Mizuka; Kawai, Takao; Kitagawa, Mikiya; Kumeda, Yuko

    2013-05-01

    The Bacillus cereus emetic toxin cereulide causes foodborne intoxication, which may occasionally result in severe disease, and even death. To differentially diagnose the emetic-type of foodborne disease caused by B. cereus and assess the safety of commercial food, we developed a rapid method to quantitate cereulide. This method was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the extraction of cereulide from food using a normal-phase silica gel cartridge. The limits of detection and quantification were 0.1 and 0.5 ng of cereulide ml(-1), respectively. Spiked cereulide was reproducibly recovered with over 67% efficiency from nine diverse foods implicated in cereulide food poisoning. The recovery rate, reproducibility, and intermediate precision for this single laboratory validation using boiled rice were 87.1%, 4.4%, and 7.0%, respectively. Further, we detected a wide range of cereulide concentrations in leftover food and vomitus samples from two emetic foodborne outbreaks. LC-MS/MS analysis correlated closely with those acquired using the HEp-2 cell assay, and quantitated cereulide from 10 food samples at least five times faster than the bioassay. This new method will provide clinicians with an improved tool for more rapidly and quantitatively determining the presence of cereulide in food and diagnosing food poisoning caused by cereulide.

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

    PubMed

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

    2015-04-01

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

  3. 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.

  4. 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.

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

    PubMed

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

    1997-09-01

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

  6. [Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

    PubMed

    Lin, Feng; Liu, Li; Hao, Gui-Jie; Cao, Zheng; Sheng, Peng-Cheng; Wu, Ying-Lei; Shen, Jin-Yu

    2014-09-01

    White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection. PMID:25562958

  7. [Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

    PubMed

    Lin, Feng; Liu, Li; Hao, Gui-Jie; Cao, Zheng; Sheng, Peng-Cheng; Wu, Ying-Lei; Shen, Jin-Yu

    2014-09-01

    White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection.

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

    PubMed

    Pittet, Alain; Royer, Delphine

    2002-01-16

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

  9. Comparison of Three Commercial Methods for Rapid Detection of Clostridium difficile Toxins A and B from Fecal Specimens▿

    PubMed Central

    Alcalá, L.; Sánchez-Cambronero, L.; Catalán, M. P.; Sánchez-Somolinos, M.; Peláez, M. T.; Marín, M.; Bouza, E.

    2008-01-01

    Three rapid enzyme immunoassays (X/pect Clostridium difficile Toxin A/B test, Wampole Tox A/B Quik Chek, and ImmunoCard Toxins A&B) were compared for the diagnosis of Clostridium difficile infection. Of the 367 stool specimens tested, 102 (27.8%) were positive for toxigenic C. difficile when a combination of direct cytotoxicity assay and cytotoxic culture was used as the gold standard. Sensitivity/specificity values were 49.0%/95.8%, 54.9%/95.5%, and 66.7%/95.1%, respectively. The median times to test five stool specimens were 28, 30, and 24 min, respectively. The ImmunoCard test was the quickest and most sensitive test of the three enzyme immunoassays evaluated. PMID:18784313

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

    PubMed

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

    2016-07-01

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

  11. 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

  12. Methods of Endotoxin Detection.

    PubMed

    Su, Wenqiong; Ding, Xianting

    2015-08-01

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

  13. 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.

  14. Rapid molecular detection of Lujo virus RNA.

    PubMed

    Atkinson, Barry; Chamberlain, John; Dowall, Stuart D; Cook, Nicola; Bruce, Christine; Hewson, Roger

    2014-01-01

    Lujo virus is an emerging arenavirus circulating in Southern Africa. Although to date there has only been a single outbreak of the novel haemorrhagic disease resulting from human infection with this virus, the case-fatality rate of exposed individuals, including nosocomial transmission, was 80%. The ability to identify viral haemorrhagic fevers accurately, especially those capable of nosocomial transmission, is of critical importance. Timely identification of these diseases allow medical professionals to isolate patients and implement barrier nursing techniques in order to prevent onward transmission of the virus. While rapid diagnostic methods are published for most viral haemorrhagic fevers, at present there are no such virus specific protocols for Lujo haemorrhagic fever. This report details the first set of diagnostic molecular assays designed to identify Lujo viral RNA rapidly, and demonstrates the potential functionality of these assays for use in the clinical setting. Although these assays have been designed and validated against a solitary isolate of Lujo virus, this represents the entirety of strains detected to date, and offer quick, cheap and easy methods for use in diagnostic laboratories.

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

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2016-01-01

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

  17. 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

  18. Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

    PubMed

    Zimmer-Faust, Amity G; Thulsiraj, Vanessa; Ferguson, Donna; Jay, Jennifer A

    2014-05-01

    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.

  19. 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

  20. 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.

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

    PubMed

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

    2016-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. 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.

  4. Human nails metabolite analysis: A rapid and simple method for quantification of uric acid in human fingernail by high-performance liquid chromatography with UV-detection.

    PubMed

    Li, Xi-Ling; Li, Gao; Jiang, Ying-Zi; Kang, Dongzhou; Jin, Cheng Hua; Shi, Qing; Jin, Toufeng; Inoue, Koichi; Todoroki, Kenichiro; Toyo'oka, Toshimasa; Min, Jun Zhe

    2015-10-01

    A rapid and simple analytical method for the quantification of uric acid (UA) in human fingernails by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. UA was extracted from human fingernail samples at 90°C for 20min, then separated on an Inertsil ODS-2 column (250×4.6mm I.D., 5.0μm, GL Sciences) by isocratic elution using methanol: 74mM phosphate buffer (pH 2.2) 2:98 (v/v). An UV detector was used to monitor at 284nm. The results indicated that under optimized measurement conditions results were achieved within 8.0min, and a good linearity was achieved from the calibration curves (r(2)>0.9999) in the range of 1.0-10000ng; the limit of detection (S/N=3) was 2.0pg, the inter-day and intra-day assay precisions were all less than 0.46% and the mean recoveries (%) of the uric acid spiked in the human fingernail were 101.95%. The amounts of UA in the fingernails of healthy volunteers were determined.

  5. Method validation of resistive heating--gas chromatography with flame photometric detection for the rapid screening of organophosphorus pesticides in fruit and vegetables.

    PubMed

    Patel, Katan; Fussell, Richard J; Macarthur, Roy; Goodall, David M; Keely, Brendan J

    2004-08-13

    A rapid method for the screening of organophosphorus (OP) pesticides in fruit and vegetables is reported. Sample extracts were analysed using resistive heating-gas chromatography (RH-GC) with flame photometric detection (FPD). A CarboFrit insert in the GC liner allowed injection of crude extracts onto the GC system. Separation of up to 20 pesticides was achieved in 4.3 min with excellent retention time stability. Signal-to-noise ratios of 5:1 or better were obtained for the majority of the pesticides at the lowest calibrated level (LCL), 0.01 microg ml(-1), with excellent linearity over the range 0.01-0.5 microg ml(-1) (0.004-0.2 mg kg(-1) equivalent). Average recoveries between 70 and 116% were obtained for pesticides spiked at 0.01 and 0.1 mg kg(-1) with associated R.S.D. values < or =20% in the majority of cases. Estimates of relative reproducibility standard deviation (R.S.D.(R)), made by combining observed R.S.D. values with estimates of uncertainty associated with mean recovery allowed the determination of HORRAT values which confirmed that the method is capable of producing results which are fit for purpose. The validated method was then used to screen peaches, grapes and sweet peppers for a total of 37 pesticides. Incurred residue results obtained using RH-GC-FPD were in good agreement with the results from analysis of the same samples using MS confirmation.

  6. Rapid auditory learning of temporal gap detection.

    PubMed

    Mishra, Srikanta K; Panda, Manasa R

    2016-07-01

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

  7. 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.

  8. Simple and rapid method for the detection of Filobasidiella neoformans in a probiotic dairy product by using loop-mediated isothermal amplification.

    PubMed

    Ishikawa, Hiroshi; Kasahara, Kohei; Sato, Sumie; Shimakawa, Yasuhisa; Watanabe, Koichi

    2014-05-16

    Yeast contamination is a serious problem in the food industry and a major cause of food spoilage. Several yeasts, such as Filobasidiella neoformans, which cause cryptococcosis in humans, are also opportunistic pathogens, so a simple and rapid method for monitoring yeast contamination in food is essential. Here, we developed a simple and rapid method that utilizes loop-mediated isothermal amplification (LAMP) for the detection of F. neoformans. A set of five specific LAMP primers was designed that targeted the 5.8S-26S rDNA internal transcribed spacer 2 region of F. neoformans, and the primer set's specificity was confirmed. In a pure culture of F. neoformans, the LAMP assay had a lower sensitivity threshold of 10(2)cells/mL at a runtime of 60min. In a probiotic dairy product artificially contaminated with F. neoformans, the LAMP assay also had a lower sensitivity threshold of 10(2)cells/mL, which was comparable to the sensitivity of a quantitative PCR (qPCR) assay. We also developed a simple two-step method for the extraction of DNA from a probiotic dairy product that can be performed within 15min. This method involves initial protease treatment of the test sample at 45°C for 3min followed by boiling at 100°C for 5min under alkaline conditions. In a probiotic dairy product artificially contaminated with F. neoformans, analysis by means of our novel DNA extraction method followed by LAMP with our specific primer set had a lower sensitivity threshold of 10(3)cells/mL at a runtime of 60min. In contrast, use of our novel method of DNA extraction followed by qPCR assay had a lower sensitivity threshold of only 10(5)cells/mL at a runtime of 3 to 4h. Therefore, unlike the PCR assay, our LAMP assay can be used to quickly evaluate yeast contamination and is sensitive even for crude samples containing bacteria or background impurities. Our study provides a powerful tool for the primary screening of large numbers of food samples for yeast contamination.

  9. Rapid methods and automation in dairy microbiology.

    PubMed

    Vasavada, P C

    1993-10-01

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

  10. 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...

  11. 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.

  12. 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.

  13. Rapid detection of Mycobacterium tuberculosis complex and rifampin resistance in smear-negative clinical samples by use of an integrated real-time PCR method.

    PubMed

    Moure, Raquel; Muñoz, Laura; Torres, Miriam; Santin, Miguel; Martín, Rogelio; Alcaide, Fernando

    2011-03-01

    Sixty-four of 85 (75.3%) smear-negative respiratory (n = 78) and nonrespiratory (n = 7) samples with positive cultures of Mycobacterium tuberculosis complex (MTC) were detected by the GeneXpert system using the Xpert MTB/RIF assay (GX). In addition, GX found rpoB mutations in all six of the rifampin-resistant strains detected. The test was negative in 20 culture-negative and 20 nontuberculous culture-positive samples (100% specificity). GX offers high potential for the diagnosis of tuberculosis due to its capacity for direct detection of MTC, its rapidity, and its simplicity.

  14. 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.

  15. A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum.

    PubMed

    Ren, Zhi-Qi; Liu, Tian-Cai; Hou, Jing-Yuan; Chen, Mei-Jun; Chen, Zhen-Hua; Lin, Guan-Feng; Wu, Ying-Song

    2014-09-01

    Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

  16. 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.

  17. 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

  18. Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea).

    PubMed

    Mao, Zhijuan; Qiu, Yangyu; Zheng, Lei; Chen, Jigang; Yang, Jifang

    2012-06-01

    In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.

  19. 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.

  20. Rapid analytical detection of microcystins using gold colloidal immunochromatographic strip.

    PubMed

    Pyo, Dongjin; Choi, Jongchon; Hong, Jonguk; Oo, Hlaing Hlaing

    2006-01-01

    Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is low and the detection method is usually complicated. We developed a rapid analytical detection method of microcystins gold colloidal immunochromatogeraphic strip. The sensitivity of the strip is about 1 ng/mL for microcystin LR; it is able to distinguish visually among different concentrations of microcystin solutions. The developed gold colloidal strip can detect microcystins within 15 min and does not require either a complicated extraction system, or trained or qualified experts.

  1. 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.

  2. Rapid and simple colorimetric detection of Escherichia coli O157:H7 in apple juice using a novel recombinant bacteriophage-based Method.

    PubMed

    Hoang, Hoang A; Dien, Le T

    2015-01-01

    In this study, a bacteriophage-based method for the colorimetric detection of E. coli O157:H7 in apple juice was investigated. Firstly, a gene encoding Cytochrome c Peroxidase (CCP) chromogenic enzyme was inserted into a wild type PP01 phage genome to construct the recombinant PP01ccp phage that was used in the production of the chromogenic enzyme through specific infection into E. coli O157:H7. The method was then examined in the colorimetric detection of E. coli O157:H7 in broth, and the appearance of E. coli O157:H7 in broth was confirmed by the color change after a few minutes of the enzyme assay. Secondly, the method was investigated in the colorimetric detection of E. coli O157:H7 in apple juice. A low E. coli O157:H7 concentration as 1 CFU mL(-1) was detected in 15 h that was in a shorter time than in previous bioluminescence phage-based methods. Moreover, the method is much simpler compared to other previous phage-based methods since it enables detection without the need for expensive apparatus.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. A rapid, reliable, and inexpensive method for detection of di- and trinucleotide repeat markers and disease loci from dried blood spots

    SciTech Connect

    Holden, J.A. |; Chalifoux, M.; Wing, M.

    1996-08-09

    We used a rapid and inexpensive method for studying the FMR1 CGG-repeat from dried blood spots, prepared from heel pricks, finger pricks, or an aliquot of blood from a venipuncture. The procedure includes a single tube for preparation of template DNA for PCR and minimal handling, avoiding opportunities for mislabelling specimens and loss of template. We extended the protocol to numerous di- and trinucleotide repeat markers and disease loci, including FRAXE, FRAXF, DXS548, DRPLA, and ZFY. The use of a highly reliable and very inexpensive method which employs blood spots as a source for target DNA means that newborn Guthrie cards can be used to establish allele frequencies for linkage disequilibrium studies, that large populations can be screened for genetic disorders, and that mapping studies can proceed rapidly even when only small amounts of blood are available from key family members. 16 refs., 5 figs.

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

    PubMed

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

    2016-09-01

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

  6. Rapid DNA detection by beacon-assisted detection amplification.

    PubMed

    Connolly, Ashley R; Trau, Matt

    2011-06-01

    This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples.

  7. 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.

  8. A rapid and sensitive method for the analysis of brain monoamine neurotransmitters using ultra-fast liquid chromatography coupled to electrochemical detection.

    PubMed

    Parrot, Sandrine; Neuzeret, Pierre-Charles; Denoroy, Luc

    2011-12-15

    Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). This paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC-electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-03-01

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

  11. Rapid methods for identification of yeasts.

    PubMed Central

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

    1975-01-01

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

  12. 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.

  13. 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.

  14. 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.

  15. qPCR is a sensitive and rapid method for detection of cytomegaloviral DNA in formalin-fixed, paraffin-embedded biopsy tissue.

    PubMed

    McCoy, Morgan H; Post, Kristin; Sen, Joyashree D; Chang, Hsim Y; Zhao, Zijin; Fan, Rong; Chen, Shaoxiong; Leland, Diane; Cheng, Liang; Lin, Jingmei

    2014-01-01

    It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

  16. qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

    PubMed Central

    McCoy, Morgan H.; Post, Kristin; Sen, Joyashree D.; Chang, Hsim Y.; Zhao, Zijin; Fan, Rong; Chen, Shaoxiong; Leland, Diane; Cheng, Liang; Lin, Jingmei

    2014-01-01

    It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies. PMID:25046572

  17. PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB.

    PubMed

    Zhou, Guangpeng; Cao, Boyang; Dou, Yan; Liu, Yanwei; Feng, Lu; Wang, Lei

    2011-08-01

    A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level. PMID:21626022

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

    PubMed

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

    2015-01-01

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

  19. 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.

  20. 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

  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

    2014-07-01

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

  2. A novel method for the rapid detection of benzo(a)pyrene in liquid milk by dimethyl sulfoxide selectively enhanced synchronous fluorescence spectrometry.

    PubMed

    Lin, Li-Rong; Luo, He-Dong; Li, Xiu-Ying; Li, Na; Zhou, Na; Jia, Yu-Zhu; Liu, Yi-Hong; Li, Yao-Qun

    2014-01-01

    Based on the high solubility efficiency and strong fluorescence response of benzo(a)pyrene (BaP) in dimethyl sulfoxide in combination with the high-performance derivative constant-energy synchronous fluorescence spectroscopic (DCESFS) technique, a simple, sensitive and economic method was developed for the determination of BaP in liquid milk. This method comprises ultrasound-assisted solvent extraction, solvent replacement and DCESFS detection. No saponification or other tedious clean-up procedures were needed. The recoveries of BaP in different milk samples were greater than 82%. Detection limits in full- and low-fat milk were 0.03 and 0.04 μg kg(-1), respectively. PMID:24827591

  3. Multigrid contact detection method

    NASA Astrophysics Data System (ADS)

    He, Kejing; Dong, Shoubin; Zhou, Zhaoyao

    2007-03-01

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

  4. 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

  5. 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.

  6. 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

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

    PubMed

    Rodríguez, J; Castañeda, G; Muñoz, L

    2013-01-15

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

  8. 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

  9. 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.

  10. 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.

  11. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    PubMed Central

    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

    2015-01-01

    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 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 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, comparable to a more labor-intensive fluorescence detection method reported previously. PMID:26097292

  12. 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.

  13. 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.

  14. Development of a loop-mediated isothermal amplification method for the rapid detection of the dioxin-degrading bacterium Ochrobactrum anthropi in soil.

    PubMed

    Chen, Hsi-Jien; Lee, Meng-Shiou; Lai, Jun-Yu; Lai, Guan-Hua

    2015-09-01

    In this study, loop-mediated isothermal amplification (LAMP) and real-time LAMP assays were developed to detect the dioxin-degrading bacterium Ochrobactrum anthropi strain BD-1 in soil. Four primers were designed to use ITS gene amplification for the strain O. anthropi BD-1. The real-time LAMP assay was found to accomplish the reaction by 1 pg of genomic DNA load when used for nucleic acid amplification. This assay was then applied to detect O. anthropi BD-1 in eight soil samples collected from a dioxin-contaminated site. The results demonstrated that these newly developed LAMP and real-time LAMP assays will not only be useful and efficient tools for detecting the target gene, but also be used as molecular tools for monitoring the growth of dioxin-degrading O. anthropi in the soil. This is the first report to demonstrate the use of LAMP assays to monitor the presence of O. anthropi in dioxin-contaminated soil. The application of this method should improve the biomonitoring of dioxin contamination.

  15. Methods of Melanoma Detection.

    PubMed

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

    2016-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  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. 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.

  19. 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.

  20. A rapid and simple method to determine the specific activities of serotonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophan in brain by HPLC with electrochemical detection

    SciTech Connect

    Bernstein, M.J.; Shea, P.A.

    1982-01-01

    The specific activities of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) have been determined in the brain of rats by HPLC using electrochemical detection. The method allows, from a single sample, the simultaneous measurement of all three compounds and collection of each peak for radioactivity determinations. Five male Wistar rats were injected i.v. with 2.0 mCi/kg of DL-5-hydroxy-(G-/sup 3/H)tryptophan (2.6 Ci/mmol) and 30 min later the animals were killed by near freezing. Whole brains were removed and homogenized in an acid medium. The content of 5-HTP, 5-HT, and 5-HIAA were determined by HPLC. Each peak of interest was immediately collected after detection in scintillation vials by use of a small dead space detector (TL-9A, Bioanalytical Systems, Inc.). The amounts of radioactivity were determined and specific activities calculated from the results. A second chromatography system (TLC) was used to check the authenticity and purity of compounds separated by the HPLC.

  1. 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.

  2. 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. PMID:18236668

  3. 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.

  4. 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)).

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

    PubMed

    Takeda, N; Gotoh, M; Matsuoka, T

    2011-09-01

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

  6. Methods for Doping Detection.

    PubMed

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

    2016-01-01

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

  7. 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.).

  8. Specific antibody detection in human aspergillosis: a GEMO* multicentre evaluation of a rapid immunoelectrophoresis method (Paragon). Group d'Etude des Mycoses Opportunistes.

    PubMed

    Persat, F; Gari-Toussaint, M; Lebeau, B; Cambon, M; Raberin, H; Addo, A; Picot, S; Piens, M A; Blancard, A; Mallié, M; Bastide, J M; Grillot, R

    1996-01-01

    A new immunoelectrophoresis system, the Paragon system, was evaluated in three different hospital centres with the aim of improving standardization of the serodiagnosis of human aspergillosis. To select the most efficient antigen, various commercial and home-made antigens were first tested on 19 sera from 19 patients with highly probable aspergillosis. The value measured using the Paragon anti-Aspergillus antibody detection system was then compared with the results obtained by conventional serological diagnostic methods (conventional immunoelectrophoresis, enzyme linked-immunosorbent assay, indirect immunofluorescence): this step was performed using the first 19 sera as well as 16 other sera from 13 patients with suspected aspergillosis. Concordant results were obtained in 28 cases. The discrepancies observed with seven sera were probably related to differences in the nature of the antigens. Paragon immunoelectrophoresis proved to be a practicable technique requiring only a small amount of serum and giving results within a shorter time than competitive methods (24-48 h). Its major drawbacks compared with conventional immunoelectrophoresis are some difficulties in reading, fewer precipitin lines and the relatively high cost of routine analysis.

  9. Rapid method for the determination of coumarin, vanillin, and ethyl vanillin in vanilla extract by reversed-phase liquid chromatography with ultraviolet detection.

    PubMed

    Ali, Laila; Perfetti, Gracia; Diachenko, Gregory

    2008-01-01

    A method is described for determining coumarin, vanillin, and ethyl vanillin in vanilla extract products. A product is diluted one-thousand-fold and then analyzed by reversed-phase liquid chromatography using a C18 column and a mobile phase consisting of 55% acetonitrile-45% aqueous acetic acid (1%) solution at a flow rate of 1.0 mL/min. Peaks are detected with a UV detector set at 275 nm. Vanilla extracts were spiked with 250, 500, and 1000 microg/g each of coumarin, vanillin, and ethyl vanillin. Recoveries averaged 97.4, 97.8, and 99.8% for coumarin, vanillin, and ethyl vanillin, respectively, with coefficient of variation values of 1.8, 1.3, and 1.3%, respectively. No significant difference was observed among the 3 spiking levels. A survey of 23 domestic and imported vanilla extract products was conducted using the method. None of the samples contained coumarin. The surveyed samples contained between 0.4 to 13.1 and 0.4 to 2.2 mg/g vanillin and ethyl vanillin, respectively.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Method for detecting biomolecules

    DOEpatents

    Huo, Qisheng; Liu, Jun

    2008-08-12

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

  13. 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.

  14. 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.

  15. Rapid and quantitative detection of hepatitis B virus

    PubMed Central

    Liu, Yue-Ping; Yao, Chun-Yan

    2015-01-01

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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. Evaluation of the Roche LightCycler: a simple and rapid method for direct detection of factor V Leiden and prothrombin G20210A genotypes from blood samples without the need for DNA extraction.

    PubMed

    Cooper, Peter C; Cooper, Susan M; Smith, Julie M; Kitchen, Steven; Makris, Michael

    2003-07-01

    The Roche LightCycler is a micro-volume thermocycler that combines extremely rapid polymerase chain reaction with fluorescence resonance energy transfer analysis of amplified products. We have evaluated the use of minimally processed blood samples for detection of two point mutations known to increase the risk of venous thromboembolism. Results from the LightCycler using the supernatant of diluted heated blood were compared with those gained by traditional methods based on polymerase chain reaction and restriction enzyme digestion. For factor V Leiden mutation, there was complete agreement between both methods in detection of wild-type (n = 82), heterozygous (n = 100) and homozygous (n = 18) genotypes. Similarly, the prothrombin G20210A mutation showed complete agreement for wild-type (n = 135), heterozygous (n = 63) and homozygous (n = 2) subjects.

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

    PubMed

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

    2016-09-01

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

  2. 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.

  3. Olfactory processing: detection of rapid changes.

    PubMed

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

    2015-06-01

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

  4. 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.

  5. A method for rapid characterization of diffusion.

    PubMed

    Song, Y-Q; Hürlimann, M D; Flaum, C

    2003-04-01

    This paper describes a method to determine molecular displacements as a function of time in just two scans: one reference scan using the Carr-Purcell-Meiboom-Gill (CPMG) sequence, a second scan using a modified CPMG sequence (KCPMG). Measurements on free diffusion in bulk fluids, and on restricted diffusion in porous rock samples are reported. This technique can also be used for rapid measurement of flow and chemical exchange. PMID:12713974

  6. 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.

  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. Monoclonal antibody technologies and rapid detection assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Rapid detection of bacteria by carbon quantum dots.

    PubMed

    Mandal, Tapas K; Parvin, Nargish

    2011-12-01

    This work demonstrated a fluorescence measurement method for rapid detection of bacteria and their counting by using water-soluble carbon quantum dots (CQDs) as a fluorescence marker while sewage water bacteria were detection target bacteria. Highly luminescent water-soluble CQDs were prepared by carbonizing waste part of rice straw materials in a furnace under in-sufficient air flow. Bacteria in a LB media with < 100 nm of water soluble CQDs were cultured for 30-40 minutes. The multi colored bacterial cell images were obtained by using fluorescence microscopy. Our results showed that CQDs prepared in water phase were highly luminescent, stable, and successfully conjugated with bacterial cell membrane only. Also we could count the total number of bacteria within a shortest time from any sample of environment.

  10. 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.

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

    PubMed

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

    2015-09-01

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

  12. 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.

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

    PubMed

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

    1976-08-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2012-03-01

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

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

    PubMed

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

    2012-03-01

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

  18. 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.

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

    PubMed

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

    2016-08-19

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

  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 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

  2. 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

  3. 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.

  4. Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification.

    PubMed

    Komatsu, Ken; Urayama, Syun-Ichi; Katoh, Yu; Fuji, Shin-Ichi; Hase, Shu; Fukuhara, Toshiyuki; Arie, Tsutomu; Teraoka, Tohru; Moriyama, Hiromitsu

    2016-02-01

    Magnaporthe oryzae chrysovirus 1 (MoCV1) is a mycovirus with a dsRNA genome that infects the rice blast fungus Magnaporthe oryzae and impairs its growth. To date, MoCV1 has only been found in Vietnamese isolates of M. oryzae, and the distribution of this virus in M. oryzae isolates from other parts of the world remains unknown. In this study, using a one-step reverse transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus in M. oryzae in Japan (named MoCV1-AK) whose sequence shares considerable similarity with that of the MoCV1 Vietnamese isolate. To establish a system for a comprehensive survey of MoCV1 infection in the field, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. The sensitivity of the RT-LAMP assay was at least as high as that of the one-step RT-PCR assay. In addition, we detected MoCV1-AK in M. oryzae-infected oatmeal agar plates and lesions on rice leaves using the RT-LAMP assay without dsRNA extraction, by simple sampling with a toothpick. Preliminary screening of MoCV1 in Japanese M. oryzae isolates indicated that MoCV1 is currently distributed in rice fields in Japan. Our results provide a first example of the application of RT-LAMP for the detection of mycoviruses, which will accelerate surveys for mycovirus infection. PMID:26547578

  5. A Coffee Ring Aptasensor for Rapid Protein Detection

    PubMed Central

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

    2013-01-01

    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 between surface hydrophobicity, concentration efficiency and signal amplification. Based on our results, we found that the increase in 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 4x diluted whole blood. Based on our results, coffee ring spots produced detection signals 40x larger than 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. Based on 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. PMID:23540796

  6. 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.

  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. 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.

  9. Rapid sequencing of DNA based on single-molecule detection

    NASA Astrophysics Data System (ADS)

    Soper, Steven A.; Davis, Lloyd M.; Fairfield, Frederick R.; Hammond, Mark L.; Harger, Carol A.; Jett, James H.; Keller, Richard A.; Marrone, Babetta L.; Martin, John C.; Nutter, Harvey L.; Shera, E. Brooks; Simpson, Daniel J.

    1991-07-01

    Sequencing the human genome is a major undertaking considering the large number of nucleotides present in the genome and the slow methods currently available to perform the task. The authors have recently reported on a scheme to sequence DNA rapidly using a non-gel based technique. The concept is based upon the incorporation of fluorescently labeled nucleotides into a strand of DNA, isolation and manipulation of a labeled DNA fragment and the detection of single nucleotides using ultra-sensitive laser-induced fluorescence detection following their cleavage from the fragment. Detection of individual fluorophores in the liquid phase was accomplished with time-gated detection following pulsed-laser excitation. The photon bursts from individual rhodamine 6G (R6G) molecules travelling through a laser beam have been observed, as have bursts from single fluorescently modified nucleotides. Using two different biotinylated nucleotides as a model system for fluorescently labeled nucleotides, the authors have observed synthesis of the complementary copy of M13 bacteriophage. Work with fluorescently labeled nucleotides is underway. Individual molecules of DNA attached to a microbead have been observed and manipulated with an epifluorescence microscope.

  10. Rapid sequencing of DNA based on single molecule detection

    SciTech Connect

    Soper, S.A.; Davis, L.M.; Fairfield, F.R.; Hammond, M.L.; Harger, C.A.; Jett, J.H.; Keller, R.A.; Marrone, B.L.; Martin, J.C.; Nutter, H.L.; Shera, E.B.; Simpson, D.J.

    1991-01-01

    Sequencing the human genome is a major undertaking considering the large number of nucleotides present in the genome and the slow methods currently available to perform the task. We have recently reported on a scheme to sequence DNA rapidly using a non-gel based technique. The concept is based upon the incorporation of fluorescently labeled nucleotides into a strand of DNA, isolation and manipulation of a labeled DNA fragment and the detection of single nucleotides using ultra-sensitive laser-induced fluorescence detection following their cleavage from the fragment. Detection of individual fluorophores in the liquid phase was accomplished with time-gated detection following pulsed-laser excitation. The photon bursts from individual rhodamine 6G (R6G) molecules travelling through a laser beam have been observed as have bursts from single fluorescently modified nucleotides. Using two different biotinylated nucleotides as a model system for fluorescently labeled nucleotides, we have observed synthesis of the complementary copy of M13 bacteriophage. Work with fluorescently labeled nucleotides is underway. We have observed and manipulated individual molecules of DNA attached to a microbead with an epifluorescence microscope. 16 refs., 3 figs., 1 tab.

  11. 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.

  12. A rapid DNA extraction method from culture and clinical samples. Suitable for the detection of human cytomegalovirus by the polymerase chain reaction.

    PubMed

    Zandotti, C; De Lamballerie, X; Guignole-Vignoli, C; Bollet, C; De Micco, P

    1993-02-01

    We propose an one-step DNA extraction method suitable for the polymerase chain reaction. This procedure utilizes Chelex 100, a chelating in exchange resin. This technique was compared with a traditional technique (proteinase K lysis, phenol-chloroform extraction and ethanol precipitation) for isolation of human cytomegalovirus DNA from clinical samples. The procedure using Chelex 100 appeared to be a simple and fast extraction method for human cytomegalovirus DNA.

  13. [Development of a rapid method of immunologic monitoring during pregnancy].

    PubMed

    Khismatullina, N A; Iusupov, R Kh; Selimov, M A; Ianbarisova, S R

    2001-01-01

    The authors validate the efficiency of enzyme-linked immunoassay (ELISA) for rapid diagnosis of rabies and titration of specific antibodies and of the method of isolation of field rabies strains in the rat Gasser's node neurinoma cells. Highly active specific diagnostic agents for ELISA and immunofluorescent test based on sheep immunoglobulins have been created for detection of the rabies agent in pathological material from different animals and humans.

  14. 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.

  15. 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.

  16. 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

  17. Development and validation of a dynamic vapor sorption-fast gas chromatography-flame ionization detection method for rapid analysis of volatile release from glassy matrices.

    PubMed

    Bohn, Dawn M; Cadwallader, Keith R; Schmidt, Shelly J

    2005-04-20

    Historically, saturated salt solutions in desiccators have been used to investigate the effects of increasing relative humidity and temperature on the volatile retention efficiency of amorphous glasses. Obtaining data using desiccators is a static process that gives the researcher discrete data points with which to draw conclusions. Dynamic vapor sorption (DVS; SMS, London, U.K.) is a humidification system that creates specific relative humidity and temperature environments within a chamber that contains the material being investigated. This study had two specific aims: (1) to develop a DVS-fast GC-FID method that dynamically evaluates the effects of humidification and temperature increases on volatile release from amorphous carbohydrate glasses and (2) to evaluate the validity of the DVS-fast GC-FID method. Artificial cherry Durarome (Firmenich, Plainsboro, NJ) was used as the model system. The DVS-fast GC-FID method proved to be an innovative, accurate, and precise technique that can be used to conduct humidification/temperature-volatile release studies. PMID:15826072

  18. 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.

  19. 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 & Treisman, 2005), or is…

  20. 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. PMID:21204700

  1. 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. PMID:27315516

  2. Homogeneous liquid-liquid extraction (HoLLE) via flotation combined with gas chromatography-flame ionization detection as a very simple, rapid and sensitive method for the determination of fenitrothion in water samples.

    PubMed

    Mashayekhi, Hossein Ali

    2013-01-01

    Homogeneous liquid-liquid extraction via flotation assistance (HoLLE-FA) and gas chromatography-flame ionization detection (GC-FID) was presented for the extraction and determination of fenitrothion in water samples. In this work, a rapid, simple and efficient HoLLE-FA method was developed based on applying low-density organic solvents without employing centrifugation. A special extraction cell was designed to facilitate the collection of low-density solvent extraction in the determination of fenitrothion in water samples. The water sample solution was added into an extraction cell that contained an appropriate mixture of extraction and homogeneous solvents. By using air flotation, the organic solvent was collected at the conical part of the designed cell. Under the optimum conditions, the method performance was studied in terms of the linear dynamic range (LDR from 1.0 up to 100 μg L⁻¹), linearity (r² > 0.998), and precision (repeatability < 8.0%). Also, limit of detection (LOD) of 0.4 μg L⁻¹ was obtained for fenitrothion. The applicability of the HoLLE-FA method was evaluated by the extraction and determination of fenitrothion in three different water samples.

  3. Homogeneous liquid-liquid extraction (HoLLE) via flotation combined with gas chromatography-flame ionization detection as a very simple, rapid and sensitive method for the determination of fenitrothion in water samples.

    PubMed

    Mashayekhi, Hossein Ali

    2013-01-01

    Homogeneous liquid-liquid extraction via flotation assistance (HoLLE-FA) and gas chromatography-flame ionization detection (GC-FID) was presented for the extraction and determination of fenitrothion in water samples. In this work, a rapid, simple and efficient HoLLE-FA method was developed based on applying low-density organic solvents without employing centrifugation. A special extraction cell was designed to facilitate the collection of low-density solvent extraction in the determination of fenitrothion in water samples. The water sample solution was added into an extraction cell that contained an appropriate mixture of extraction and homogeneous solvents. By using air flotation, the organic solvent was collected at the conical part of the designed cell. Under the optimum conditions, the method performance was studied in terms of the linear dynamic range (LDR from 1.0 up to 100 μg L⁻¹), linearity (r² > 0.998), and precision (repeatability < 8.0%). Also, limit of detection (LOD) of 0.4 μg L⁻¹ was obtained for fenitrothion. The applicability of the HoLLE-FA method was evaluated by the extraction and determination of fenitrothion in three different water samples. PMID:23934566

  4. Rapid Multiplex Nested PCR for Detection of Respiratory Viruses▿

    PubMed Central

    Lam, W. Y.; Yeung, Apple C. M.; Tang, Julian W.; Ip, Margaret; Chan, Edward W. C.; Hui, Mamie; Chan, Paul K. S.

    2007-01-01

    Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken. PMID:17804659

  5. 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.

  6. An overview of transducers as platform for the rapid detection of foodborne pathogens.

    PubMed

    Arora, Pooja; Sindhu, Annu; Kaur, Harmanmeet; Dilbaghi, Neeraj; Chaudhury, Ashok

    2013-03-01

    The driving advent of portable, integrated biosensing ways for pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques. The miniaturization and automation of integrated detection systems present a significant advantage for rapid, portable detection of foodborne microbes. In this review, we have highlighted current developments and directions in foodborne pathogen detection systems. Recent progress in the biosensor protocols toward the detection of specific microbes has been elaborated in detail. It also includes strategies and challenges for the implementation of a portable platform toward rapid foodborne sensing systems.

  7. 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. PMID:26873217

  8. 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.

  9. Rapid ratiometric biomarker detection with topically applied SERS nanoparticles

    PubMed Central

    Wang, Yu “Winston”; Khan, Altaz; Som, Madhura; Wang, Danni; Chen, Ye; Leigh, Steven Y.; Meza, Daphne; McVeigh, Patrick Z.; Wilson, Brian C.; Liu, Jonathan T.C.

    2014-01-01

    Multiplexed surface-enhanced Raman scattering (SERS) nanoparticles (NPs) offer the potential for rapid molecular phenotyping of tissues, thereby enabling accurate disease detection as well as patient stratification to guide personalized therapies or to monitor treatment outcomes. The clinical success of molecular diagnostics based on SERS NPs would be facilitated by the ability to accurately identify tissue biomarkers under time-constrained staining and detection conditions with a portable device. In vitro, ex vivo and in vivo experiments were performed to optimize the technology and protocols for the rapid detection (0.1-s integration time) of multiple cell-surface biomarkers with a miniature fiber-optic spectral-detection probe following a brief (5 min) topical application of SERS NPs on tissues. Furthermore, we demonstrate that the simultaneous detection and ratiometric quantification of targeted and nontargeted NPs allows for an unambiguous assessment of molecular expression that is insensitive to nonspecific variations in NP concentrations. PMID:25045721

  10. 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.

  11. 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.

  12. 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.

  13. A field-practical assay for rapid detection of chlorophenols

    SciTech Connect

    Washburn, K.S.; Phillips, T.D.

    1994-12-31

    Water-solvated chlorophenols (CPs) are environmental toxins associated with wood preservation and pesticide synthesis and usage. Their toxicity and association with dioxin-contaminated wastes are well-documented, as is their stability in most environmental settings. Several analytical procedures, mainly HPLC and GC/MS, are currently used to detect and quantify CPs, but these procedures are based on expensive equipment and technical expertise in a laboratory setting. The authors have developed an inexpensive, field-practical method for CPs, utilizing a small, packed glass minicolumn and derivatization of target CP molecules with dansyl chloride (5-dimethylaminonaphthalene-1sulfonyl chloride), or DsCl. A nonfluorescent borosilicate glass tube was used to house an array of inorganic sorbent materials, including preparative layers and a reactive neutral alumina interface separated by sand. DsCl is a substituted naphthalene with a conjugated X system that is responsible for its fluorescent complexation. Amines that reacted with DsCl were removed with a small amount of phyllosilicate clay to avoid interference. A neutral alumina/sand interface was used to strongly bind and immobilize the dansylated CPs. Activities greater than 3.0 for the alumina were avoided to prevent loss of selectivity, intensity and color of the fluorescence at the reactive interface. The results indicated that this assay was capable of rapidly screening potable water samples and detecting CP contamination at very low concentrations (i.e., 1.0 ppb of pentachlorophenol in drinking water).

  14. 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.

  15. Rapid method for SV40 titration.

    PubMed

    Drayman, Nir; Kler, Slava; Ben-nun-Shaul, Orly; Oppenheim, Ariella

    2010-03-01

    SV40 titer is determined traditionally by the conventional plaque assay. Plaques appear after several rounds of infection and the assay takes around two weeks, which may delay research. A simpler assay was developed, based on detection of T-antigen in the infected cells by flow cytometry. Cells grown in 6-well plates are infected with serial dilutions of the viral stock, harvested 48h post-infection, stained and analyzed for T-antigen using a flow cytometer. The viral titer is calculated based on the percentage of T-antigen positive cells. The procedure is accomplished in 2 days. Unexpectedly we found that titers on different permissive African Green Monkey kidney cell lines were consistently different, suggesting variable susceptibility to SV40 infection. The method described, optimized for SV40 titration, may be adapted readily to other viruses.

  16. Rapid Detection of Ophiostoma piceae and O. quercus in Stained Wood by PCR

    PubMed Central

    Kim, Seong Hwan; Uzunovic, Adnan; Breuil, Colette

    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. PMID:9872792

  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. Colorimetric method for rapid determination of bacteriuria.

    PubMed

    Wallis, C; Melnick, J L; Longoria, C J

    1981-09-01

    An inexpensive, rapid, and simple colorimetric test for detection of bacteriuria is described. This test does not require bacterial growth and has the marked advantage of being able to quantify bacteria, even when the organisms are present in the urine of bacteriuric patients who are being treated with antibiotics. The test is carried out with 1 ml of urine, which is processed through a 10-mm-diameter filter than entraps the bacteria on its surface. Safranine dye is passed through the filter to stain the bacteria and the filter fibers. A decolorizer, which removes the dye from the filter fibers but not from the bacteria, is then passed through the filter. If there are greater than or equal to 10(5) colony-forming units of bacteria per ml in the sample, the 10-mm filter disk manifests a pink to red color. If there are less than 10(5)colony-forming units of bacteria per ml, the filter disk remains white or becomes slightly yellow. The entire procedure has been adapted to a semiautomated instrument and the time required per test is less than 1 min. The results obtained on the test card are a permanent record to be filled with the patient's chart. The bacteria can be quickly classified as gram positive or gram negative by selective staining of a second milliliter of urine on the filter. Of 441 urine specimens tested, 430 (98%) were correctly classified as containing more or less than 10(5) colony-forming units per ml. A total of 62 urine specimens were positive by bacterial plating (greater than or equal to 10(5) colony-forming units per ml), and 59 were positive by the colorimetric test. Eight false-positives (colorimetric test positive, plate counts less than 10(5)) were encountered in patients (bacteriuric) being prescribed antibiotics. Removal of the antibiotics from these urine specimens, with subsequent replating of the samples, indicated the presence of greater than or equal to 10(5) colony-forming units of bacteria per ml in three representative cases tested

  19. 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.

  20. A Rapid Method for Determination of the Main Conjugated Linoleic Acid Precursors (C18:2 n-6 and C18:3 n-3) in Forage by Capillary Zone Electrophoresis with Ultraviolet Detection Using Gas Chromatography with Flame Ionization Detection as a Comparative Method.

    PubMed

    de Jesus Coelho Castro, Renata; Sobrinho, Fausto Souza; Sundfeld da Gama, Marco Antônio; Takabayashi Sato, Renata; Lavorato Lima, Larissa; Leal de Oliveira, Marcone Augusto

    2015-01-01

    A rapid method has been proposed for determination of the main conjugated linoleic acid precursors such as linoleic (C18:2 n-6) and linolenic (C18:3 n-3) acids in forages by capillary zone electrophoresis (CZE) with direct UV detection at 200 nm. Among the fatty acids found in forages, C18:2 n-6 and C18:3 n-3 have received particular attention due to their roles as precursors for the synthesis of conjugated linoleic acid, a class of health-enhancing compounds that is predominantly found in dairy products. The electrolyte background consisted of 12.0 mmol/L tetraborate buffer (pH 9.2) added to 12.0 mmol/L Brij 35®, 17% acetonitrile, and 33% methanol. Under the optimized conditions, the baseline separation of C18:2 n-6 and C18:3 n-3 was achieved within 4 min. The CZE-UV method was compared to GC with a flame ionization detector, which is the American Oil Chemists' Society (AOCS 996.06) official method for fatty acid analysis. The methods did not show any evidence of significant differences within 95% confidence interval (P>0.05). The CZE-UV method was successfully applied to the analysis of 80 genotypes of Brachiaria ruzizienses clones submitted to a genetic improvement program in agricultural research. PMID:26651572

  1. 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.

  2. 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.

  3. Method for detecting leaks

    SciTech Connect

    Baillie, L.A.; Senese, F.J.

    1988-09-27

    This patent describes a method for determining leaks in a storage tank containing a liquid. The method consists of: locating a float means initially at a depth in the liquid approximately equal to V/sub L//S/sub L/ where V/sub L/ is the volume of the liquid in the tank and S/sub L/ is free surface area of the liquid in the tank; saturating vapor above the liquid in the tank with vapor of the liquid by atomizing so as to fill the vapor above the liquid with small drops of liquid or coating surfaces of the tank above the liquid with a thin layer of the liquid so as to hasten saturation of the vapor above the liquid, so that temperature variation of the liquid in the tank does not change the depth at which the float means floats in the liquid; and determining changes of location of the float means in the tank.

  4. An FPGA-based rapid wheezing detection system.

    PubMed

    Lin, Bor-Shing; Yen, Tian-Shiue

    2014-02-01

    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.

  5. 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

  6. Rapid enzymatic method for pectin methyl esters determination.

    PubMed

    Lękawska-Andrinopoulou, Lucyna; Vasiliou, Efstathios G; Georgakopoulos, Dimitrios G; Yialouris, Constantinos P; Georgiou, Constantinos A

    2013-01-01

    Pectin is a natural polysaccharide used in food and pharma industries. Pectin degree of methylation is an important parameter having significant influence on pectin applications. A rapid, fully automated, kinetic flow method for determination of pectin methyl esters has been developed. The method is based on a lab-made analyzer using the reverse flow-injection/stopped flow principle. Methanol is released from pectin by pectin methylesterase in the first mixing coil. Enzyme working solution is injected further downstream and it is mixed with pectin/pectin methylesterase stream in the second mixing coil. Methanol is oxidized by alcohol oxidase releasing formaldehyde and hydrogen peroxide. This reaction is coupled to horse radish peroxidase catalyzed reaction, which gives the colored product 4-N-(p-benzoquinoneimine)-antipyrine. Reaction rate is proportional to methanol concentration and it is followed using Ocean Optics USB 2000+ spectrophotometer. The analyzer is fully regulated by a lab written LabVIEW program. The detection limit was 1.47 mM with an analysis rate of 7 samples h(-1). A paired t-test with results from manual method showed that the automated method results are equivalent to the manual method at the 95% confidence interval. The developed method is rapid and sustainable and it is the first application of flow analysis in pectin analysis. PMID:24455426

  7. Rapid Enzymatic Method for Pectin Methyl Esters Determination

    PubMed Central

    Łękawska-Andrinopoulou, Lucyna; Vasiliou, Efstathios G.; Georgakopoulos, Dimitrios G.; Yialouris, Constantinos P.; Georgiou, Constantinos A.

    2013-01-01

    Pectin is a natural polysaccharide used in food and pharma industries. Pectin degree of methylation is an important parameter having significant influence on pectin applications. A rapid, fully automated, kinetic flow method for determination of pectin methyl esters has been developed. The method is based on a lab-made analyzer using the reverse flow-injection/stopped flow principle. Methanol is released from pectin by pectin methylesterase in the first mixing coil. Enzyme working solution is injected further downstream and it is mixed with pectin/pectin methylesterase stream in the second mixing coil. Methanol is oxidized by alcohol oxidase releasing formaldehyde and hydrogen peroxide. This reaction is coupled to horse radish peroxidase catalyzed reaction, which gives the colored product 4-N-(p-benzoquinoneimine)-antipyrine. Reaction rate is proportional to methanol concentration and it is followed using Ocean Optics USB 2000+ spectrophotometer. The analyzer is fully regulated by a lab written LabVIEW program. The detection limit was 1.47 mM with an analysis rate of 7 samples h−1. A paired t-test with results from manual method showed that the automated method results are equivalent to the manual method at the 95% confidence interval. The developed method is rapid and sustainable and it is the first application of flow analysis in pectin analysis. PMID:24455426

  8. 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.

  9. 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.

  10. Local method for detecting communities

    NASA Astrophysics Data System (ADS)

    Bagrow, James P.; Bollt, Erik M.

    2005-10-01

    We propose a method of community detection that is computationally inexpensive and possesses physical significance to a member of a social network. This method is unlike many divisive and agglomerative techniques and is local in the sense that a community can be detected within a network without requiring knowledge of the entire network. A global application of this method is also introduced. Several artificial and real-world networks, including the famous Zachary karate club, are analyzed.

  11. Rapid identification of salmonella serotypes with stereo and hyperspectral microscope imaging Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Rapid Identification of Salmonella Serotypes with Stereo and Hyperspectral Microscope Imaging Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Sensitive and rapid detection of staphylococcus aureus in milk via cell binding domain of lysin.

    PubMed

    Yu, Junping; Zhang, Yun; Zhang, Yun; Li, Heng; Yang, Hang; Wei, Hongping

    2016-03-15

    Staphylococcus aureus (S. aureus) is an important food-borne pathogen in dairy products contaminated through raw ingredients or improper food handling. Rapid detection of S. aureus with high sensitivity is of significance for food quality and safety. In this study, a new method was developed for detecting S. aureus in milk by coupling immunomagnetic separation with enzyme linked cell wall binding domain (CBD) of lysin plyV12, which can bind to S. aureus with high affinity. There are millions of binding sites present on the cell surface of S. aureus for the CBD attachment, which greatly improves the detection sensitivity. The method has the overall testing time of only 1.5h with the detection limit of 4 × 10(3)CFU/mL in spiked milk. Because it is simple, rapid and sensitive, this method could be used for the detection of S. aureus in various food samples.

  14. 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. PMID:25703096

  15. Rapid sampling for rapid analysis: A new method for VOCs in drinking water

    SciTech Connect

    Gardner, B.D.; Johnson, J.A.; Holland, J.F.

    1995-12-31

    Until recently, there has been only moderate emphasis placed on the time consumption of the sampling process in many gas chromatography/mass spectrometry (GC/MS) analyses. Historically, the total analysis time has been primarily dependent upon the chromatographic separation and detection steps, and significant decreases in sample preparation time have had little impact on overall sample throughput. The recent maturation of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) using time array detection has decreased chromatographic separation and mass spectrometric detection times by factors of 10 or more, leaving the sampling process as a significant and often limiting factor in the total analysis time. Current methods for the analysis of volatile organic compounds (VOCs) in drinking water utilize headspace or purge and trap techniques requiring 20-30 minute sampling times to extract these compounds for analysis by GC/MS. Solid-phase microextraction (SPME) has recently been presented as a viable alternative to the above methods. Sampling and desorption times of less than 10 minutes have been reported for part-per-billion levels of volatile organics in aqueous solution. These rapid times suggest a potential for combining SPME and GC/TOFMS technologies to create a methodology for high sample throughput.

  16. 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.

  17. Rapid and reliable diagnostic algorithm for detection of Clostridium difficile.

    PubMed

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

    2008-01-01

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

  18. Rapid method for the quantification of hydroquinone concentration: chemiluminescent analysis.

    PubMed

    Chen, Tung-Sheng; Liou, Show-Yih; Kuo, Wei-Wen; Wu, Hsi-Chin; Jong, Gwo-Ping; Wang, Hsueh-Fang; Shen, Chia-Yao; Padma, V Vijaya; Huang, Chih-Yang; Chang, Yen-Lin

    2015-11-01

    Topical hydroquinone serves as a skin whitener and is usually available in cosmetics or on prescription based on the hydroquinone concentration. Quantification of hydroquinone content therefore becomes an important issue in topical agents. High-performance liquid chromatography (HPLC) is the commonest method for determining hydroquinone content in topical agents, but this method is time-consuming and uses many solvents that can become an environmental issue. We report a rapid method for quantifying hydroquinone content by chemiluminescent analysis. Hydroquinone induces the production of hydrogen peroxide in the presence of basic compounds. Hydrogen peroxide induced by hydroquinone oxidized light-emitting materials such as lucigenin, resulted in the production of ultra-weak chemiluminescence that was detected by a chemiluminescence analyzer. The intensity of the chemiluminescence was found to be proportional to the hydroquinone concentration. We suggest that the rapid (measurement time, 60 s) and virtually solvent-free (solvent volume, <2 mL) chemiluminescent method described here for quantifying hydroquinone content may be an alternative to HPLC analysis. PMID:25693839

  19. 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. PMID:22079620

  20. 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.

  1. Rapid and sensitive detection of Staphylococcus species in milk by ELISA based on monodisperse magnetic particles.

    PubMed

    Yazdankhah, S P; Hellemann, A L; Rønningen, K; Olsen, E

    1998-04-30

    Staphylococcus spp. are the single most important cause of mastitis in dairy cows. Aiming for a rapid and sensitive method for detection of staphylococci, a magnetic bead-based enzyme-linked immunosorbent assay (ELISA) employing MAb for the detection of staphylococci in milk was developed. Pretreatment of the sample with lysostaphin increased the sensitivity of the assay. The method is specific for most of staphylococci which cause mastitis in bovines, detects between 10(4)-10(5) organisms per ml and takes three hours. Thus, the principle for staphylococci ELISA based on monodisperse magnetic beads is a way for rapid detection of staphylococci in milk and possibly also for detecting staphylococci by direct testing of other clinical specimens. The system may be automated.

  2. Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB).

    PubMed

    Han, Sang-Hoon; Lee, Do-Bu; Lee, Dong-Woo; Kim, Eul-Hwan; Yoon, Byoung-Su

    2008-09-01

    A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene of P. larvae with a micro-scale chip-based real-time PCR system, GenSpector TMC-1000, which has uncommonly fast heating and cooling rates (10 degrees C per second) and small reaction volume (6microl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools. PMID:18571197

  3. Development of a rapid and comprehensive proteomics-based arboviruses detection system.

    PubMed

    Okamoto, Kenta; Endo, Yushirou; Inoue, Shingo; Nabeshima, Takeshi; Nga, Phan Thi; Guillermo, Posadas H; Yu, Fuxun; Loan, Do Phuong; Trang, Bui Minh; Natividad, Filipinas F; Hasebe, Futoshi; Morita, Kouichi

    2010-07-01

    A rapid and comprehensive protocol, which combines simple purification and liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI/MS/MS), was developed for identification of arboviruses in infected culture fluid. Using this protocol, various arboviruses were detected including uncommon viruses that were described previously as Banna virus and Yunnan orbivirus. This approach is useful for the rapid screening of viral samples that cannot be identified by conventional gene amplification or immunological methods.

  4. Methods and compositions for rapid thermal cycling

    SciTech Connect

    Beer, Neil Reginald; Benett, William J.; Frank, James M.; Deotte, Joshua R.; Spadaccini, Christopher

    2015-10-27

    The rapid thermal cycling of a material is targeted. A microfluidic heat exchanger with an internal porous medium is coupled to tanks containing cold fluid and hot fluid. Fluid flows alternately from the cold tank and the hot tank into the porous medium, cooling and heating samples contained in the microfluidic heat exchanger's sample wells. A valve may be coupled to the tanks and a pump, and switching the position of the valve may switch the source and direction of fluid flowing through the porous medium. A controller may control the switching of valve positions based on the temperature of the samples and determined temperature thresholds. A sample tray for containing samples to be thermally cycled may be used in conjunction with the thermal cycling system. A surface or internal electrical heater may aid in heating the samples, or may replace the necessity for the hot tank.

  5. Rapid and highly sensitive detection of lead ions in drinking water based on a strip immunosensor.

    PubMed

    Kuang, Hua; Xing, Changrui; Hao, Changlong; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

    2013-03-28

    In this study, we have first developed a rapid and sensitive strip immunosensor based on two heterogeneously-sized gold nanoparticles (Au NPs) probes for the detection of trace lead ions in drinking water. The sensitivity was 4-fold higher than that of the conventional LFA under the optimized conditions. The visual limit of detection (LOD) of the amplified method for qualitative detection lead ions was 2 ng/mL and the LOD for semi-quantitative detection could go down to 0.19 ng/mL using a scanning reader. The method suffered from no interference from other metal ions and could be used to detect trace lead ions in drinking water without sample enrichment. The recovery of the test samples ranged from 96% to 103%. As the detection method could be accomplished within 15 min, this method could be used as a potential tool for preliminary monitoring of lead contamination in drinking water.

  6. Rapid and Highly Sensitive Detection of Lead Ions in Drinking Water Based on a Strip Immunosensor

    PubMed Central

    Kuang, Hua; Xing, Changrui; Hao, Changlong; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

    2013-01-01

    In this study, we have first developed a rapid and sensitive strip immunosensor based on two heterogeneously-sized gold nanoparticles (Au NPs) probes for the detection of trace lead ions in drinking water. The sensitivity was 4-fold higher than that of the conventional LFA under the optimized conditions. The visual limit of detection (LOD) of the amplified method for qualitative detection lead ions was 2 ng/mL and the LOD for semi-quantitative detection could go down to 0.19 ng/mL using a scanning reader. The method suffered from no interference from other metal ions and could be used to detect trace lead ions in drinking water without sample enrichment. The recovery of the test samples ranged from 96% to 103%. As the detection method could be accomplished within 15 min, this method could be used as a potential tool for preliminary monitoring of lead contamination in drinking water. PMID:23539028

  7. Rapid detection of enteric fever by coagglutination and countercurrent immunoelectrophoresis.

    PubMed

    Mishra, M; Thakar, Y S; Chande, C; Tankhiwale, N S; Saoji, A M

    1998-10-01

    A total of 463 patients clinically suspected of enteric fever and 100 healthy individuals were investigated by coagglutination (COAG) and countercurrent immunoelectrophoresis (CIEP) for rapid diagnosis of enteric fever. The S. typhi was grown in blood culture in 32 (6.91%) and Widal test was positive in 126 (27.21%) cases. The serum COAG with local antiserum was positive in 165 (35.64%), serum COAG with standard antiserum in 163 (35.21%), Blood culture supernatant (BCS) COAG in 153 (33.05%), serum CIEP for antigen (Ag) in 118 (25.09%), BCS CIEP in 99 (21.39%) while serum CIEP for antibody (Ab) was positive in 34 (7.34%) cases. Only two healthy controls revealed positive COAG result with local antiserum. The sensitivity of all antigen detection tests was 100% except BCS CIEP (98.25%) in the first week of fever and declined rapidly to 75.79% for serum COAG tests, 69.47% for BCS COAG and dramatically to 37.89% for serum CIEP for Ag and 22.11% for BCS CIEP tests during the second week whereas the sensitivity of serum CIEP for Ab detection rose from 17.54% to 23.16% from first to second week of illness. In view of the resulting data, it is suggested that both COAG and CIEP may be employed for the rapid diagnosis of enteric fever in the routine clinical setup.

  8. Rapid detection of Naegleria fowleri in water distribution pipeline biofilms and drinking water samples.

    PubMed

    Puzon, Geoffrey J; Lancaster, James A; Wylie, Jason T; Plumb, Iason J

    2009-09-01

    Rapid detection of pathogenic Naegleria fowler in water distribution networks is critical for water utilities. Current detection methods rely on sampling drinking water followed by culturing and molecular identification of purified strains. This culture-based method takes an extended amount of time (days), detects both nonpathogenic and pathogenic species, and does not account for N. fowleri cells associated with pipe wall biofilms. In this study, a total DNA extraction technique coupled with a real-time PCR method using primers specific for N. fowleri was developed and validated. The method readily detected N. fowleri without preculturing with the lowest detection limit for N. fowleri cells spiked in biofilm being one cell (66% detection rate) and five cells (100% detection rate). For drinking water, the detection limit was five cells (66% detection rate) and 10 cells (100% detection rate). By comparison, culture-based methods were less sensitive for detection of cells spiked into both biofilm (66% detection for <10 cells) and drinking water (0% detection for <10 cells). In mixed cultures of N. fowleri and nonpathogenic Naegleria, the method identified N. fowleri in 100% of all replicates, whereastests with the current consensus primers detected N. fowleri in only 5% of all replicates. Application of the new method to drinking water and pipe wall biofilm samples obtained from a distribution network enabled the detection of N. fowleri in under 6 h, versus 3+ daysforthe culture based method. Further, comparison of the real-time PCR data from the field samples and the standard curves enabled an approximation of N. fowleri cells in the biofilm and drinking water. The use of such a method will further aid water utilities in detecting and managing the persistence of N. fowleri in water distribution networks.

  9. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi, Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2006-08-01

    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM, on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA, on the 5' end.

  10. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi,Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2011-07-05

    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

  11. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, J.P.

    1997-07-29

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

  12. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, James P.

    1997-01-01

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

  13. Rapid vegetative propagation method for carob

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many fruit species are propagated by vegetative methods such as budding, grafting, cutting, suckering, layering etc. to avoid heterozygosity. Carob trees (Ceratonia siliqua L.) are of highly economical value and it is among the most difficult-to-propagate fruit species. In this study, air-layering p...

  14. Method for rapid, high sensitivity tritiated water extraction

    SciTech Connect

    Failor, R.; Belovodsky, L.; Gaevoy, V.; Golubev, A.

    1997-04-20

    We have developed a thermal vacuum desorption process to rapidly extract water from environmental samples for tritium analysis. Thermal vacuum desorption allows for extraction of the moisture from the sample within a few hours in a form and quantity suitable for liquid scintillation counting and allows detection of tritium at the levels of <2 Bq/L of milk, <0.5 Bq/gm of vegetation, and < 0.5 Bq/gin of soil. We developed a prototype unit that can process batches of twenty or more samples within 24 hours. Early data shows that a high percentage of water is extracted reproducibly without enrichment or depletion of the tritium content. The quench coefficient of the extracted water is low allowing for accurate, direct liquid scintillation counting. Excellent comparison has been observed with results using freeze-dry lypholization as the water extraction method.

  15. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1990-10-09

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  16. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1987-10-07

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  17. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Moyzis, Robert K.; Ratliff, Robert L.; Shera, E. Brooks; Stewart, Carleton C.

    1990-01-01

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

  18. Method and system rapid piece handling

    DOEpatents

    Spletzer, Barry L.

    1996-01-01

    The advent of high-speed fabric cutters has made necessary the development of automated techniques for the collection and sorting of garment pieces into collated piles of pieces ready for assembly. The present invention enables a new method for such handling and sorting of garment parts, and to apparatus capable of carrying out this new method. The common thread is the application of computer-controlled shuttling bins, capable of picking up a desired piece of fabric and dropping it in collated order for assembly. Such apparatus with appropriate computer control relieves the bottleneck now presented by the sorting and collation procedure, thus greatly increasing the overall rate at which garments can be assembled.

  19. Evaluation of the diagnostic accuracy of a kit for the rapid detection of group A streptococci.

    PubMed

    Savoia, D; Francesetti, C; Millesimo, M; Dotti, G; Gatti, G; Rurali, C

    1994-01-01

    A rapid immunoassay method using an Event Test Strip Strep A experimental kit (Boehringer Mannheim) was evaluated. Results obtained were compared with culture results to evaluate the accuracy of detection of group A streptococci directly from throat swabs or from artificial swabs containing various bacterial concentrations. A good diagnostic accuracy was obtained with a sensitivity of 96.9% in the assay of throat swabs which provided more than ten group A Streptococcus colonies per plate. Since a low level of micro-organisms may indicate infection, it is recommended that a culture be performed when the rapid test based on antigen detection is negative. PMID:8208140

  20. Method for detecting biological toxins

    SciTech Connect

    Ligler, F.S.; Campbell, J.R.

    1992-01-01

    Biological toxins are indirectly detected by using polymerase chain reaction to amplify unique nucleic acid sequences coding for the toxins or enzymes unique to toxin synthesis. Buffer, primers coding for the unique nucleic acid sequences and an amplifying enzyme are added to a sample suspected of containing the toxin. The mixture is then cycled thermally to exponentially amplify any of these unique nucleic acid sequences present in the sample. The amplified sequences can be detected by various means, including fluorescence. Detection of the amplified sequences is indicative of the presence of toxin in the original sample. By using more than one set of labeled primers, the method can be used to simultaneously detect several toxins in a sample.

  1. Dipsticks for rapid detection of plasmodium in vectoring anopheles mosquitoes.

    PubMed

    Ryan, J R; Dav, K; Emmerich, E; Garcia, L; Yi, L; Coleman, R E; Sattabongkot, J; Dunton, R F; Chan, A S; Wirtz, R A

    2001-06-01

    Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

  2. A rapid improved method for sexing embryo of water buffalo.

    PubMed

    Zoheir, K M A; Allam, A A

    2011-07-01

    The objective of the experiment of this paper is to develop and improve in the sexing method for preimplantation embryos of water buffalo (Bubalus bubalis) using loop-mediated isothermal amplification (LAMP) reaction. Embryo sexing has been recognized to control effectively the sex of offspring in the embryo transfer industry. A rapid and simple detection system was established by adding ethidium bromide (EB) or 5 μl of CuSO4 (3M) to the product of LAMP reaction. The result of these additions after 2 min was a color change and a precipitate. It could be employed as an alternative method in the detection of the reaction products in place of the time consuming electrophoresis or the turbidity meter. The in vitro produced buffalo embryos were divided into one to eight pieces using a microblade attached to a micromanipulator. The cell number in each piece was counted before sexing. Sexing of DNA samples extracted from one to five biopsies cells was performed by LAMP. After biopsy, the remaining part of the embryos was used to confirm the sex by polymerase chain reaction (PCR). Fifty buffalo embryos were used and the accuracy of sex prediction was 100% when the blastomeres dissociated from a morula exceeds three. In conclusion, the present procedure without turbidity meter and electrophoresis was reliable and applicable for sexing the water buffalo embryos.

  3. A rapid, inexpensive high throughput screen method for neurite outgrowth.

    PubMed

    Yeyeodu, Susan T; Witherspoon, Sam M; Gilyazova, Nailya; Ibeanu, Gordon C

    2010-01-01

    Neurite outgrowth assays are the most common phenotypic screen to assess chemical effects on neuronal cells. Current automated assays involve expensive equipment, lengthy sample preparation and handling, costly reagents and slow rates of data acquisition and analysis. We have developed a high throughput screen (HTS) for neurite outgrowth using a robust neuronal cell model coupled to fast and inexpensive visualization methods, reduced data volume and rapid data analysis. Neuroscreen-1 (NS-1) cell, a subclone of PC12, possessing rapid growth and enhanced sensitivity to NGF was used as a model neuron. This method reduces preparation time by using cells expressing GFP or native cells stained with HCS CellMask(™) Red in a multiplexed 30 min fixation and staining step. A 2x2 camera binning process reduced both image data files and analysis times by 75% and 60% respectively, compared to current protocols. In addition, eliminating autofocus steps during montage generation reduced data collection time. Pharmacological profiles for stimulation and inhibition of neurite outgrowth by NGF and SU6656 were comparable to current standard method utilizing immunofluorescence detection of tubulin. Potentiation of NGF-induced neurite outgrowth by members of a 1,120-member Prestwick compound library as assayed using this method identified six molecules, including etoposide, isoflupredone acetate, fludrocortisone acetate, thioguanosine, oxyphenbutazone and gibberellic acid, that more than doubled the neurite mass primed by 2 ng/ml NGF. This simple procedure represents an important routine approach in high throughput screening of large chemical libraries using the neurite outgrowth phenotype as a measure of the effects of chemical molecules on neuronal cells. PMID:21347208

  4. Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses.

    PubMed

    Takahashi, Tadanobu; Takano, Maiko; Kurebayashi, Yuuki; Agarikuchi, Takashi; Suzuki, Chihiro; Fukushima, Keijo; Takahashi, Shunsaku; Otsubo, Tadamune; Ikeda, Kiyoshi; Minami, Akira; Suzuki, Takashi

    2015-01-01

    Human parainfluenza virus type 1 (hPIV1) does not form clear plaque by the conventional plaque formation assay because of slightly a cytopathic effects in many cell lines infected with hPIV1, thus making in virus titration, isolation and inhibitor evaluation difficult. We have succeeded in fluorescent histochemical visualization of sialidase activities of influenza A and B viruses, Newcastle disease virus and Sendai virus by using a novel fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). In this study, we applied the BTP3-Neu5Ac assay for rapid detection of hPIV1 and hPIV type 3. The BTP3-Neu5Ac assay could histochemically visualize dot-blotted hPIVs on a membrane and hPIV-infected cells as local fluorescence under UV irradiation. We succeeded in distinct fluorescent visualization of hPIV1-infected cells in only 3 d using the BTP3-Neu5Ac assay. Due to there being no fixation, hPIV1 was isolated directly from fluorescent stained focus cells by the BTP3-Neu5Ac assay. Establishment of a sensitive, easy, and rapid fluorescent focus detection assay for hPIV, hPIV1 in particular will contribute greatly to progress in hPIV studies.

  5. Reusable fiber optic immunofluorosensor for rapid detection of pesticides

    NASA Astrophysics Data System (ADS)

    Anis, Nabil A.; Valdes, James J.; Thompson, Roy G.; Menking, Darrell E.; Wong, Rosie B.; Eldefrawi, Mohyee E.

    1993-05-01

    Quartz fibers coated with acetylcholinesterase (AChE) or antibody (Ab) are used as biosensors utilizing total reflectance fluorescence for the rapid detection of pesticides. The enzyme biosensor was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ AChE on quartz fibers. The fluorescent signal was generated by hydrolysis of acetylcholine (ACh) that is present in the perfusate. Organophosphate (OP) and carbamate anticholinesterase (AntiChE) insecticides inhibited AChE and reduced the fluorescent quenching resulting from AChE hydrolysis. A parathion biosensor was constructed by immobilizing casein-parathion on the quartz fibers, that bound rabbit antiparathion antibody. The optical signal was generated by perfusing the fibers with fluorescein-labeled goat antirabbit IgG. Free parathion inhibited the binding of antiparathion Abs and reduced the optical signal and provided the basis for detection of parathion. Another immunosensor developed detected the herbicide PursuitR by utilizing the reversible binding of a fluorescein-Pursuit derivative to antiPursuit Abs immobilized on the fiber. Unlabeled Pursuit competed effectively and displaced the bound fluorescent compound in a dose-dependent manner. The sensor discriminated effectively between Pursuit-like and structurally unrelated herbicides. The immunosensor offers the advantage of continuous monitoring, ease of operation, speed of detection, low cost, stability, specificity, matrix transparency, and reusability.

  6. Rapid and Robust Damage Detection using Radar Remote Sensing

    NASA Astrophysics Data System (ADS)

    Yun, S.; Fielding, E. J.; Simons, M.; Webb, F.; Rosen, P. A.; Owen, S. E.

    2012-12-01

    Under ARIA (Advanced Rapid Imaging and Analysis) project at JPL and Caltech, we developed a prototype algorithm and data system to rapidly detect surface change caused by natural or man-made damage using a radar remote sensing technique of InSAR coherence. We tested the algorithm with a building demolition site in the City of Pasadena, California. The results show clear signal at the demolition site, with about 150% SNR improvement compared to conventional approach. Out of fourteen strongest detected signals, we confirmed that at least eleven of them were associated with real demolition and construction projects. We applied the algorithm to the February 2011 M6.3 Christchurch earthquake in New Zealand, which killed 185 people and caused financial damage of US $16-24 billion. We produced a damage proxy map (DPM) using radar data from ALOS satellite (Figure A), where red pixels identify regions where there may have been earthquake induced building damage, landslides, and liquefaction. The distribution of the red regions agrees well with the post-earthquake assessment performed on the ground by inspectors from the New Zealand government and summarized in their damage assessment zone map (Figure B). The DPM was derived from radar data acquired 3 days after the earthquake, whereas the ground truth zone map was first published 4 months after the earthquake. In addition to all-weather and day-and-night capability of radar, the sensitivity of radar signal to surface property change is high enough for reliable damage assessment. Current and future satellite and airborne missions should keep the expected composite data acquisition latency within a day. Rapidly produced accurate damage assessment maps will help saving people, assisting effective prioritization of rescue operations at early stage of response, and significantly improve timely situational awareness for emergency management and national / international assessment for response and recovery.

  7. Rapid Brightness Variations as a Tool to Enhance Satellite Detectability

    NASA Astrophysics Data System (ADS)

    Laas-Bourez, Myrtille; Klotz, Alain; Ducrotte, Etienne; Boer, Michel; Blanchet, Gwendoline

    2009-03-01

    To preserve the space environment for future generations and ensure the safety of space missions, we have to improve our knowledge of the debris at all altitudes. Since 2004, we have started to observe and study satellites and debris on the geostationary orbit. We use a network of robotic telescopes called TAROT (Télescopes Action Rapide pour les Objets Transitoires - Rapid Action Telescope for Transient Objects) which are located in France and Chile. This system processes the data in real time. Its wide field of view is useful for detection, systematic survey and to follow both catalogued and uncatalogued objects. The TAROTs are 25 cm telescopes with a wide field of view of 1.86deg x 1.86deg. It can detect objects up to 17th magnitude with an integration time of 30 seconds, corresponding to an object of 50cm in the geostationary belt with a 0.2 albedo. Tiny debris are also dangerous for space mission and satellites. To detect them, we need either to increase the TAROT sensitivity or to observe them in optimal light conditions.Last year we detected very important magnitude variations from several geostationary satellites during observations close to equinoxes. The brightness of a geostationary satellite evolves during the night and during the year, depending on the angle between the observer, the satellite and the sun. Geostationary satellites will be brighter near March 1st and of October 10th, at their exit of the shade. In this period the sun crosses the equatorial plan of the Earth, the enlightened surface will reach a maximum during a limited periods of time (about 30 minutes), provoking a short, bright flash. This phenomenon is used in two ways: first, it allows to detect smaller objects, which are usually below the detection limit, enhancing the sensitivity of the survey. Secondly, for longer objects the light curve during and outside the °ash contains information on the object intrinsic geometry and reflectivity. In this paper we discuss how the various

  8. Development of a double-antibody sandwich ELISA for rapid detection of Bacillus Cereus in food.

    PubMed

    Zhu, Longjiao; He, Jing; Cao, Xiaohan; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-01-01

    Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 10(4)-2.8 × 10(6) cells/mL with a detection limit (LOD) of 0.9 × 10(3) cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens. PMID:26976753

  9. Development of a double-antibody sandwich ELISA for rapid detection of Bacillus Cereus in food.

    PubMed

    Zhu, Longjiao; He, Jing; Cao, Xiaohan; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-01-01

    Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 10(4)-2.8 × 10(6) cells/mL with a detection limit (LOD) of 0.9 × 10(3) cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens.

  10. Use of Rapid-Scan EPR to Improve Detection Sensitivity for Spin-Trapped Radicals

    PubMed Central

    Mitchell, Deborah G.; Rosen, Gerald M.; Tseitlin, Mark; Symmes, Breanna; Eaton, Sandra S.; Eaton, Gareth R.

    2013-01-01

    The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1–6.0 μM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR. PMID:23870255

  11. Rapid Detection and Identification of Respiratory Viruses by Direct Immunofluorescence

    PubMed Central

    D'Alessio, Donn; Williams, Stanley; Dick, Elliot C.

    1970-01-01

    The use of fluorescein-conjugated antiserum against respiratory syncytial (RS) and parainfluenza 1 and 3 viruses was compared with conventional techniques in the rapid detection of virus in tissue cultures inoculated with pharyngeal specimens known to contain these viruses. Twenty-three specimens were tested: 9 RS, 8 parainfluenza 1, and 6 parainfluenza 3. The fluorescent-antibody technique (FA) detected virus in 52% of the tissue cultures in 24 hr, and, by 72 hr, 22 of the 23 cultures were FA-positive whereas only 5 were positive by conventional techniques. Additionally, conjugated antisera were prepared against herpes simplex, influenza A2, and adenovirus type 5. All conjugates stained only the homologous virus and were 100- to 10,000-fold more sensitive than conventional techniques in detecting descending dilutions of virus inocula by 24 hr. With the procedures described, several antisera could be conjugated and ready for use within 24 hr. Serum fractionation was by ammonium sulfate precipitation, and with the procedure outlined virtually complete recovery of the globulin fraction and elimination of all of the albumin were accomplished. Images PMID:4098101

  12. Rapid molecular strategy for orbivirus detection and characterization.

    PubMed

    Palacios, Gustavo; Cowled, Chris; Bussetti, Ana V; Savji, Nazir; Weir, Richard; Wick, Ivan; Travassos da Rosa, Amelia; Calisher, Charles H; Tesh, Robert B; Boyle, David; Lipkin, W Ian

    2011-06-01

    Orbiviruses infect a wide range of hosts, including humans. The ability to detect them has been hampered by their diversity. Here we present a simple consensus reverse transcription (RT)-PCR method targeting the polymerase gene for orbivirus recognition and characterization. Phylogenetic assignment is achieved by automated Web-based sequence analysis of amplification products.

  13. Rapid detection of porcine kobuvirus in feces by reverse transcription loop-mediated isothermal amplification

    PubMed Central

    2014-01-01

    Background PKV is a new emerging pathogen detected in diarrhea pigs. At present, no more detection methods were reported except RT-PCR method. this study was to develop a fast diagnostic method based on the LAMP reaction for rapid detection of PKV nucleic acid in fecal samples. Findings Two pairs of primers were designed to amplify the conservative 3D gene of PKV genome. The PKV RT-LAMP method possessed well specificity and had 100 times higher sensitivity than common reverse transcription PCR (RT-PCR), which could detect up to 10 RNA copies of the target gene. Conclusions The results showed that the optimal reaction condition for RT-LAMP was achieved at 64°C for 50 min. Furthermore, the RT-LAMP procedure does not demand special equipment and is time-saving. PMID:24755372

  14. A novel Love Wave biosensor for rapid and sensitive detection of marine toxins.

    PubMed

    Zhang, Xi; Fang, Jiaru; Zou, Yingchang; Zou, Ling; Hu, Ning; Wang, Ping

    2015-01-01

    Marine toxins are produced by plankton and do a great harm to human through food chain by accumulating in shellfishes and fishes. It is highly required and favorable to develop novel methods for the rapid and efficient detection of marine toxins to avoid the poisoning cases that have occurred frequently in many countries. This study presents a real-time Love Wave biosensor for the rapid detection of okadaic acid (OA), which used HepG2 cell lines as the sensing elements. The results indicate that this cell-based biosensor can provide real-time information of cellular activities induced by okadaic acid and has a higher sensitivity than the conventional cell-based assay. It is suggested that this cell-based biosensor can be used as a convenient and efficient method for marine toxin detection, which has a great potential to contribute to avoid the harmful effects of marine toxins on the human health.

  15. A novel Love Wave biosensor for rapid and sensitive detection of marine toxins.

    PubMed

    Zhang, Xi; Fang, Jiaru; Zou, Yingchang; Zou, Ling; Hu, Ning; Wang, Ping

    2015-01-01

    Marine toxins are produced by plankton and do a great harm to human through food chain by accumulating in shellfishes and fishes. It is highly required and favorable to develop novel methods for the rapid and efficient detection of marine toxins to avoid the poisoning cases that have occurred frequently in many countries. This study presents a real-time Love Wave biosensor for the rapid detection of okadaic acid (OA), which used HepG2 cell lines as the sensing elements. The results indicate that this cell-based biosensor can provide real-time information of cellular activities induced by okadaic acid and has a higher sensitivity than the conventional cell-based assay. It is suggested that this cell-based biosensor can be used as a convenient and efficient method for marine toxin detection, which has a great potential to contribute to avoid the harmful effects of marine toxins on the human health. PMID:26736968

  16. Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification.

    PubMed

    Wang, Xin-Ru; Wu, Li-Fen; Wang, Yan; Ma, Ying-Ying; Chen, Feng-Hua; Ou, Hong-Ling

    2015-01-01

    Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 °C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg/μl DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg/μl and 147 pg/μl DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity.

  17. Survey of Anomaly Detection Methods

    SciTech Connect

    Ng, B

    2006-10-12

    This survey defines the problem of anomaly detection and provides an overview of existing methods. The methods are categorized into two general classes: generative and discriminative. A generative approach involves building a model that represents the joint distribution of the input features and the output labels of system behavior (e.g., normal or anomalous) then applies the model to formulate a decision rule for detecting anomalies. On the other hand, a discriminative approach aims directly to find the decision rule, with the smallest error rate, that distinguishes between normal and anomalous behavior. For each approach, we will give an overview of popular techniques and provide references to state-of-the-art applications.

  18. Method for detecting toxic gases

    DOEpatents

    Stetter, J.R.; Zaromb, S.; Findlay, M.W. Jr.

    1991-10-08

    A method is disclosed which is capable of detecting low concentrations of a pollutant or other component in air or other gas. This method utilizes a combination of a heating filament having a catalytic surface of a noble metal for exposure to the gas and producing a derivative chemical product from the component. An electrochemical sensor responds to the derivative chemical product for providing a signal indicative of the product. At concentrations in the order of about 1-100 ppm of tetrachloroethylene, neither the heating filament nor the electrochemical sensor is individually capable of sensing the pollutant. In the combination, the heating filament converts the benzyl chloride to one or more derivative chemical products which may be detected by the electrochemical sensor. 6 figures.

  19. Rapid Bacterial Detection via an All-Electronic CMOS Biosensor

    PubMed Central

    Nikkhoo, Nasim; Cumby, Nichole; Gulak, P. Glenn; Maxwell, Karen L.

    2016-01-01

    The timely and accurate diagnosis of infectious diseases is one of the greatest challenges currently facing modern medicine. The development of innovative techniques for the rapid and accurate identification of bacterial pathogens in point-of-care facilities using low-cost, portable instruments is essential. We have developed a novel all-electronic biosensor that is able to identify bacteria in less than ten minutes. This technology exploits bacteriocins, protein toxins naturally produced by bacteria, as the selective biological detection element. The bacteriocins are integrated with an array of potassium-selective sensors in Complementary Metal Oxide Semiconductor technology to provide an inexpensive bacterial biosensor. An electronic platform connects the CMOS sensor to a computer for processing and real-time visualization. We have used this technology to successfully identify both Gram-positive and Gram-negative bacteria commonly found in human infections. PMID:27618185

  20. Rapid Bacterial Detection via an All-Electronic CMOS Biosensor.

    PubMed

    Nikkhoo, Nasim; Cumby, Nichole; Gulak, P Glenn; Maxwell, Karen L

    2016-01-01

    The timely and accurate diagnosis of infectious diseases is one of the greatest challenges currently facing modern medicine. The development of innovative techniques for the rapid and accurate identification of bacterial pathogens in point-of-care facilities using low-cost, portable instruments is essential. We have developed a novel all-electronic biosensor that is able to identify bacteria in less than ten minutes. This technology exploits bacteriocins, protein toxins naturally produced by bacteria, as the selective biological detection element. The bacteriocins are integrated with an array of potassium-selective sensors in Complementary Metal Oxide Semiconductor technology to provide an inexpensive bacterial biosensor. An electronic platform connects the CMOS sensor to a computer for processing and real-time visualization. We have used this technology to successfully identify both Gram-positive and Gram-negative bacteria commonly found in human infections. PMID:27618185

  1. Rapid detection of HIV-1 p24 antigen using magnetic immuno-chromatography (MICT).

    PubMed

    Workman, Shon; Wells, Susan K; Pau, Chou-Pong; Owen, S Michele; Dong, X Fan; LaBorde, Ron; Granade, Timothy C

    2009-09-01

    Detection of human immunodeficiency virus (HIV) infections has been enhanced by incorporating p24 antigen detection with current HIV antibody detection using enzyme immunoassays (EIAs). However, screening for HIV antibodies has increased through the use of rapid, lateral-flow HIV antibody detection assays that currently do not have the capability to detect HIV p24 antigen. In this report, a lateral-flow based assay using super-paramagnetic particles as the detection marker was developed for the detection of HIV-1 p24 antigen. This magnetic immuno-chromatographic test (MICT) uses an inexpensive, low-maintenance instrument that detects the magnetic moment of the super-paramagnetic particles in a magnetic field. MICT is simple to perform, provides a numerical output for easier determination of reactive results and can be completed in 40min. The lower limit of detection for HIV-1 p24 spiked into assay sample buffer and 50% plasma was 30pg/ml for both. Detection of HIV-1 p24 antigen at 50pg/ml was reproducible in both inter-run and intra-run assays with coefficients of variation of <13%. Furthermore, the MICT p24 assay was able to detect intact virus spiked into 50% plasma (lower detection limit of approximately 250,000 viral RNA copies/ml). MICT detection of increasing HIV-1 p24 levels in commercially available seroconversion panels by MICT was only slightly later than that detected by much more complex EIAs. MICT could provide a simple, low-cost, and portable method for rapid HIV-1 p24 detection in a variety of testing environments. PMID:19482361

  2. Rapid and specific SPRi detection of L. pneumophila in complex environmental water samples.

    PubMed

    Foudeh, Amir M; Trigui, Hana; Mendis, Nilmini; Faucher, Sebastien P; Veres, Teodor; Tabrizian, Maryam

    2015-07-01

    Legionellosis is a very devastating disease worldwide mainly due to unpredictable outbreaks in man-made water systems. Developing a highly specific and sensitive rapid detection system that detects only metabolically active bacteria is a main priority for water quality assessment. We previously developed a versatile technique for sensitive and specific detection of synthetic RNA. In the present work, we further investigated the performance of the developed biosensor for detection of Legionella pneumophila in complex environmental samples, particularly those containing protozoa. The specificity and sensitivity of the detection system were verified using total RNA extracted from L. pneumophila in spiked water co-cultured with amoebae. We demonstrated that the expression level of ribosomal RNA (rRNA) is extremely dependent on the environmental conditions. The presence of amoebae with L. pneumophila, especially in nutrition-deprived samples, increased the amount of L. pneumophila 15-fold after 1 week as measured through the expression of 16s rRNA. Using the developed surface plasmon resonance imaging (SPRi) detection method, we were also able to successfully detect L. pneumophila within 3 h, both in the presence and absence of amoebae in the complex environmental samples obtained from a cooling water tower. These findings suggest that the developed biosensing system is a viable method for rapid, real-time and effective detection not only for L. pneumophila in environmental samples but also to assess the risk associated with the use of water contaminated with other pathogens. PMID:25935681

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

    PubMed

    Boyle, David S; McNerney, Ruth; Teng Low, Hwee; Leader, Brandon Troy; Pérez-Osorio, Ailyn C; Meyer, Jessica C; O'Sullivan, Denise M; Brooks, David G; Piepenburg, Olaf; Forrest, Matthew S

    2014-01-01

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

  4. [Rapid quantitative detection of sulfate reducing bacteria in oil field].

    PubMed

    Wei, Li; Ma, Fang; Wang, Ji-Hua; Zhao, Li-Jun

    2007-02-01

    It take long time and high cost to measure sulfate reducing bacteria (SRB) in wastewater of oil field. A rapid quantitative method was developed by combining polymerase chain reaction(PCR) and most probable number (MPN) to measure sulfate reducing bacteria (SRB) in wastewater of oil field. The bacterium solution was directly prepared from wastewater for PCR amplification, which ensured quantitative accuracy. Reaction system and amplification condition were designed using universal primers DSR1F and DSR5R of dissimilatory sulfite reductase in SRB. The result show that the accuracy of this method is two magnitude higher than that of MPN. The whole measuring process take 3 - 4 hours and the reproducibility of this method is extremely stable, being fit to practical process.

  5. Rapid pathogen detection with bacterial-assembled magnetic mesoporous silica.

    PubMed

    Lee, Soo Youn; Lee, Jiho; Lee, Hye Sun; Chang, Jeong Ho

    2014-03-15

    We report rapid and accurate pathogen detection by coupling with high efficiency magnetic separation of pathogen by Ni(2+)-heterogeneous magnetic mesoporous silica (Ni-HMMS) and real time-polymerase chain reaction (RT-PCR) technique. Ni-HMMS was developed with a significant incorporation of Fe particles within the silica mesopores by programmed thermal hydrogen reaction and functionalized with Ni(2+) ion on the surface by the wet impregnation process. High abundant Ni(2+) ions on the Ni-HMMS surface were able to assemble with cell wall component protein NikA (nickel-binding membrane protein), which contains several pathogenic bacteria including Escherichia coli O157:H7. NikA protein expression experiment showed the outstanding separation rate of the nikA gene-overexpressed E. coli (pSY-Nik) when comparing with wild-type E. coli (44.5 ± 13%) or not over-expressed E. coli (pSY-Nik) (53.2 ± 2.7%). Moreover, Ni-HMMS showed lower obstacle effect by large reaction volume (10 mL) than spherical core/shell-type silica magnetic nanoparticles functionalized with Ni(2+) (ca. 40 nm-diameters). Finally, the Ni-HMMS was successfully assessed to separate pathogenic E. coli O157:H7 and applied to direct and rapid RT-PCR to quantitative detection at ultralow concentration (1 Log10 cfu mL(-1)) in the real samples (milk and Staphylococcus aureus culture broth) without bacterial amplification and DNA extraction step.

  6. Rapid and automated detection of salmonella by electrical measurements.

    PubMed Central

    Easter, M. C.; Gibson, D. M.

    1985-01-01

    A rapid method for determining the presence of salmonella in food is described. It consists of pre-enrichment in buffered peptone water modified by the addition of dulcitol and trimethylamine oxide, followed by selective enrichment in a selenite-cystine broth with similar modifications. Changes in the conductance of the selective enrichment broth are monitored continuously using a suitable impediometric instrument. Most of the Salmonella spp. tested gave a fast (approximately 100 microS/h) and large (greater than 600 microS) change in conductance, other enteric bacteria much less or no change. The assay is usually complete within 24 h. Samples of foodstuffs, naturally and artificially contaminated with Salmonella spp., were all correctly classified. Some strains of Citrobacter freundii produced a false positive conductance response, and they could not be selectively eliminated using antibiotics or cyanide. The conductance method is simple and easy to use, gives rapid results and involves less media and subculturing than is required for traditional methods. PMID:3891846

  7. Development of a rapid capture-cum-detection method for Escherichia coli O157 from apple juice comprising nano-immunomagnetic separation in tandem with surface enhanced Raman scattering.

    PubMed

    Najafi, Roya; Mukherjee, Shubhasish; Hudson, Jim; Sharma, Anup; Banerjee, Pratik

    2014-10-17

    A combined capture and detection method comprising of nano-immunomagnetic separation (NIMS) and surface enhanced Raman spectroscopy (SERS) was developed to detect Escherichia coli O157 from liquid media including apple juice. The capture antibodies (cAbs) were immobilized on magnetite-gold (Fe3O4/Au) magnetic nanoparticles (MNPs) which were used for separation and concentration of the E. coli O157 cells from model liquid food matrix. The capture efficiency (CE) for E. coli O157 using MNP was found to be approximately 84-94%. No cross reactivity was observed with background non-target organisms. There was a significant difference in the mean CE of bacteria captured by MNP and commercially sourced immunomagnetic microbeads (p<0.05). For the detection of target pathogen, SERS labels were prepared by conjugating gold nanoparticles with Raman reporter molecules and the detector antibody (dAb). Au-Raman label-dAb was interacted with gold coated MNP-cAb-E. coli O157 complex. The ability of this immunoassay to detect E. coli O157 in apple juice was investigated. We have successfully applied the synthesized Fe3O4/Au nanoclusters to E. coli O157 detection in apple juice using the SERS method. The lowest detectable bacterial cell concentration in apple juice was 10(2)CFU/mL with a total analysis time of less than an hour. This method presents a convenient way of preconcentration, separation, and detection of low levels of target pathogen from liquid food matrix.

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

  9. Rapid Method for Escherichia coli in the Cuyahoga River

    USGS Publications Warehouse

    Brady, Amie M.G.

    2007-01-01

    This study is a continuation of a previous U.S. Geological Survey (USGS) project in cooperation with the National Park Service at Cuyahoga Valley National Park in Brecksville, Ohio. A rapid (1-hour) method for detecting Escherichia coli (E. coli) in water was tested and compared to the standard (24-hour) method for determining E. coli concentrations. Environmental data were collected to determine turbidity, rainfall, and streamflow at the time of sampling. In the previous study (2004-5), data collected were used to develop predictive models to determine recreational water quality in the river at two sites within the park. Data collected during this continued study (2006) were used to test these models. At Jaite, a centrally located site within the park, the model correctly predicted exceedances or nonexceedances of the Ohio Environmental Protection Agency maximum for recreational water quality in 80 percent of samples. At Old Portage, a site near the upstream boundary of the park, the model correctly predicted recreational water quality in 58 percent of samples. All of the data collected in 2004-6 will be used to develop more accurate models for use in future studies. Analysis and discussion of model results are scheduled to be included in an upcoming USGS Scientific Investigations Report.

  10. Object-based rapid change detection for disaster management

    NASA Astrophysics Data System (ADS)

    Thunig, Holger; Michel, Ulrich; Ehlers, Manfred; Reinartz, Peter

    2011-11-01

    Rapid change detection is used in cases of natural hazards and disasters. This analysis lead to quick information about areas of damage. In certain cases the lack of information after catastrophe events is obstructing supporting measures within disaster management. Earthquakes, tsunamis, civil war, volcanic eruption, droughts and floods have much in common: people are directly affected, landscapes and buildings are destroyed. In every case geospatial data is necessary to gain knowledge as basement for decision support. Where to go first? Which infrastructure is usable? How much area is affected? These are essential questions which need to be answered before appropriate, eligible help can be established. This study presents an innovative strategy to retrieve post event information by use of an object-based change detection approach. Within a transferable framework, the developed algorithms 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 normalized temporal change index (NTCI) panchromatic datasets are used to extract areas which are destroyed, areas which were not affected and in addition areas which are developing new for cases where rebuilding has already started. The results of the study are also feasible for monitoring urban growth.

  11. REVIEW OF RAPID METHODS FOR ASSESSING WETLAND CONDITION

    EPA Science Inventory

    We evaluated over 40 wetland rapid assessment methods developed for a variety of purposes for their use in the assessment of ecological integrity or ecosystem condition. Four criteria were used to screen methods: 1) the method can be used to measure condition, 2) it is truly rap...

  12. Rapid detection of acetamiprid in foods using surface-enhanced Raman spectroscopy (SERS).

    PubMed

    Wijaya, Wisiani; Pang, Shintaro; Labuza, Theodore P; He, Lili

    2014-04-01

    Acetamiprid is a neonicotinoid pesticide that is commonly used in modern farming. Acetamiprid residue in food commodities can be a potential harm to human and has been implicated in the honey bee hive die off crisis. In this study, we developed rapid, simple, and sensitive methods to detect acetamiprid in apple juice and on apple surfaces using surface-enhanced Raman spectroscopy (SERS). No pretreatment of apple juice sample was performed. A simple surface swab method was used to recover acetamiprid from the apple surface. Samples were incubated with silver dendrites for several minutes and SERS spectra were taken directly from the silver surface. Detection of a set of 5 apple juice samples can be done within 10 min. The swab-SERS method took 15 min for a set of 5 samples. Resulting spectral data were analyzed using principal component analysis. The highest acetamiprid peak at 634 cm(-1) was used to detect and quantify the amount of acetamiprid spiked in 1:1 water-methanol solvent, apple juice, and on apple surface. The SERS method was able to successfully detect acetamiprid at 0.5 μg/mL (0.5 ppm) in solvent, 3 μg/mL (3 ppm) in apple juice, and 0.125 μg/cm(2) on apple surfaces. The SERS methods provide simple, rapid, and sensitive ways to detect acetamiprid in beverages and on the surfaces of thick skinned fruits and vegetables. PMID:24620941

  13. Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to Duchenne muscula dystrophy carrier detection

    SciTech Connect

    Prior, T.W.; Wenger, G.D.; Moore, J.

    1994-09-01

    A new strategy has been developed for rapid haplotype analysis. It is based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. The method is simple, rapid, does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using 12 commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. As a result of expanding the number of detectable polymorphisms throughout the dystrophin gene, we show how the method can easily be combined with dinucleotide analysis to improve the accuracy of carrier detection in the nondeletion cases. The technique is also shown to be used as an effective screen for improving carrier detection in several families with deletions. The finding of heterozygosity within the deletion identifies the at-risk female as a noncarrier. Using this method, we have identified and incorporated 3 new dystrophin polymorphisms (one of which in exon 16 is unique to African Americans). The method may be used other genetic diseases when mutations are unknown, or there are few dinucleotide markers in the gene proximity, or for the identification of haplotype backgrounds of mutant alleles.

  14. A Novel Self-Assembling DNA Nano Chip for Rapid Detection of Human Papillomavirus Genes

    PubMed Central

    Li, Xin; Li, Yanbo; Hong, Li

    2016-01-01

    Rapid detection of tumor-associated DNA such as Human Papillomavirus (HPV) has important clinical value for the early screening of tumors. By attaching oligonucleotides or cDNA onto the chip surface, DNA chip technology provides a rapid method to analyze gene expression. However, challenges remain regarding increasing probe density and improving detection time. To address these challenges, we proposed a DNA chip that was self-assembled from single stranded DNA in combination with high probe density and a rapid detection method. Over 200 probes could be attached to the surface of this 100-nm diameter DNA chip. For detection, the chips were adsorbed onto a mica surface and then incubated for ten minutes with HPV-DNA; the results were directly observable using atomic force microscopy (AFM). This bottom-up fabricated DNA nano chip combined with high probe density and direct AFM detection at the single molecule level will likely have numerous potential clinical applications for gene screening and the early diagnosis of cancer. PMID:27706184

  15. Biosensor for dengue virus detection: sensitive, rapid, and serotype specific.

    PubMed

    Baeumner, Antje J; Schlesinger, Nicole A; Slutzki, Naomi S; Romano, Joseph; Lee, Eun Mi; Montagna, Richard A

    2002-03-15

    A serotype-specific RNA biosensor was developed for the rapid detection of Dengue virus (serotypes 1-4) in blood samples. After RNA amplification, the biosensor allows the rapid detection of Dengue virus RNA in only 15 min. In addition, the biosensor is portable, inexpensive, and very easy to use, making it an ideal detection system for point-of-care and field applications. The biosensor is coupled to the isothermal nucleic acid sequence-based amplification (NASBA) technique with which small amounts of virus RNA are amplified using a simple water bath. During the NASBA reaction, a generic sequence is attached to all RNA molecules as described earlier (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). It has been shown earlier that Dengue virus can be detected specifically using two DNA probes: a first probe hybridized with the attached generic sequence and, therefore, bound to every amplified RNA molecule; and a second probe either bound to all four Dengue virus serotypes or chosen to be specific for only one serotype. These probes were utilized in the biosensor described in this publication. For a generic Dengue virus biosensor, the second probe is complementary to a conserved region found in all Dengue serotypes. For identification of the individual Dengue virus serotypes, four serotype-specific probes were developed (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtiff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The generic DNA probe (reporter probe) is coupled to the outside of dye-encapsulating liposomes. The conserved or Dengue serotype specific probes (capture probes) are immobilized on a polyethersulfone membrane strip

  16. A field-deployable device for the rapid detection of cyanide poisoning in whole blood

    NASA Astrophysics Data System (ADS)

    Boehringer, Hans; Tong, Winnie; Chung, Roy; Boss, Gerry; O'Farrell, Brendan

    2012-06-01

    Feasibility of a field-deployable device for the rapid and early diagnosis of cyanide poisoning in whole blood using the spectral shift of the vitamin B12 precursor cobinamide upon binding with cyanide as an indicator is being assessed. Cyanide is an extremely potent and rapid acting poison with as little as 50 mg fatal to humans. Cyanide poisoning has been recognized as a threat from smoke inhalation and potentially through weapons of mass destruction. Currently, no portable rapid tests for the detection of cyanide in whole blood are available. Cobinamide has an extremely high affinity for cyanide and captures hemoglobin associated cyanide from red blood cells. Upon binding of cyanide, cobinamide undergoes a spectral shift that can be measured with a spectrophotometer. We have combined the unique cyanide-binding properties of cobinamide with blood separation technology, sample transport and a detection system, and are developing a rapid, field deployable, disposable device which will deliver an intuitive result to a first responder, allowing for rapid response to exposure events. Feasibility of the cobinamide-Cyanide chemistry in a rapid test using a whole blood sample from a finger-stick has been demonstrated with an assay time from sample collection to a valid result of under 5 minutes. Data showing the efficacy of the diagnostic method and initial device design concepts will be shown.

  17. Method for detecting toxic gases

    DOEpatents

    Stetter, Joseph R.; Zaromb, Solomon; Findlay, Jr., Melvin W.

    1991-01-01

    A method capable of detecting low concentrations of a pollutant or other component in air or other gas, utilizing a combination of a heating filament having a catalytic surface of a noble metal for exposure to the gas and producing a derivative chemical product from the component, and an electrochemical sensor responsive to the derivative chemical product for providing a signal indicative of the product. At concentrations in the order of about 1-100 ppm of tetrachloroethylene, neither the heating filament nor the electrochemical sensor is individually capable of sensing the pollutant. In the combination, the heating filament converts the benzyl chloride to one or more derivative chemical products which may be detected by the electrochemical sensor.

  18. Rapid in vivo detection of isoniazid-sensitive Mycobacterium tuberculosis by breath test

    PubMed Central

    Maiga, Mariama C.; Atudorei, Viorel; Sharp, Zachary D.; Bishai, William R.; Timmins, Graham S.

    2014-01-01

    There is urgent need for rapid, point of care diagnostic tools for tuberculosis (TB) and drug sensitivity. Current methods based on in vitro growth take weeks, while DNA amplification can neither differentiate live from dead organisms nor determine phenotypic drug resistance. Here we show the development and evaluation of a rapid breath test for isoniazid (INH)-sensitive TB based on detection of labeled N2 gas formed specifically from labeled INH by mycobacterial KatG enzyme. In vitro data shows the assay is specific, dependent on mycobacterial abundance, and discriminates between INH-sensitive and resistant (S315T mutant KatG) TB. In vivo, the assay is rapid with maximal detection of 15N2 in exhaled breath of infected rabbits within five to ten minutes. No increase in 15N2 is detected in un-infected animals, and the increases in 15N2 are dependent on infection dose. This test may allow rapid detection of INH-sensitive TB. PMID:25247851

  19. Rapid Detection of the Varicella Zoster Virus in Saliva

    NASA Technical Reports Server (NTRS)

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

    2011-01-01

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

  20. Bacteria detection instrument and method

    NASA Technical Reports Server (NTRS)

    Renner, W.; Fealey, R. D. (Inventor)

    1972-01-01

    A method and apparatus for screening a sample fluid for bacterial presence are disclosed wherein the fluid sample is mixed with culture media of sufficient quantity to permit bacterial growth in order to obtain a test solution. The concentration of oxygen dissolved in the test solution is then monitored using the potential difference between a reference electrode and a noble metal electrode which are in contact with the test solution. The change in oxygen concentration which occurs during a period of time as indicated by the electrode potential difference is compared with a detection criterion which exceeds the change which would occur absent bacteria.

  1. A rapid survey technique for Tropilaelaps mite (Mesostigmata: Laelapidae) detection.

    PubMed

    Pettis, Jeffery S; Rose, Robyn; Lichtenberg, Elinor M; Chantawannakul, Panuwan; Buawangpong, Ninat; Somana, Weeraya; Sukumalanand, Prachaval; Vanengelsdorp, Dennis

    2013-08-01

    Parasitic Tropilaelaps (Delfinado and Baker) mites are a damaging pest of European honey bees (Apis mellifera L.) in Asia. These mites represent a significant threat if introduced to other regions of the world, warranting implementation of Tropilaelaps mite surveillance in uninfested regions. Current Tropilaelaps mite-detection methods are unsuitable for efficient large scale screening. We developed and tested a new bump technique that consists of firmly rapping a honey bee brood frame over a collecting pan. Our method was easier to implement than current detection tests, reduced time spent in each apiary, and minimized brood destruction. This feasibility increase overcomes the test's decreased rate of detecting infested colonies (sensitivity; 36.3% for the bump test, 54.2% and 56.7% for the two most sensitive methods currently used in Asia). Considering this sensitivity, we suggest that screening programs sample seven colonies per apiary (independent of apiary size) and 312 randomly selected apiaries in a region to be 95% sure of detecting an incipient Tropilaelaps mite invasion. Further analyses counter the currently held view that Tropilaelaps mites prefer drone bee brood cells. Tropilaelaps mite infestation rate was 3.5 +/- 0.9% in drone brood and 5.7 +/- 0.6% in worker brood. We propose the bump test as a standard tool for monitoring of Tropilaelaps mite presence in regions thought to be free from infestation. However, regulators may favor the sensitivity of the Drop test (collecting mites that fall to the bottom of a hive on sticky boards) over the less time-intensive Bump test.

  2. A rapid survey technique for Tropilaelaps mite (Mesostigmata: Laelapidae) detection.

    PubMed

    Pettis, Jeffery S; Rose, Robyn; Lichtenberg, Elinor M; Chantawannakul, Panuwan; Buawangpong, Ninat; Somana, Weeraya; Sukumalanand, Prachaval; Vanengelsdorp, Dennis

    2013-08-01

    Parasitic Tropilaelaps (Delfinado and Baker) mites are a damaging pest of European honey bees (Apis mellifera L.) in Asia. These mites represent a significant threat if introduced to other regions of the world, warranting implementation of Tropilaelaps mite surveillance in uninfested regions. Current Tropilaelaps mite-detection methods are unsuitable for efficient large scale screening. We developed and tested a new bump technique that consists of firmly rapping a honey bee brood frame over a collecting pan. Our method was easier to implement than current detection tests, reduced time spent in each apiary, and minimized brood destruction. This feasibility increase overcomes the test's decreased rate of detecting infested colonies (sensitivity; 36.3% for the bump test, 54.2% and 56.7% for the two most sensitive methods currently used in Asia). Considering this sensitivity, we suggest that screening programs sample seven colonies per apiary (independent of apiary size) and 312 randomly selected apiaries in a region to be 95% sure of detecting an incipient Tropilaelaps mite invasion. Further analyses counter the currently held view that Tropilaelaps mites prefer drone bee brood cells. Tropilaelaps mite infestation rate was 3.5 +/- 0.9% in drone brood and 5.7 +/- 0.6% in worker brood. We propose the bump test as a standard tool for monitoring of Tropilaelaps mite presence in regions thought to be free from infestation. However, regulators may favor the sensitivity of the Drop test (collecting mites that fall to the bottom of a hive on sticky boards) over the less time-intensive Bump test. PMID:24020263

  3. Explosives detection system and method

    DOEpatents

    Reber, Edward L.; Jewell, James K.; Rohde, Kenneth W.; Seabury, Edward H.; Blackwood, Larry G.; Edwards, Andrew J.; Derr, Kurt W.

    2007-12-11

    A method of detecting explosives in a vehicle includes providing a first rack on one side of the vehicle, the rack including a neutron generator and a plurality of gamma ray detectors; providing a second rack on another side of the vehicle, the second rack including a neutron generator and a plurality of gamma ray detectors; providing a control system, remote from the first and second racks, coupled to the neutron generators and gamma ray detectors; using the control system, causing the neutron generators to generate neutrons; and performing gamma ray spectroscopy on spectra read by the gamma ray detectors to look for a signature indicative of presence of an explosive. Various apparatus and other methods are also provided.

  4. Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

    PubMed

    Muniroh, M S; Sariah, M; Zainal Abidin, M A; Lima, N; Paterson, R R M

    2014-05-01

    Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations. PMID:24681306

  5. Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

    PubMed

    Muniroh, M S; Sariah, M; Zainal Abidin, M A; Lima, N; Paterson, R R M

    2014-05-01

    Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.

  6. Field's stain--a rapid staining method for Acanthamoeba spp.

    PubMed

    Pirehma, M; Suresh, K; Sivanandam, S; Anuar, A K; Ramakrishnan, K; Kumar, G S

    1999-10-01

    Acanthamoeba sp. is a free-living amoeba known to cause chronic central nervous system infection or eye infection in humans. Many cases remain undetected for want of a good detection system. We report for the first time a rapid staining method to facilitate the identification of Acanthamoeba sp. using the modified Field's staining technique. A. castellanii, which was used in the present experiment, is maintained in our laboratory in mycological peptone medium (Gibco). The cultures were pooled together and smears were made on glass slides for staining purposes. Different types of stains such as Field's stain, modified Field's stain, Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and trichrome stain were used to determine the best stain for the identification of this amoeba. The concentration of various stains and the duration of staining were varied to provide the best color and contrast for each stain. Acanthamoeba was also obtained from the brain of experimentally infected mice and was stained with various stains as mentioned above to determine the best stain for use in identifying the presence of this parasite in experimentally infected animals. The modified Field's stain gives a very good color contrast as compared with other stains. Furthermore, it takes only 20 s to be carried out using the least number of reagents, making it suitable for both laboratory and field use.

  7. Rapid determination of ampicillin in bovine milk by liquid chromatography with fluorescence detection

    SciTech Connect

    Ang, C.Y.W.; Luo, Wenhong

    1997-01-01

    A rapid and sensitive liquid chromatographic (LC) method was developed for the determination of ampicillin residues in raw bovine milk, processed skim milk, and pasteurized, homogenized whole milk with vitamin D. Milk samples were deproteinized with trichloroacetic acid (TCA) and acetonictrile. After centrifugation, the clear supernatant was reacted with formaldehyde and TCA under heat. The major fluorescent derivative of ampicillin was then determined by reversed-phase LC with fluorescence detection. Average recoveries of ampicillin fortified at 5, 10, and 20 ppb (ng/mL) were all >85% with coefficients of variation <10%. Limits of detection ranged from 0.31 to 0.51 ppb and limits of quantitation, from 0.66 to 1.2 ppb. After appropriate validation, this method should be suitable for rapid analysis of milk for ampicillin residues at the tolerance level of 10 ppb. 16 refs., 4 figs., 3 tabs.

  8. Rapid detection of feline morbillivirus by a reverse transcription loop-mediated isothermal amplification

    PubMed Central

    KOIDE, Rie; SAKAGUCHI, Shoichi; OGAWA, Makoto; MIYAZAWA, Takayuki

    2015-01-01

    Feline morbillivirus (FmoPV) is an emerging virus in domestic cats and considered to be one of the causes of chronic renal failure in cats. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of FmoPV. The results indicated that the detection limit of the assay was 10 50% tissue culture infective dose (TCID50)/ml in the original sample, and sensitivity of the assay was calculated as 0.12 TCID50 per one RT-LAMP reaction. We also detected FmoPV in clinical urine samples from cats infected with FmoPV. The FmoPV RT-LAMP assay is rapid, simple and highly specific for the detection of FmoPV, and thus, it would be a reliable detection method for FmoPV. PMID:26268665

  9. Gold nanoparticle-based colorimetric aptasensor for rapid detection of six organophosphorous pesticides.

    PubMed

    Bai, Wenhui; Zhu, Chao; Liu, Jinchuan; Yan, Mengmeng; Yang, Shuming; Chen, Ailiang

    2015-10-01

    Fast immunoassay-based screening methods are unavailable for most small-molecule pesticides because of a lack of immunogenicity and the difficulty in obtaining antibodies by animal immunization. Aptamers are single-stranded DNA molecules selected through an in vitro process, which can bind to any target including nonimmunogenic small molecules with high affinity and specificity. Although various aptamer-based sensing methods have been developed for antibiotics, microorganisms, heavy metal ions, and biotoxins, there are few reports on aptamer-based methods for quick detection of organophosphorous pesticides. The gold (Au) nanoparticle (AuNP) colorimetric assay is a widely utilized rapid detection method because of properties such as easy operation and visualized results. In the present study, organophosphorous pesticide aptamers were adsorbed on the surface of AuNPs to stabilize the AuNP solution against high concentrations of salt to prevent AuNP aggregation. After the addition of targets, the aptamers binding to the targets are detached from the AuNPs, resulting in aggregation of AuNPs and a color change from red to purple-blue. The proposed method can detect 6 organophosphorous pesticides with good recoveries from 72% to 135% in environmental river water samples. The present study provides a new way for simple, rapid, and multiplex detection of organophosphorous pesticides.

  10. A gold nanoparticle-based colorimetric probe for rapid detection of 1-hydroxypyrene in urine.

    PubMed

    Hu, Yaqi; Du, Chunyan; Li, Yunchao; Fan, Louzhen; Li, Xiaohong

    2015-07-01

    Direct and rapid detection of 1-hydroxypyrene (1-OHP) is of great importance owing to its high carcinogenicity, teratogenicity and toxicity. In this study, a simple colorimetric assay for rapid determination of 1-OHP is reported, which is based on non-crosslinking aggregation of gold nanoparticles (Au NPs) induced by 1-OHP in the presence of formic acid (FA). Initially, Au NPs were synthesized with citrate as the capping agent and exhibited red color. Subsequently, the addition of FA did not cause aggregation of Au NPs, but a proton transfer process occurred from FA to carboxylic anions on the surface of Au NPs with a decreased zeta potential. The subsequent addition of 1-OHP resulted in a further decreased zeta potential and an intensely hydrophobic environment, which led to a strong and rapid non-crosslinking aggregation of Au NPs within 5 min with the color changing from red to violet blue. Based on this principle, sensitive and selective detection of 1-OHP was achieved. The detection limit was 3.3 nM. Finally, the colorimetric assay was successfully applied to detect 1-OHP in a urine sample. This strategy provides new insights into developing colorimetric methods for on-site and real-time detection of polycyclic aromatic hydrocarbons. PMID:25988203

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

    PubMed

    Tan, Hsih Yin; Loke, Weng Keong; Nguyen, Nam-Trung; Tan, Swee Ngin; Tay, Nam Beng; Wang, Wei; Ng, Sum Huan

    2014-04-01

    This paper reports a lab-on-a-chip for the detection of Sarin nerve agent based on rapid electrochemical detection. The chemical warfare agent Sarin (C₄H₁₀FO₂P, O-isopropyl methylphosphonofluoridate) is a highly toxic organophosphate that induces rapid respiratory depression, seizures and death within minutes of inhalation. As purified Sarin is colourless, odourless, water soluble and a easily disseminated nerve agent, it has been used as a weapon in terrorist or military attacks. To ascertain whether potable water supplies have been adulterated with this extremely potent poison, an inexpensive, sensitive and easy to use portable test kit would be of interest to first responders investigating such attacks. We report here an amperometric-based approach for detecting trace amounts of Sarin in water samples using a screen-printed electrode (SPE) integrated in a microfluidic chip. Enzymatic inhibition was obtained by exposing the immobilised biosensor in the microfluidic platform to Sarin in water samples. With the aid of cobalt phthalocyanine modified SPE, the device could detect Sarin at part-per-billion levels with concentration as low as 1 nM. The detection method reported here represents a significant improvement over the authors'previous optical-based detection method. PMID:24288016

  12. Rapid collection, detection, and assessment of environmental petroleum spills

    SciTech Connect

    Johnson, B.T.; Huckins, J.N.; Petty, J.D.

    1995-12-31

    The bioavailability and toxicity of petroleum oil spills were determined by a new tandem monitoring approach with a passive sorptive device for collection and concentration, and luminescent bacteria for detection. Crude oil, fuel oil, jet fuel, gasoline, and recycled motor oil, as model contaminants, were collected over eight weeks from sediment:water microcosms simulating environmental petroleum spills. Bioremediation strategies for removal of oil contaminants with organic and inorganic nutrients, heterotrophic bacterial strains, and detergent were monitored for efficacy. Air and waterborne oil contaminants was collected with a semipermeable membrane device (SPMD) that contains a thin film of triolein. Analytes were extracted from SPMDs by either dialysis or diluted with organic solvents. Acute toxicity and genotoxicity of analytes were determined with the luminescent bacterial assays Microtox{reg_sign} and Mutatox{reg_sign}. Changes in the bioavailability and toxicity of different oil products as well as influences of different bioremediation treatments were successfully monitored in aquatic microcosms over time. SPMDs and the Microtox-Mutatox systems used in combination offered a sensitive, rapid and cost-effective toxicological screening tool to assess the bioavailability of hazardous environmental oil contaminants.

  13. Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection.

    PubMed

    Hu, Hongmei; Guo, Yuanming; Li, Tiejun

    2015-01-01

    Nonaqueous microchip electrophoresis (NAMCE), which makes use of an organic medium instead of a conventional aqueous buffer solution, is a promising separation method for analytical chemistry due to the enhanced solubility of hydrophobic analytes and tailored selectivity of separation. Here, we describe an NAMCE with LIF detection combined with a pump-free negative pressure sampling device for rapid determination of catecholamines (CAs) in urine samples, and the whole analysis time (including sampling time and separation time) was less than 1 min.

  14. Nucleic acid detection system and method for detecting influenza

    DOEpatents

    Cai, Hong; Song, Jian

    2015-03-17

    The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

  15. Rapid Molecular Assays for the Detection of Yellow Fever Virus in Low-Resource Settings

    PubMed Central

    Escadafal, Camille; Faye, Oumar; Sall, Amadou Alpha; Faye, Ousmane; Weidmann, Manfred; Strohmeier, Oliver; von Stetten, Felix; Drexler, Josef; Eberhard, Michael; Niedrig, Matthias; Patel, Pranav

    2014-01-01

    Background Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. Methodology The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. Conclusion/Significance The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in

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

    NASA Astrophysics Data System (ADS)

    2001-11-01

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

  17. The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

    PubMed Central

    Carranza-Rodríguez, Cristina; Pérez-Arellano, José Luis; Vicente, Belén; López-Abán, Julio; Muro, Antonio

    2015-01-01

    Background Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis. Methodology/Principal Findings A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%). Conclusions/Significance We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid

  18. Methods and systems for rapid prototyping of high density circuits

    DOEpatents

    Palmer, Jeremy A.; Davis, Donald W.; Chavez, Bart D.; Gallegos, Phillip L.; Wicker, Ryan B.; Medina, Francisco R.

    2008-09-02

    A preferred embodiment provides, for example, a system and method of integrating fluid media dispensing technology such as direct-write (DW) technologies with rapid prototyping (RP) technologies such as stereolithography (SL) to provide increased micro-fabrication and micro-stereolithography. A preferred embodiment of the present invention also provides, for example, a system and method for Rapid Prototyping High Density Circuit (RPHDC) manufacturing of solderless connectors and pilot devices with terminal geometries that are compatible with DW mechanisms and reduce contact resistance where the electrical system is encapsulated within structural members and manual electrical connections are eliminated in favor of automated DW traces. A preferred embodiment further provides, for example, a method of rapid prototyping comprising: fabricating a part layer using stereolithography and depositing thermally curable media onto the part layer using a fluid dispensing apparatus.

  19. Immunofluorescence detection methods using microspheres

    NASA Astrophysics Data System (ADS)

    Szurdoki, Ferenc; Michael, Karri L.; Agrawal, Divya; Taylor, Laura C.; Schultz, Sandra L.; Walt, David R.

    1999-01-01

    Microsphere-based immunoassays were devised for compounds of agricultural and biomedical interest (e.g., digoxin, theophylline, and zearalenone). Commercially available microspheres with surface functional groups for chemical derivatization were used as solid carriers. After immobilizing the target substances, the surface of the haptenized microspheres was blocked by a protein to reduce aspecific binding. Competitive immunoassays were performed using the functionalized microspheres and antibodies labeled with horseradish peroxidase. Immunofluorescence signal amplification was achieved by enzyme-catalyzed reporter deposition (CARD). An epifluorescence microscope, a CCD camera interfaced with a computer, and microscopy image analysis software were employed for quantitative detection of fluorescent light emitted from individual microspheres. Integration of several such immunoassays and application of an optical encoding method enabled multianalyte determination. These immunoassays can also be utilized in an immunosensor array format. This immunoarray format could facilitate miniaturization and automation of multianalyte immunoassays.

  20. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test

    PubMed Central

    Bruning, A.H.L.; Susi, P.; Toivola, H.; Christensen, A.; Söderlund-Venermo, M.; Hedman, K.; Aatola, H.; Zvirbliene, A.; Koskinen, J.O.

    2016-01-01

    Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days. PMID:27014463

  1. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test.

    PubMed

    Bruning, A H L; Susi, P; Toivola, H; Christensen, A; Söderlund-Venermo, M; Hedman, K; Aatola, H; Zvirbliene, A; Koskinen, J O

    2016-05-01

    Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days. PMID:27014463

  2. Rapid detection of Escherichia coli and Salmonella typhimurium by surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Su, Lan; Zhang, Ping; Zheng, Da-wei; Wang, Yang-jun-qi; Zhong, Ru-gang

    2015-03-01

    In this paper, the surface-enhanced Raman scattering (SERS) is used as an analytical tool for the detection and identification of pathogenic bacteria of Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium). Compared with normal Raman signal, the intensity of SERS signal is greatly enhanced. After processing all SERS data, the obvious differences between the SERS spectra of two species are determined. And applying the chemometric tools of principal component analysis and hierarchical cluster analysis (PCA-HCA), the SERS spectra of two species are distinguished more accurately. The results indicate that SERS analysis can provide a rapid and sensitive method for the detection of pathogenic bacteria.

  3. Development of an ultra-high-performance liquid chromatography coupled to high-resolution quadrupole-Orbitrap mass spectrometry method for the rapid detection and confirmation of illegal adulterated sedative-hypnotics in dietary supplements.

    PubMed

    Jiang, Shuyin; Tan, Huijie; Guo, Changchuan; Gong, Liping; Shi, Feng

    2015-01-01

    A novel method using ultra-high performance liquid chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap) was developed and validated for the simultaneous screening, identification and quantification of sedative-hypnotics in dietary supplements. Chromatographic conditions were optimised and a full data-dependent MS(2) scan (MS/dd-MS(2)) in positive and negative ion mode was used. A single injection was sufficient to perform the simultaneous screening and identification/quantification of samples. The response showed a good linear relationship with analyte concentrations over wide ranges (e.g., 1.0-1000 ng g(-1) for diazepam) with all the determination coefficients (r(2)) > 0.9985. The method was validated, obtaining accuracy (intra- and inter-day) in the range of 94.5-105.3% and precision (intra- and inter-day) in the range of 0.4-8.9%, respectively. The detection limits (LODs) were in the range of 0.3-1.0 ng g(-1) for different analytes. Recoveries were performed and ranged from 74.1% to 90.2%, while all matrix effects were over the range of 85.4-93.6%. Finally, this method was used to detect sedative-hypnotics in commercial dietary supplements. Of a total of 45 batches of dietary supplements, only three batches were found to be positive samples with concentrations of diazepam, clonazepam and alprazolam at high levels (≥ 8.22 mg g(-1)).

  4. Rapid detection of bacteria with miniaturized pyrolysis-gas chromatographic analysis

    NASA Astrophysics Data System (ADS)

    Mowry, Curtis; Morgan, Catherine H.; Baca, Quentin; Manginell, Ronald P.; Kottenstette, Richard J.; Lewis, Patrick; Frye-Mason, Gregory C.

    2002-02-01

    Rapid detection and identification of bacteria and other pathogens is important for many civilian and military applications. The profiles of biological markers such as fatty acids can be used to characterize biological samples or to distinguish bacteria at the gram-type, genera, and even species level. Common methods for whole cell bacterial analysis are neither portable nor rapid, requiring lengthy, labor intensive sample preparation and bench-scale instrumentation. These methods chemically derivatize fatty acids to produce more volatile fatty acid methyl esters (FAMEs) that can be separated and analyzed by a gas chromatograph (GC)/mass spectrometer. More recent publications demonstrate decreased sample preparation time with in situ derivatization of whole bacterial samples using pyrolysis/derivatization. Ongoing development of miniaturized pyrolysis/GC instrumentation by this department capitalizes on Sandia advances in the field of microfabricated chemical analysis systems ((mu) ChemLab). Microdevices include rapidly heated stages capable of pyrolysis or sample concentration, gas chromatography columns, and surface acoustic wave (SAW) sensor arrays. We will present results demonstrating the capabilities of these devices toward fulfilling the goal of portable, rapid detection and early warning of the presence of pathogens in air or water.

  5. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

  6. Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples.

    PubMed

    Ling, Sumei; Wang, Rongzhi; Gu, Xiaosong; Wen, Can; Chen, Lingling; Chen, Zhibin; Chen, Qing-Ai; Xiao, Shiwei; Yang, Yanling; Zhuang, Zhenhong; Wang, Shihua

    2015-12-15

    Fumonisin B1 (FB1) is the most common and highest toxic of fumonisins species, exists frequently in corn and corn-based foods, leading to several animal and human diseases. Furthermore, FB1 was reported that it was associated with the human esophageal cancer. In view of the harmful of FB1, it is urgent to develop a feasible and accuracy method for rapid detection of FB1. In this study, a competitive immunoassay for FB1 detection was developed based on colloidal gold-antibody conjugate. The FB1-keyhole limpet hemoeyanin (FB1-KLH) conjugate was embedded in the test line, and goat anti-mouse IgG antibody embedded in the control line. The color density of the test line correlated with the concentration of FB1 in the range from 2.5 to 10 ng/mL, and the visual limit detection of test for FB1 was 2.5 ng/mL. The results indicated that the test strip is specific for FB1, and no cross-reactivity to other toxins. The quantitative detection for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time of visual evaluation was less than 5 min. Hence, the developed colloidal gold-antibody assay can be used as a feasible method for FB1 rapid and quantitative detection in corn samples.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    PubMed

    Mangal, Manisha; Bansal, Sangita; Sharma, Satish K; Gupta, Ram K

    2016-07-01

    Food safety is a global health concern. For the prevention and recognition of problems related to health and safety, detection of foodborne pathogen is of utmost importance at all levels of food production chain. For several decades, a lot of research has been targeted at the development of rapid methodology as reducing the time needed to complete pathogen detection tests has been the primary goal of food microbiologists. With the result, food microbiology laboratories now have a wide array of detection methods and automated technologies such as enzyme immunoassay, polymerase chain reaction, and microarrays, which can cut test times considerably. Nucleic acid amplification strategies and advances in amplicon detection methodologies have been the key factors in the progress of molecular microbiology. A comprehensive literature survey has been carried out to give an overview in the field of foodborne pathogen detection. In this paper, we describe the conventional methods, as well as recent developments in food pathogen detection, identification, and quantification, with a major emphasis on molecular detection methods.

  9. Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity

    PubMed Central

    Jiménez, Kenia Barrantes; McCoy², Clyde B.; Achí, Rosario

    2010-01-01

    A Multiplex Polymerase Chain Reaction (PCR) assay to be used as an alternative to the conventional culture method in detecting Shigella and enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce was developed. Efficacy and rapidity of the molecular method were determined as compared to the conventional culture. Lettuce samples were inoculated with different Shigella flexneri concentrations (from 10 CFU/ml to 107 CFU/ml). DNA was extracted directly from lettuce after inoculation (direct-PCR) and after an enrichment step (enrichment PCR). Multiplex PCR detection limit was 104CFU/ml, diagnostic sensitivity and specificity were 100% accurate. An internal amplification control (IAC) of 100 bp was used in order to avoid false negative results. This method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. Also, the culture method detection limit was 106 CFU/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. In this study a Multiplex PCR method for detection of virulence genes in Shigella and EIEC was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to Shigella conventional culture. PMID:24031579

  10. A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents.

    PubMed

    Mechaly, Adva; Cohen, Hila; Cohen, Ofer; Mazor, Ohad

    2016-08-01

    Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein-protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10(4) pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner. PMID:27156814

  11. Immunoliposome-based immunomagnetic concentration and separation assay for rapid detection of Cronobacter sakazakii.

    PubMed

    Shukla, Shruti; Lee, Gibaek; Song, Xinjie; Park, Sunhyun; Kim, Myunghee

    2016-03-15

    This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii (C. sakazakii), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti-C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti-C. sakazakii IgG to the magnetic nanoparticles was 86.23 ± 0.59%. The assay developed in this study detected as few as 3.3 × 10(3) CFUmL(-1) of C. sakazakii in pure culture within 2h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2 × 10(5) CFUmL(-1) of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10(3) CFUmL(-1) of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii. PMID:26547009

  12. Rapid detection of economic adulterants in fresh milk by liquid chromatography-tandem mass spectrometry.

    PubMed

    Abernethy, Grant; Higgs, Kerianne

    2013-05-01

    A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods. PMID:23540766

  13. Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses

    PubMed Central

    2013-01-01

    Background The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses. Methods A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively. Results Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays. Conclusion The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis. PMID:23410000

  14. Rapid detection of shrimp white spot syndrome virus by real time, isothermal recombinase polymerase amplification assay.

    PubMed

    Xia, Xiaoming; Yu, Yongxin; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2014-01-01

    White spot syndrome virus (WSSV) causes large economic losses to the shrimp aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. In this study, we present the development of a novel real time isothermal recombinase polymerase amplification (RPA) assay for WSSV detection on a small ESEQuant Tube Scanner device. The RPA sensitivity, specificity and rapidity were evaluated by using a plasmid standard as well as viral and shrimp genomic DNAs. Compared with qPCR, the RPA assay revealed more satisfactory performance. It reached a detection limit up to 10 molecules in 95% of cases as determined by probit analysis of 8 independent experiments within 6.41 ± 0.17 min at 39 °C. Consequently, this rapid RPA method has great application potential for field use or point of care diagnostics.

  15. Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time, Isothermal Recombinase Polymerase Amplification Assay

    PubMed Central

    Xia, Xiaoming; Yu, Yongxin; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2014-01-01

    White spot syndrome virus (WSSV) causes large economic losses to the shrimp aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. In this study, we present the development of a novel real time isothermal recombinase polymerase amplification (RPA) assay for WSSV detection on a small ESEQuant Tube Scanner device. The RPA sensitivity, specificity and rapidity were evaluated by using a plasmid standard as well as viral and shrimp genomic DNAs. Compared with qPCR, the RPA assay revealed more satisfactory performance. It reached a detection limit up to 10 molecules in 95% of cases as determined by probit analysis of 8 independent experiments within 6.41±0.17 min at 39°C. Consequently, this rapid RPA method has great application potential for field use or point of care diagnostics. PMID:25121957

  16. Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhao, Yong-Qiang; Song, Jian; Wang, Hong-Li; Xu, Bin; Liu, Feng; He, Kun; Wang, Na

    2016-04-01

    The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity. In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice.

  17. Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry.

    PubMed

    Zhao, Yong-Qiang; Song, Jian; Wang, Hong-Li; Xu, Bin; Liu, Feng; He, Kun; Wang, Na

    2016-04-01

    The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity. In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice. Graphical Abstract ᅟ. PMID:26873724

  18. Evaluation of a complete denture trial method applying rapid prototyping.

    PubMed

    Inokoshi, Masanao; Kanazawa, Manabu; Minakuchi, Shunsuke

    2012-02-01

    A new trial method for complete dentures using rapid prototyping (RP) was compared with the conventional method. Wax dentures were fabricated for 10 edentulous patients. Cone-beam CT was used to scan the wax dentures. Using 3D computer-aided design software, seven 3D denture images with different artificial teeth arrangements were made and seven trial dentures per patient were fabricated accordingly. Two prosthodontists performed a denture try-in for one patient using both conventional and RP methods. The prosthodontists and patients rated satisfaction for both methods using a visual analogue scale. Satisfaction ratings with both conventional and RP methods were compared using the Wilcoxon signed-rank test. Regarding prosthodontist's ratings, esthetics and stability were rated significantly higher with the conventional method than with the RP method, whereas chair time was rated significantly longer with the RP method than with the conventional method. Although further improvements are needed, the trial method applying RP seems promising.

  19. RAPID SEPARATION METHOD FOR ACTINIDES IN EMERGENCY SOIL SAMPLES

    SciTech Connect

    Maxwell, S.; Culligan, B.; Noyes, G.

    2009-11-09

    A new rapid method for the determination of actinides in soil and sediment samples has been developed at the Savannah River Site Environmental Lab (Aiken, SC, USA) that can be used for samples up to 2 grams in emergency response situations. The actinides in soil method utilizes a rapid sodium hydroxide fusion method, a lanthanum fluoride soil matrix removal step, and a streamlined column separation process with stacked TEVA, TRU and DGA Resin cartridges. Lanthanum was separated rapidly and effectively from Am and Cm on DGA Resin. Vacuum box technology and rapid flow rates are used to reduce analytical time. Alpha sources are prepared using cerium fluoride microprecipitation for counting by alpha spectrometry. The method showed high chemical recoveries and effective removal of interferences. This new procedure was applied to emergency soil samples received in the NRIP Emergency Response exercise administered by the National Institute for Standards and Technology (NIST) in April, 2009. The actinides in soil results were reported within 4-5 hours with excellent quality.

  20. Rapid and automatic detection of brain tumors in MR images

    NASA Astrophysics Data System (ADS)

    Wang, Zhengjia; Hu, Qingmao; Loe, KiaFock; Aziz, Aamer; Nowinski, Wieslaw L.

    2004-04-01

    An algorithm to automatically detect brain tumors in MR images is presented. The key concern is speed in order to process efficiently large brain image databases and provide quick outcomes in clinical setting. The method is based on study of asymmetry of the brain. Tumors cause asymmetry of the brain, so we detect brain tumors in 3D MR images using symmetry analysis of image grey levels with respect to the midsagittal plane (MSP). The MSP, separating the brain into two hemispheres, is extracted using our previously developed algorithm. By removing the background pixels, the normalized grey level histograms are calculated for both hemispheres. The similarity between these two histograms manifests the symmetry of the brain, and it is quantified by using four symmetry measures: correlation coefficient, root mean square error, integral of absolute difference (IAD), and integral of normalized absolute difference (INAD). A quantitative analysis of brain normality based on 42 patients with tumors and 55 normals is presented. The sensitivity and specificity of IAD and INAD were 83.3% and 89.1%, and 85.7% and 83.6%, respectively. The running time for each symmetry measure for a 3D 8bit MR data was between 0.1 - 0.3 seconds on a 2.4GHz CPU PC.

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

    SciTech Connect

    Xu, Y.; Dunn, J.; Gao, S.; Bruno, J. F.; Luft, B. J.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

  2. Development of a sensitive DNA microarray suitable for rapid detection of Campylobacter spp.

    PubMed

    Keramas, Georgios; Bang, Dang Duong; Lund, Marianne; Madsen, Mogens; Rasmussen, Svend Erik; Bunkenborg, Henrik; Telleman, Pieter; Christensen, Claus Bo Vöge

    2003-08-01

    Campylobacter is the most common cause of human acute bacterial gastroenteritis worldwide, widely distributed and isolated from human clinical samples as well as from many other different sources. To comply with the demands of consumers for food safety, there is a need for development of a rapid, sensitive and specific detection method for Campylobacter. In this study, we present the development of a novel sensitive DNA-microarray based detection method, evaluated on Campylobacter and non-Campylobacter reference strains, to detect Campylobacter directly from the faecal cloacal swabs. The DNA-microarray method consists of two steps: first, both universal bacterial sequences and specific Campylobacter sequences (size range: 149-307 bp) are amplified and fluorescently labeled using multiplex-PCR, targeting the 16S rRNA, the 16S-23S rRNA intergenic region and specific Campylobacter genes. Secondly, the Cy5 labeled PCR-amplicons are hybridised to immobilised capture probes on the microarray. The method allows detection of three to thirty genome equivalents (6-60 fg DNA) of Campylobacter within 3 h, with a hands on time of only 15 min. Using the DNA-microarrays, two closely related Campylobacter species, Campylobacter jejuni and Campylobacter coli could be detected and differentiated directly from chicken faeces. The DNA-microarray method has a high potential for automation and incorporation into a dedicated mass screening microsystem.

  3. Procedure for rapid concentration and detection of enteric viruses from berries and vegetables.

    PubMed

    Butot, S; Putallaz, T; Sánchez, G

    2007-01-01

    Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID(50)), 54 RT-PCR units, and 0.02 TCID(50) per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.

  4. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Posner, Richard G.; Marrone, Babetta L.; Hammond, Mark L.; Simpson, Daniel J.

    1995-01-01

    Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

  5. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

    1995-04-11

    A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

  6. Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

    PubMed Central

    Moss, C. Wayne; Lambert, M. A.; Merwin, W. H.

    1974-01-01

    When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF3-CH3OH, BCl3-CH3OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described. PMID:4844271

  7. Rapid detection of infectious rotavirus group A using a molecular beacon assay.

    PubMed

    Bertol, Jéssica Wildgrube; Gatti, Maria Silvia Viccari

    2016-08-01

    Rapid, sensitive and specific methods are necessary to detect and quantify infectious viruses. Cultivating and detecting enteric viruses in cell culture are difficult, thus impairing the advancement of knowledge regarding virus-induced diarrhea. Rotavirus (RV) detection has been conducted by serological or molecular biology methods, which do not provide information regarding viral infectivity. Molecular beacons (MBs) have demonstrated efficacy for viral detection in cell culture. We propose a MB assay to detect human rotavirus group A (HuRVA) in cell culture. MA104 cells were mock-infected or infected with HuRVA strains (RotaTeq(®) vaccine and K8 strains), and a specific MB for the HuRVA VP6 gene was used for virus detection. Mock-infected cells showed basal fluorescence, while infected cells exhibited increased fluorescence emission. MB hybridization to the viral mRNA target of HuRVA was confirmed. Fluorescence increased according to the increase in the number of infectious viral particles per cell (MOI 0.5-MOI 1). This technique provides quick and efficient HuRVA detection in cell culture without a need for viral culture for several days or many times until cytopathic effects are visualized. This methodology could be applied in the selection of samples for developing RV vaccines. PMID:27131514

  8. Rapid detection of infectious rotavirus group A using a molecular beacon assay.

    PubMed

    Bertol, Jéssica Wildgrube; Gatti, Maria Silvia Viccari

    2016-08-01

    Rapid, sensitive and specific methods are necessary to detect and quantify infectious viruses. Cultivating and detecting enteric viruses in cell culture are difficult, thus impairing the advancement of knowledge regarding virus-induced diarrhea. Rotavirus (RV) detection has been conducted by serological or molecular biology methods, which do not provide information regarding viral infectivity. Molecular beacons (MBs) have demonstrated efficacy for viral detection in cell culture. We propose a MB assay to detect human rotavirus group A (HuRVA) in cell culture. MA104 cells were mock-infected or infected with HuRVA strains (RotaTeq(®) vaccine and K8 strains), and a specific MB for the HuRVA VP6 gene was used for virus detection. Mock-infected cells showed basal fluorescence, while infected cells exhibited increased fluorescence emission. MB hybridization to the viral mRNA target of HuRVA was confirmed. Fluorescence increased according to the increase in the number of infectious viral particles per cell (MOI 0.5-MOI 1). This technique provides quick and efficient HuRVA detection in cell culture without a need for viral culture for several days or many times until cytopathic effects are visualized. This methodology could be applied in the selection of samples for developing RV vaccines.

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

    PubMed

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

    2016-02-01

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

  10. Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

    PubMed Central

    Tissier, Adeline; Denis, Martine; Hartemann, Philippe

    2012-01-01

    Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter. PMID:22138985

  11. Development of a rapid and sensitive method combining a cellulose ester microfilter and a real-time quantitative PCR assay to detect Campylobacter jejuni and Campylobacter coli in 20 liters of drinking water or low-turbidity waters.

    PubMed

    Tissier, Adeline; Denis, Martine; Hartemann, Philippe; Gassilloud, Benoît

    2012-02-01

    Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.

  12. Rapid-estimation method for assessing scour at highway bridges

    USGS Publications Warehouse

    Holnbeck, Stephen R.

    1998-01-01

    A method was developed by the U.S. Geological Survey for rapid estimation of scour at highway bridges using limited site data and analytical procedures to estimate pier, abutment, and contraction scour depths. The basis for the method was a procedure recommended by the Federal Highway Administration for conducting detailed scour investigations, commonly referred to as the Level 2 method. Using pier, abutment, and contraction scour results obtained from Level 2 investigations at 122 sites in 10 States, envelope curves and graphical relations were developed that enable determination of scour-depth estimates at most bridge sites in a matter of a few hours. Rather than using complex hydraulic variables, surrogate variables more easily obtained in the field were related to calculated scour-depth data from Level 2 studies. The method was tested by having several experienced individuals apply the method in the field, and results were compared among the individuals and with previous detailed analyses performed for the sites. Results indicated that the variability in predicted scour depth among individuals applying the method generally was within an acceptable range, and that conservatively greater scour depths generally were obtained by the rapid-estimation method compared to the Level 2 method. The rapid-estimation method is considered most applicable for conducting limited-detail scour assessments and as a screening tool to determine those bridge sites that may require more detailed analysis. The method is designed to be applied only by a qualified professional possessing knowledge and experience in the fields of bridge scour, hydraulics, and flood hydrology, and having specific expertise with the Level 2 method.

  13. Aptasensors for rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium

    NASA Astrophysics Data System (ADS)

    Wu, Wen-he; Li, Min; Wang, Yue; Ouyang, Hou-xian; Wang, Lin; Li, Ci-xiu; Cao, Yu-chen; Meng, Qing-he; Lu, Jian-xin

    2012-11-01

    Herein we reported the development of aptamer-based biosensors (aptasensors) based on label-free aptamers and gold nanoparticles (AuNPs) for detection of Escherichia coli ( E. coli) O157:H7 and Salmonella typhimurium. Target bacteria binding aptamers are adsorbed on the surface of unmodified AuNPs to capture target bacteria, and the detection was accomplished by target bacteria-induced aggregation of the aptasensor which is associated as red-to-purple color change upon high-salt conditions. By employing anti- E. coli O157:H7 aptamer and anti- S. typhimurium aptamer, we developed a convenient and rapid approach that could selectively detect bacteria without specialized instrumentation and pretreatment steps such as cell lysis. The aptasensor could detect as low as 105colony-forming units (CFU)/ml target bacteria within 20 min or less and its specificity was 100%. This novel method has a great potential application in rapid detection of bacteria in the near future.

  14. Simple and rapid detection of Tilletia horrida causing rice kernel smut in rice seeds

    PubMed Central

    Chen, Yu; Yang, Xue; Yao, Jian; Kyaw, Ei Phyu; Zhang, Ai-Fang; Li, Yun-Fei; Gu, Chun-Yan; Zang, Hao-Yu; Gao, Tong-Chun

    2016-01-01

    A simple and rapid method for the detection of Tilletia horrida, the causal agent of rice kernel smut, in rice seeds is developed based on specific polymerase chain reaction (PCR). To design the specific primers for the detection of T. horrida, partial sequences of internal transcribed spacer (ITS) DNA region of T. horrida, T. controversa, T. walkeri, T. ehrhartae, T. indica and T. caries were analyzed and compared. A 503-bp fragment was amplified with the designed primers from the T. horrida genomic DNA. However, no PCR product was obtained from the DNA of other five Tilletia species and 22 fungal plant pathogens tested in the present work indicating the specificity of the primers for the detection of T. horrida. The PCR was performed by directly using the spores, isolated from the 21 different rice seed samples, as template DNA. The T. horrida was detected in 6 of the samples, indicating that 28.6% of the rice samples were contaminated with the kernel smut pathogen. This simple PCR based diagnostic assay can be applied for the direct and rapid detection and identification of T. horrida to screen large numbers of rice seed samples. PMID:27624858

  15. Rapid detection of bacteriophages in starter culture using water-in-oil-in-water emulsion microdroplets.

    PubMed

    Wang, Min S; Nitin, Nitin

    2014-10-01

    Bacteriophage contamination of starter culture and raw material poses a major problem in the fermentation industry. In this study, a rapid detection of lytic phage contamination in starter culture using water-in-oil-in-water (W/O/W) emulsion microdroplets was described. A model bacteria with varying concentrations of lytic phages were encapsulated in W/O/W emulsion microdroplets using a simple needle-in-tube setup. The detection of lytic phage contamination was accomplished in 1 h using the propidium iodide labeling of the phage-infected bacteria inside the W/O/W emulsion microdroplets. Using this approach, a detection limit of 10(2) PFU/mL of phages was achieved quantitatively, while 10(4) PFU/mL of phages could be detected qualitatively based on visual comparison of the fluorescence images. Given the simplicity and sensitivity of this approach, it is anticipated that this method can be adapted to any strains of bacteria and lytic phages that are commonly used for fermentation, and has potential for a rapid detection of lytic phage contamination in the fermentation industry.

  16. Simple and rapid detection of Tilletia horrida causing rice kernel smut in rice seeds.

    PubMed

    Chen, Yu; Yang, Xue; Yao, Jian; Kyaw, Ei Phyu; Zhang, Ai-Fang; Li, Yun-Fei; Gu, Chun-Yan; Zang, Hao-Yu; Gao, Tong-Chun

    2016-01-01

    A simple and rapid method for the detection of Tilletia horrida, the causal agent of rice kernel smut, in rice seeds is developed based on specific polymerase chain reaction (PCR). To design the specific primers for the detection of T. horrida, partial sequences of internal transcribed spacer (ITS) DNA region of T. horrida, T. controversa, T. walkeri, T. ehrhartae, T. indica and T. caries were analyzed and compared. A 503-bp fragment was amplified with the designed primers from the T. horrida genomic DNA. However, no PCR product was obtained from the DNA of other five Tilletia species and 22 fungal plant pathogens tested in the present work indicating the specificity of the primers for the detection of T. horrida. The PCR was performed by directly using the spores, isolated from the 21 different rice seed samples, as template DNA. The T. horrida was detected in 6 of the samples, indicating that 28.6% of the rice samples were contaminated with the kernel smut pathogen. This simple PCR based diagnostic assay can be applied for the direct and rapid detection and identification of T. horrida to screen large numbers of rice seed samples. PMID:27624858

  17. Reverse Transcription-PCR-Enzyme-Linked Immunosorbent Assay for Rapid Detection and Differentiation of Alphavirus Infections▿

    PubMed Central

    Wang, Eryu; Paessler, Slobodan; Aguilar, Patricia V.; Carrara, Anne-Sophie; Ni, Haolin; Greene, Ivorlyne P.; Weaver, Scott C.

    2006-01-01

    Due to the lack of a rapid, simple, and inexpensive assay for detecting alphavirus infections, we combined a reverse transcription-PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) to identify human pathogenic alphaviruses that are endemic in the New World. By combining the sensitivity of PCR, the detection simplicity of ELISA, and the specificities of DNA probes, this method rapidly detected and differentiated closely related species and subtypes of several medically important alphaviruses. After an amplification using RT-PCR with primers targeting conserved sequences in the nonstructural protein 1 gene, sequence-specific, biotin-labeled probes targeted against Venezuelan, eastern, and western equine encephalitis or Mayaro virus genes were used for the detection of amplicons using ELISA. The assay is simple, fast, and easy to perform in an ordinary diagnostic laboratory or clinical setting. Nucleic acid derived from cell cultures infected with several alphaviruses, clinical specimens, and mosquito pools as well as frozen and paraffin-embedded animal tissues were detected and identified within 6 to 7 h in a sensitive and specific manner. PMID:16957044

  18. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

    PubMed

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2016-07-01

    In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control. PMID:27118244

  19. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

    PubMed

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2016-07-01

    In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control.

  20. Electrochemical Biosensor for Rapid and Sensitive Detection of Magnetically Extracted Bacterial Pathogens

    PubMed Central

    Setterington, Emma B.; Alocilja, Evangelyn C.

    2012-01-01

    Biological defense and security applications demand rapid, sensitive detection of bacterial pathogens. This work presents a novel qualitative electrochemical detection technique which is applied to two representative bacterial pathogens, Bacillus cereus (as a surrogate for B. anthracis) and Escherichia coli O157:H7, resulting in detection limits of 40 CFU/mL and 6 CFU/mL, respectively, from pure culture. Cyclic voltammetry is combined with immunomagnetic separation in a rapid method requiring approximately 1 h for presumptive positive/negative results. An immunofunctionalized magnetic/polyaniline core/shell nano-particle (c/sNP) is employed to extract target cells from the sample solution and magnetically position them on a screen-printed carbon electrode (SPCE) sensor. The presence of target cells significantly inhibits current flow between the electrically active c/sNPs and SPCE. This method has the potential to be adapted for a wide variety of target organisms and sample matrices, and to become a fully portable system for routine monitoring or emergency detection of bacterial pathogens. PMID:25585629

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

    PubMed

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

    2013-05-01

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

  2. A Rapid and Sensitive HPLC Method for Quantitation of Paclitaxel in Biological Samples using Liquid-Liquid Extraction and UV Detection: Application to Pharmacokinetics and Tissues Distribution Study of Paclitaxel Loaded Targeted Polymeric Micelles in Tumor Bearing Mice.

    PubMed

    Rezazadeh, Mahboubeh; Emami, Jaber; Mostafavi, Abolfazl; Rostami, Mahboubeh; Hassanzadeh, Farshid; Sadeghi, Hojjat; Minaiyan, Mohsen; Lavasanifar, Afsaneh

    2015-01-01

    A simple, rapid, and sensitive reversed-phase HPLC method was developed and validated for determination of paclitaxel (PTX) in plasma, various organs and tumor tissues of tumor-bearing mice. Tissue specimens of liver, kidneys, spleen, lungs, heart and tumor were separately homogenized in normal saline. Plasma or tissue homogenate (250 µl) containing PTX and internal standard (diazepam) were extracted by diethyl ether (6 ml). The separation was achieved on a µ-Bondapak C18 HPLC column using sodium acetate buffer solution (0.01 M)/acetonitrile (58/42 v/v) at pH 5 ± 0.1 and flow rate of 1.9 mL/min. The effluent was monitored at 227 nm and column temperature was adjusted at 58ºC. The internal standard and PTX were eluted at 4.2 and 5.2 min, respectively and no interfering peaks were observed. Calibration curves were linear over the concentration range of 0.25-10 µg/ml of PTX in plasma and 0.3-20 µg/ml PTX in tissue homogenates with acceptable precision and accuracy (<15%). The mean recoveries of the drug after plasma extraction was 87.4% ± 3.6 while those of tissue homogenates ranged from 62.1± 4.5 to 75.5± 3.2 depending on the type of tissues studied. PTX was stable in samples with no evidence of degradation during 3 freeze-thaw cycles and 3 months storage at -70 °C. The developed HPLC method was applied to quantify PTX in the mouse plasma and tissues after intravenous administration of 10 mg equivalent PTX/Kg dose of PTX-loaded tocopherol succinate-chitosan-polyethylene glycol-folate (TS-CS-PEG-FA) micelles formulation or Anzatax® (Cremophor® EL- based formulation of PTX) to female Balb/c mice.

  3. Development of a Rapid Assimilable Organic Carbon Method for Water

    PubMed Central

    LeChevallier, Mark W.; Shaw, Nancy E.; Kaplan, Louis A.; Bott, Thomas L.

    1993-01-01

    A rapid method for measurement of assimilable organic carbon (AOC) is proposed. The time needed to perform the assay is reduced by increasing the incubation temperature and increasing the inoculum density. The ATP luciferin-luciferase method quickly enumerates the test organisms without the need for plate count media or dilution bottles. There was no significant difference between AOC values determined with strain P17 for the ATP and plate count procedures. For strain NOX, the plate count procedure underestimated bacterial levels in some samples. Comparison of AOC values obtained by the Belleville laboratory (by the ATP technique) and the Stroud Water Research Center (by plate counts) showed that values were significantly correlated and not significantly different. The study concludes that the rapid AOC method can quickly determine the bacterial growth potential of water within 2 to 4 days. PMID:16348936

  4. Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

    PubMed Central

    Cohen, Roy; Lata, James P.; Lee, Yurim; Hernández, Jean C. Cruz; Nishimura, Nozomi; Schaffer, Chris B.; Mukai, Chinatsu; Nelson, Jacquelyn L.; Brangman, Sharon A.; Agrawal, Yash; Travis, Alexander J.

    2015-01-01

    Background Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices. Methods and findings We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA. Conclusions Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using

  5. [Comparison of detection sensitivity in rapid-diagnosis influenza virus kits].

    PubMed

    Tokuno, Osamu; Fujiwara, Miki; Nakajoh, Yoshimi; Yamanouchi, Sumika; Adachi, Masayo; Ikeda, Akiko; Kitayama, Shigeo; Takahashi, Toshio; Kase, Tetsuo; Kinoshita, Shouhiro; Kumagai, Shunichi

    2009-09-01

    Rapid-diagnosis kits able to detect influenza A and B virus by immunochromatography developed by different manufacturers, while useful in early diagnosis, may vary widely in detection sensitivity. We compared sensitivity results for eight virus-detection kits in current use--Quick Chaser FluA, B (Mizuho Medy), Espline Influenza A & B-N (Fujirebio), Capilia Flu A + B (Nippon Beckton Dickinson & Alfesa Pharma), Poctem Influenza A/B (Otsuka Pharma & Sysmex), BD Flu Examan (Nippon Beckton Dickinson), Quick Ex-Flu "Seiken" (Denka Seiken), Quick Vue Rapid SP Influ (DP Pharma Biomedical), and Rapid Testa FLU stick (Daiichi Pure Chemicals)--against influenza virus stocks, contained five vaccination strains (one A/H1N1, two A/H3N2, and two B) and six clinical strains (two A/H1N1, two A/H3N2, and two B). Minimum detection concentrations giving immunologically positive signals in serial dilution and RNA copies in positive dilution in real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were assayed for all kits and virus stock combinations. RNA log10 copy numbers/mL in dilutions within detection limits yielded 5.68-7.02, 6.37-7,17, and 6.5-8.13 for A/H1N1, A/H3N2, and B. Statistically significant differences in sensitivity were observed between some kit combinations. Detection sensitivity tended to be relatively higher for influenza A than B virus. This is assumed due to different principles in kit methods, such as monoclonal antibodies, specimen-extraction conditions, and other unknown factors.

  6. Rapid Short-Read Sequencing and Aneuploidy Detection Using MinION Nanopore Technology

    PubMed Central

    Wei, Shan; Williams, Zev

    2016-01-01

    MinION is a memory stick–sized nanopore-based sequencer designed primarily for single-molecule sequencing of long DNA fragments (>6 kb). We developed a library preparation and data-analysis method to enable rapid real-time sequencing of short DNA fragments (<1 kb) that resulted in the sequencing of 500 reads in 3 min and 40,000–80,000 reads in 2–4 hr at a rate of 30 nt/sec. We then demonstrated the clinical applicability of this approach by performing successful aneuploidy detection in prenatal and miscarriage samples with sequencing in <4 hr. This method broadens the application of nanopore-based single-molecule sequencing and makes it a promising and versatile tool for rapid clinical and research applications. PMID:26500254

  7. Rapid measurement of free cyanide in liquor by ion chromatography with pulsed amperometric detection.

    PubMed

    Wu, Wenlin; Xiao, Quanwei; Zhang, Ping; Ye, Mei; Wan, Yuping; Liang, Hengxing

    2015-04-01

    This study investigated the measurement of free cyanide in liquor by ion chromatography coupled with pulsed amperometric detection (IC-PAD). Eluent concentration, interferent evaluation and method performance were discussed. Results show that free cyanide in liquor can be rapidly determined by the optimised IC-PAD method. A sample requires only 1:100 dilution and simple filtration before being subjected to IC-PAD. The linear range is 1-5000 μg/L with an R value of 0.9998. The detection limit is 1 μg/L for a 25 μL injection loop. The overall relative standard deviation (RSD) of the method is less than 5%, and the recovery range is from 98.1% to 105.0%. This study has been proven significant and may have potential applications in liquors analysis.

  8. Particle detection systems and methods

    DOEpatents

    Morris, Christopher L.; Makela, Mark F.

    2010-05-11

    Techniques, apparatus and systems for detecting particles such as muons and neutrons. In one implementation, a particle detection system employs a plurality of drift cells, which can be for example sealed gas-filled drift tubes, arranged on sides of a volume to be scanned to track incoming and outgoing charged particles, such as cosmic ray-produced muons. The drift cells can include a neutron sensitive medium to enable concurrent counting of neutrons. The system can selectively detect devices or materials, such as iron, lead, gold, uranium, plutonium, and/or tungsten, occupying the volume from multiple scattering of the charged particles passing through the volume and can concurrently detect any unshielded neutron sources occupying the volume from neutrons emitted therefrom. If necessary, the drift cells can be used to also detect gamma rays. The system can be employed to inspect occupied vehicles at border crossings for nuclear threat objects.

  9. Use of RapidChek® SELECT™ Salmonella to detect shedding of live attenuated Salmonella enterica serovar Typhi vaccine strains.

    PubMed

    Brenneman, Karen E; McDonald, Caitlin; Kelly-Aehle, Sandra M; Roland, Kenneth L; Curtiss, Roy

    2012-05-01

    Identification of individuals shedding Salmonella enterica serovar Typhi in stool is imperative during clinical trial safety evaluations. Recovery of live attenuated S. Typhi vaccine strains can be difficult because the mutations necessary for safety in humans often compromise survival in stringent selective enrichment media. RapidChek® SELECT™ Salmonella is a highly sensitive detection method for S. enterica species which utilizes a bacteriophage cocktail designed to reduce the growth of competitor microbes in mildly selective enrichment medium. Detection of Salmonella is enhanced by means of a Salmonella-specific antibody strip targeted to lipopolysaccharide. The RapidChek® SELECT™ Salmonella method was compared to conventional enrichment and plating methods to determine the most sensitive method for detecting attenuated S. Typhi strains in human stool samples. Although traditional enrichment strategies were more sensitive to the presence of wild-type S. Typhi, RapidChek® SELECT™ Salmonella was superior at detecting attenuated strains of S. Typhi. Strains containing a wide variety of attenuating mutations were detected with equal sensitivity as the wild type by RapidChek® SELECT™ Salmonella. The presence of Vi capsule or mutations which affected O-antigen synthesis (Δpmi, ΔgalE) did not decrease the sensitivity of the RapidChek® SELECT™ Salmonella assay. PMID:22425882

  10. Evaluation of an inhouse rapid ELISA test for detection of giardia in domestic sheep (Ovis aries).

    PubMed

    Wilson, Jolaine M; Hankenson, F Claire

    2010-11-01

    Sheep (Ovis aries) are increasingly used at our institution as models of human disease. Within the research environment, routine husbandry and handling of sheep has potential for transmission of zoonotic agents, including Giardia. The prevalence of Giardia in sheep may approach 68%. Classic diagnostic testing involves microscopic examination for fecal cysts or trophozoites; however, limitations of microscopy include time, labor, and potential false-negative results due to intermittent shedding. We wished to determine whether a commercial rapid ELISA used for Giardia detection in dogs and cats could be used in sheep. Fecal samples collected from sheep (n = 93) were tested with a combination of 6 methods: reference laboratory fecal flotation, reference laboratory ELISA, inhouse fecal flotation, and commercially available tests (enzyme immunoassay, direct fluorescence antibody assay, and rapid ELISA). Prevalence of Giardia infection in facility sheep was 11.8% (11 of 93 animals). Of the 11 samples considered positive, 3 were confirmed by multiple testing methods, and 5 were positive by microscopy alone. Inhouse fecal flotation for 8 samples was positive on only 1 of 2 consecutive testing days. The rapid ELISA test exhibited 0% sensitivity for sheep giardiasis. Overall, the examined methods had low sensitivities and low positive predictive values. Despite limitations, microscopic analysis of repeat fecal samples remained the most accurate diagnostic method for ovine giardiasis among the methods tested.

  11. Field Evaluation of a Semiautomated Method for Rapid and Simple Analysis of Recreational Water Microbiological Quality

    PubMed Central

    Anglès d'Auriac, Marc B.; Roberts, Hildegarde; Shaw, Terri; Sirevåg, Reidun; Hermansen, Leonila Fajardo; Berg, James D.

    2000-01-01

    An early warning system using a rapid enzymatic semiautomated method suitable for fecal coliform detection in recreational waters within 8 h was developed further and evaluated in this study. This rapid method was compared to the standard method followed in the United Kingdom. We used 1,011 samples originating from 206 different locations in Wales. When we assessed the presence or absence of fecal coliforms, targeting very low levels of contamination, we obtained 83.9% agreement between the rapid method and the lauryl sulfate broth-membrane filtration technique, whereas direct confirmation of the samples processed by the rapid method showed 89.3% agreement. Environmental enzymatic background activity was found to be the main limiting factor for this method. Owing to a specific and integrated handling of the results by the software of the instrument, the percentage of false-positive results (a consequence of enzymatic background) was successfully limited to 2.9% by the direct confirmation evaluation. However, 7.8% false-negative results due to “late-growers” had to be accepted in order to produce results within a working day. At present, the method can be used in a more conservative way to assess the environmental threshold of 100 CFU of fecal coliforms per 100 ml in recreational waters. The implications of our findings with regard to the applicability of rapid enzymatic methods are discussed. PMID:11010890

  12. RAPID SEPARATION METHOD FOR ACTINIDES IN EMERGENCY AIR FILTER SAMPLES

    SciTech Connect

    Maxwell, S.; Noyes, G.; Culligan, B.

    2010-02-03

    A new rapid method for the determination of actinides and strontium in air filter samples has been developed at the Savannah River Site Environmental Lab (Aiken, SC, USA) that can be used in emergency response situations. The actinides and strontium in air filter method utilizes a rapid acid digestion method and a streamlined column separation process with stacked TEVA, TRU and Sr Resin cartridges. Vacuum box technology and rapid flow rates are used to reduce analytical time. Alpha emitters are prepared using cerium fluoride microprecipitation for counting by alpha spectrometry. The purified {sup 90}Sr fractions are mounted directly on planchets and counted by gas flow proportional counting. The method showed high chemical recoveries and effective removal of interferences. This new procedure was applied to emergency air filter samples received in the NRIP Emergency Response exercise administered by the National Institute for Standards and Technology (NIST) in April, 2009. The actinide and {sup 90}Sr in air filter results were reported in {approx}4 hours with excellent quality.

  13. RAPID FUSION METHOD FOR DETERMINATION OF PLUTONIUM ISOTOPES IN LARGE RICE SAMPLES

    SciTech Connect

    Maxwell, S.

    2013-03-01

    A new rapid fusion method for the determination of plutonium in large rice samples has been developed at the Savannah River National Laboratory (Aiken, SC, USA) that can be used to determine very low levels of plutonium isotopes in rice. The recent accident at Fukushima Nuclear Power Plant in March, 2011 reinforces the need to have rapid, reliable radiochemical analyses for radionuclides in environmental and food samples. Public concern regarding foods, particularly foods such as rice in Japan, highlights the need for analytical techniques that will allow very large sample aliquots of rice to be used for analysis so that very low levels of plutonium isotopes may be detected. The new method to determine plutonium isotopes in large rice samples utilizes a furnace ashing step, a rapid sodium hydroxide fusion method, a lanthanum fluoride matrix removal step, and a column separation process with TEVA Resin cartridges. The method can be applied to rice sample aliquots as large as 5 kg. Plutonium isotopes can be determined using alpha spectrometry or inductively-coupled plasma mass spectrometry (ICP-MS). The method showed high chemical recoveries and effective removal of interferences. The rapid fusion technique is a rugged sample digestion method that ensures that any refractory plutonium particles are effectively digested. The MDA for a 5 kg rice sample using alpha spectrometry is 7E-5 mBq g{sup -1}. The method can easily be adapted for use by ICP-MS to allow detection of plutonium isotopic ratios.

  14. Method For Detecting Biological Agents

    DOEpatents

    Chen, Liaohai; McBranch, Duncan W.; Wang, Hsing-Lin; Whitten, David G.

    2005-12-27

    A sensor is provided including a polymer capable of having an alterable measurable property from the group of luminescence and electrical conductivity, the polymer having an intermediate combination of a recognition element, a tethering element and a property-altering element bound thereto and capable of altering the measurable property, the intermediate combination adapted for subsequent separation from the polymer upon exposure to an agent having an affinity for binding to the recognition element whereupon the separation of the intermediate combination from the polymer results in a detectable change in the alterable measurable property, and, detecting said detectable change in the alterable measurable property.

  15. Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor.

    PubMed

    Weng, Xuan; Gaur, Gautam; Neethirajan, Suresh

    2016-01-01

    The risks associated with the presence of hidden allergens in food have increased the need for rapid, sensitive, and reliable methods for tracing food allergens in commodities. Conventional enzyme immunosorbent assay (ELISA) has usually been performed in a centralized lab, requiring considerable time and sample/reagent consumption and expensive detection instruments. In this study, a microfluidic ELISA platform combined with a custom-designed optical sensor was developed for the quantitative analysis of the proteins wheat gluten and Ara h 1. The developed microfluidic ELISA biosensor reduced the total assay time from hours (up to 3.5 h) to 15-20 min and decreased sample/reagent consumption to 5-10 μL, compared to a few hundred microliters in commercial ELISA kits, with superior sensitivity. The quantitative capability of the presented biosensor is a distinctive advantage over the commercially available rapid methods such as lateral flow devices (LFD) and dipstick tests. The developed microfluidic biosensor demonstrates the potential for sensitive and less-expensive on-site determination for rapidly detecting food allergens in a complex sample system. PMID:27338488

  16. Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor.

    PubMed

    Weng, Xuan; Gaur, Gautam; Neethirajan, Suresh

    2016-06-07

    The risks associated with the presence of hidden allergens in food have increased the need for rapid, sensitive, and reliable methods for tracing food allergens in commodities. Conventional enzyme immunosorbent assay (ELISA) has usually been performed in a centralized lab, requiring considerable time and sample/reagent consumption and expensive detection instruments. In this study, a microfluidic ELISA platform combined with a custom-designed optical sensor was developed for the quantitative analysis of the proteins wheat gluten and Ara h 1. The developed microfluidic ELISA biosensor reduced the total assay time from hours (up to 3.5 h) to 15-20 min and decreased sample/reagent consumption to 5-10 μL, compared to a few hundred microliters in commercial ELISA kits, with superior sensitivity. The quantitative capability of the presented biosensor is a distinctive advantage over the commercially available rapid methods such as lateral flow devices (LFD) and dipstick tests. The developed microfluidic biosensor demonstrates the potential for sensitive and less-expensive on-site determination for rapidly detecting food allergens in a complex sample system.

  17. Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor

    PubMed Central

    Weng, Xuan; Gaur, Gautam; Neethirajan, Suresh

    2016-01-01

    The risks associated with the presence of hidden allergens in food have increased the need for rapid, sensitive, and reliable methods for tracing food allergens in commodities. Conventional enzyme immunosorbent assay (ELISA) has usually been performed in a centralized lab, requiring considerable time and sample/reagent consumption and expensive detection instruments. In this study, a microfluidic ELISA platform combined with a custom-designed optical sensor was developed for the quantitative analysis of the proteins wheat gluten and Ara h 1. The developed microfluidic ELISA biosensor reduced the total assay time from hours (up to 3.5 h) to 15–20 min and decreased sample/reagent consumption to 5–10 μL, compared to a few hundred microliters in commercial ELISA kits, with superior sensitivity. The quantitative capability of the presented biosensor is a distinctive advantage over the commercially available rapid methods such as lateral flow devices (LFD) and dipstick tests. The developed microfluidic biosensor demonstrates the potential for sensitive and less-expensive on-site determination for rapidly detecting food allergens in a complex sample system. PMID:27338488

  18. Rapid method for measuring rotenone in water at piscicidal concentrations

    USGS Publications Warehouse

    Dawson, V.K.; Harman, P.D.; Schultz, D.P.; Allen, J.L.

    1983-01-01

    A high-performance liquid chromatography (HPLC) procedure that is rapid, specific, and sensitive (limit of detection <0.005 mg/liter) was developed for monitoring application and degradation rates of rotenone. For analysis, a water sample is buffered to pH 5 and injected through a Sep Pak(R) C18 disposable cartridge. The cartridge adsorbs and retains the rotenone which then can be eluted quantitatively from the cartridge with a small volume of methanol. This step effectively concentrates the sample and provides sample cleanup. The methanol extract is analyzed directly by HPLC on an MCH 10 reverse-phase column; methanol: water (75:25, volume : volume) is the mobile phase and flow rate is 1.5 ml/minute. The rotenone is detected by ultraviolet spectrophotometry at a wavelength of 295 nm.

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

    PubMed

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

    2014-01-01

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

  20. Screen-printed carbon electrode-based electrochemical immunosensor for rapid detection of microalbuminuria.

    PubMed

    Tsai, Jang-Zern; Chen, Ching-Jung; Settu, Kalpana; Lin, Yu-Feng; Chen, Chien-Lung; Liu, Jen-Tsai

    2016-03-15

    A urinary microalbumin test is used to detect early signs of kidney damage in people who have a risk of chronic kidney disease, such as those with diabetes or hypertension. In this study, we developed a screen-printed carbon electrode-based immunosensor for the detection of microalbumin in urine. Anti-human albumin antibodies were immobilized on the screen-printed sensor surface by the covalent immobilization method. Cyclic voltammetry (CV) and scanning electron microscopy with an energy dispersive spectroscopical (SEM-EDS) analysis demonstrated that the modification process was well performed. Chronoamperometric (CA) electrochemical measurement technique was employed for the quantitative detection of albumin. The electrochemical measurements performed with some possible interfering compounds normally present in urine (ascorbic acid, uric acid, glucose and creatinine samples) demonstrated a high specificity and selectivity of this immunosensor in albumin detection. Under optimum conditions, the immunosensor can detect urinary albumin in a wide linear range from 10 µg/ml to 300 µg/ml with a detection limit of 9.7 µg/ml. The excellent performance of this immunosensor was confirmed by analyzing microalbumin in urine samples; the results were in good agreement with those obtained by the standard immunoturbidimetric method. The biosensor proposed herein is easy to prepare and can be used for low-cost, rapid, and sensitive screening of microalbuminuria. This approach provides a promising platform for developing clinical point-of-care diagnostic applications. PMID:26579935

  1. A rapid method for the determination of honey diastase activity.

    PubMed

    Sakač, Nikola; Sak-Bosnar, Milan

    2012-05-15

    A new rapid method for the determination of honey diastase activity using direct potentiometric principles has been proposed. A platinum redox sensor has been used to quantify the amount of free triiodide released from a starch triiodide complex after starch hydrolysis by honey diastase. The method was tested on honey samples with varying diastase activities. The first 5 min of data for each sample were used for linear regression analysis in order to calculate diastase activity. The new method was compared with classical Schade and commercial Phadebas procedures. The results showed good correlations with both methods and offered a simple method for unit conversion to DN units for diastase activity, making the method suitable for routine analysis.

  2. Rapid and Sensitive Detection of Rotavirus Molecular Signatures Using Surface Enhanced Raman Spectroscopy

    PubMed Central

    Driskell, Jeremy D.; Zhu, Yu; Kirkwood, Carl D.; Zhao, Yiping; Dluhy, Richard A.; Tripp, Ralph A.

    2010-01-01

    Human enteric virus infections range from gastroenteritis to life threatening diseases such as myocarditis and aseptic meningitis. Rotavirus is one of the most common enteric agents and mortality associated with infection can be very significant in developing countries. Most enteric viruses produce diseases that are not distinct from other pathogens, and current diagnostics is limited in breadth and sensitivity required to advance virus detection schemes for disease intervention strategies. A spectroscopic assay based on surface enhanced Raman scattering (SERS) has been developed for rapid and sensitive detection of rotavirus. The SERS method relies on the fabrication of silver nanorod array substrates that are extremely SERS-active allowing for direct structural characterization of viruses. SERS spectra for eight rotavirus strains were analyzed to qualitatively identify rotaviruses and to classify each according to G and P genotype and strain with >96% accuracy, and a quantitative model based on partial least squares regression analysis was evaluated. This novel SERS-based virus detection method shows that SERS can be used to identify spectral fingerprints of human rotaviruses, and suggests that this detection method can be used for pathogen detection central to human health care. PMID:20419101

  3. Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

    NASA Astrophysics Data System (ADS)

    Gao, Hongwei; Li, Fuhua; Zhang, Xiaojun; Wang, Bing; Xiang, Jianhai

    2010-01-01

    Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase ( empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non- V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.

  4. Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification.

    PubMed

    Kuan, Cheng-Ping; Wu, Min-Tze; Lu, Yi-Lin; Huang, Hung-Chang

    2010-10-01

    A loop-mediated isothermal amplification (LAMP) assay was employed to develop a simple and efficient system for the detection of squash leaf curl virus (SLCV) in diseased plants of squash (Cucurbita pepo) and melon (Cucumis melo). Completion of LAMP assay required 30-60 min under isothermal conditions at 65 degrees C by employing a set of four primers targeting SLCV. Although the sensitivity of the LAMP assay and the polymerase chain reaction (PCR) assay was comparable at high virus concentrations, the LAMP assay was by a 10-fold dilution factor more sensitive than the PCR assay for the detection of SLCV in diseased plants. No reaction was detected in the tissues of healthy plants by either the LAMP or the PCR. The LAMP products can be visualized by staining directly in the tube with SYBR Safe DNA gel stain dye. The sensitivity of the SYBR Safe DNA gel stain is similar to analysis by gel electrophoresis. Although both the LAMP and the PCR methods were capable of detecting SLCV in infected tissues of squash and melon, the LAMP method would be more useful than the PCR method for detection of SLCV infection in cucurbitaceous plants because it is more rapid, simple, accurate and sensitive.

  5. Rapid detection of circulating fibrocytes by flowcytometry in idiopathic pulmonary fibrosis

    PubMed Central

    Alhamad, Esam H.; Shakoor, Zahid; Al-Kassimi, Feisal A.; Almogren, Adel; Gad ElRab, Mohamed O.; Maharaj, Shyam; Kolb, Martin

    2015-01-01

    BACKGROUND: Current protocols for detection of circulating fibrocytes (CFs) in peripheral blood described in various pulmonary and nonpulmonary disorders involve complex and time consuming, non standardized techniques. OBJECTIVE: Testing a method to rapidly detect and quantify CFs using whole blood lysis flow cytometry-based assay in patients with idiopathic pulmonary fibrosis (IPF) and healthy controls. METHODS: One milliliter of venous blood sample in ethylenediaminetetraacetic acid (EDTA) from 33 IPF patients and 35 healthy control subjects was collected. Using whole blood lysis method peripheral blood leukocytes were labeled with monoclonal antibodies for cell surface (CD34 and CD45) and intracellular markers (collagen-1) for flow cytometric analysis. CFs were defined as CD45+ cells coexpressing collagen-I and CD34 molecules. RESULTS: In 29 (87.8%) IPF patients and 10 (28.5%) control subjects, a well-defined highly granular CD45+ cell population was detected in dot plots generated by side scatter properties of CD45+ cells. These CD45+ cells were identified as CFs on the basis of coexpression of collagen-I and CD34; none of the other cell types in the peripheral blood were labeled with these monoclonal antibodies. In IPF patients the percentage of CFs was significantly higher compared to healthy controls (median (range): 1.37% (0.52-5.65) and 1.04% (0.1-1.84), respectively; P = 0.03). CONCLUSIONS: Whole blood lysis method combined with fluorescence-activated cell sorting (FACS) allows detecting a well-defined homogeneous population of CFs. This method is simple, reproducible, and provides an accurate and rapid estimation of CFs. PMID:26664567

  6. Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

    PubMed Central

    Gorka, Caroline A.

    2014-01-01

    A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes. PMID:24637334

  7. Rapid measurement of phytosterols in fortified food using gas chromatography with flame ionization detection.

    PubMed

    Duong, Samantha; Strobel, Norbert; Buddhadasa, Saman; Stockham, Katherine; Auldist, Martin; Wales, Bill; Orbell, John; Cran, Marlene

    2016-11-15

    A novel method for the measurement of total phytosterols in fortified food was developed and tested using gas chromatography with flame ionization detection. Unlike existing methods, this technique is capable of simultaneously extracting sterols during saponification thus significantly reducing extraction time and cost. The rapid method is suitable for sterol determination in a range of complex fortified foods including milk, cheese, fat spreads, oils and meat. The main enhancements of this new method include accuracy and precision, robustness, cost effectiveness and labour/time efficiencies. To achieve these advantages, quantification and the critical aspects of saponification were investigated and optimised. The final method demonstrated spiked recoveries in multiple matrices at 85-110% with a relative standard deviation of 1.9% and measurement uncertainty value of 10%.

  8. Rapid measurement of phytosterols in fortified food using gas chromatography with flame ionization detection.

    PubMed

    Duong, Samantha; Strobel, Norbert; Buddhadasa, Saman; Stockham, Katherine; Auldist, Martin; Wales, Bill; Orbell, John; Cran, Marlene

    2016-11-15

    A novel method for the measurement of total phytosterols in fortified food was developed and tested using gas chromatography with flame ionization detection. Unlike existing methods, this technique is capable of simultaneously extracting sterols during saponification thus significantly reducing extraction time and cost. The rapid method is suitable for sterol determination in a range of complex fortified foods including milk, cheese, fat spreads, oils and meat. The main enhancements of this new method include accuracy and precision, robustness, cost effectiveness and labour/time efficiencies. To achieve these advantages, quantification and the critical aspects of saponification were investigated and optimised. The final method demonstrated spiked recoveries in multiple matrices at 85-110% with a relative standard deviation of 1.9% and measurement uncertainty value of 10%. PMID:27283669

  9. An Immuno-Magnetic Nanobead Probe Competitive Assay for Rapid Detection of Salmonella choleraesuis.

    PubMed

    Liu, Daofeng; Yu, Zhibiao; Huang, Yanmei; Wang, Shuying; Wang, Jingyun; Guo, Qi; Xu, Chaolian; Xia, Shiqi; Lai, Weihua

    2016-03-01

    A competitive lateral flow assay for the rapid detection of Salmonella choleraesuis was developed. Immuno-magnetic nanobeads were produced by covalently coupling anti-Salmonella choleraesuis antibody to magnetic nanobeads. These immuno-magnetic nanobeads were used as visually detected probes in the subsequent assay. Compared with the traditional sandwich assay, which is used for detecting macro-molecules, this new method was developed based on the competitive relationship between S. choleraesuis in the inspected sample and the outer membrane protein immobilized on the T line. Thus, only one antibody was necessary in the new assay, whereas a pair of rigorously selected antibodies were required in the sandwich assay. The sensitivity of the competitive assay for S. choleraesuis was 1.2 x 10(7) cfu/mL. In addition, no cross reactions were found in the 17 common non-Salmonella bacteria strains and in the 4 Salmonella strains of other serotypes. Thus, with satisfactory sensitivity and specificity, the assay can be applied for the rapid detection of pre-enriched culture that may contain S. choleraesuis. PMID:27455631

  10. An Immuno-Magnetic Nanobead Probe Competitive Assay for Rapid Detection of Salmonella choleraesuis.

    PubMed

    Liu, Daofeng; Yu, Zhibiao; Huang, Yanmei; Wang, Shuying; Wang, Jingyun; Guo, Qi; Xu, Chaolian; Xia, Shiqi; Lai, Weihua

    2016-03-01

    A competitive lateral flow assay for the rapid detection of Salmonella choleraesuis was developed. Immuno-magnetic nanobeads were produced by covalently coupling anti-Salmonella choleraesuis antibody to magnetic nanobeads. These immuno-magnetic nanobeads were used as visually detected probes in the subsequent assay. Compared with the traditional sandwich assay, which is used for detecting macro-molecules, this new method was developed based on the competitive relationship between S. choleraesuis in the inspected sample and the outer membrane protein immobilized on the T line. Thus, only one antibody was necessary in the new assay, whereas a pair of rigorously selected antibodies were required in the sandwich assay. The sensitivity of the competitive assay for S. choleraesuis was 1.2 x 10(7) cfu/mL. In addition, no cross reactions were found in the 17 common non-Salmonella bacteria strains and in the 4 Salmonella strains of other serotypes. Thus, with satisfactory sensitivity and specificity, the assay can be applied for the rapid detection of pre-enriched culture that may contain S. choleraesuis.

  11. Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips

    PubMed Central

    Cheng, Xianglin; Pu, Xu; Jun, Pen; Zhu, XiaoBo; Zhu, Di; Chen, Ming

    2014-01-01

    Background Rapid immunochromatographic tests can detect disease markers in 10–15 minutes, which facilitates clinical diagnosis and treatment programs. However, most immunochromatographic tests employ gold nanoparticles as reporters, and these have only moderate sensitivity and act as qualitative methods for analyzing high biomarker concentrations. Methods In this study, we introduce quantum dots (QDs) as fluorescent probes and immunochromatographic strips to develop quantitative fluorescence point-of-care tests (QF-POCT) to analyze C-reactive protein (CRP) levels. Goat anti-rabbit IgG and rabbit IgG were used as control antibodies, and mouse monoclonal CRP antibody pairs were used for disease marker detection. One monoclonal CRP antibody was conjugated with QDs and served as a signal antibody, and the other monoclonal CRP antibody was dispensed onto the nitrocellulose membrane and served as a capturing antibody. In the presence of CRP, the fluorescence intensity of the monoclonal antibody-CRP-monoclonal antibody sandwich complex captured on the nitrocellulose membrane was determined using the fluorescence strip reader. Results QF-POCT assays could quantitatively analyze the concentration of CRP in 15 minutes had a detection limit of 0.25 mg/L, and had a wide detection linearity range (0.5–300 mg/L). The intra-assay and interassay coefficients of variation were 8.95% and 9.86% at 0.5 mg/L, 6.47% and 8.66% at 10 mg/L, and 6.81% and 9.10% at 60 mg/L, respectively. In a comparison between clinical samples, the results of this QD-based assay of CRP levels were significantly correlated with those of an Immulite 2000 assay (R=0.993, P<0.001). Conclusion Our results demonstrated that the QD-based immunochromatographic test is a rapid, sensitive, accurate, and quantitative method for the detection of disease biomarkers. PMID:25506215

  12. Rapid and Sensitive Detection of Single Cryptosporidium Oocysts from Archived Glass Slides

    PubMed Central

    Sunnotel, O.; Snelling, W. J.; Xiao, L.; Moule, K.; Moore, J. E.; Millar, B. Cherie; Dooley, J. S. G.; Lowery, C. J.

    2006-01-01

    In this study we report on the development and application of a novel method for efficiently extracting and detecting single Cryptosporidium oocysts from archived glass slides. Laser capture microscopy was used to extract low numbers of oocysts from archived glass slides. Highly sensitive real-time PCR methods were then developed to enable the rapid detection and identification of Cryptosporidium oocysts from these samples. The method was applied to fecal smears stained with a variety of standard oocyst stains and water samples. This application, with samples derived from both public health and water service laboratories, highlighted the strong potential of this method to be used as a rapid high-throughput screening tool for the routine monitoring of Cryptosporidium and other medically important pathogens from clinical, veterinary, and environmental water samples. Importantly, the application of our protocol could be used to type Cryptosporidium and other pathogens from stored archived glass slides in public health and water service laboratories, providing vital epidemiological updates and helping to identify and trace pathogens and their routes of infection and ultimately improve their control. PMID:16954262

  13. Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes.

    PubMed

    Tsui, Clement K M; Wang, Bin; Khadempour, Lily; Alamouti, Sepideh Massoumi; Bohlmann, Jörg; Murray, Brent W; Hamelin, Richard C

    2010-10-01

    Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a