Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques.

Directory of Open Access Journals (Sweden)

Valentina di Rienzo

Full Text Available In tomato, resistance to Tomato spotted wilt virus (TSWV is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke and summer (tomato crops, in the same cultivated areas of Southern Italy.

Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matrices.

Science.gov (United States)

Pearson, Brooke; Mills, Alexander; Tucker, Madeline; Gao, Siyue; McLandsborough, Lynne; He, Lili

2018-06-01

Bacterial foodborne illness continues to be a pressing issue in our food supply. Rapid detection methods are needed for perishable foods due to their short shelf lives and significant contribution to foodborne illness. Previously, a sensitive and reliable surface-enhanced Raman spectroscopy (SERS) sandwich assay based on 3-mercaptophenylboronic acid (3-MBPA) as a capturer and indicator molecule was developed for rapid bacteria detection. In this study, we explored the advantages and constraints of this assay over the conventional aerobic plate count (APC) method and further developed methods for detection in real environmental and food matrices. The SERS sandwich assay was able to detect environmental bacteria in pond water and on spinach leaves at higher levels than the APC method. In addition, the SERS assay appeared to have higher sensitivity to quantify bacteria in the stationary phase. On the other hand, the APC method was more sensitive to cell viability. Finally, a method to detect bacteria in a challenging high-sugar juice matrix was developed to enhance bacteria capture. This study advanced the SERS technique for real applications in environment and food matrices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

Science.gov (United States)

Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

2012-06-01

There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

OpenAIRE

Richardson, M D; Stubbins, J M; Warnock, D W

1982-01-01

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen ...

Evaluation of three gentamicin serum assay techniques

International Nuclear Information System (INIS)

Matzke, G.R.; Gwizdala, C.; Wery, J.; Ferry, D.; Starnes, R.

1982-01-01

This investigation was designed to compare the enzyme-modified immunoassay (Syva--EMIT) with a radioimmunoassay (New England Nuclear--RIA) and the radiometric assay (Johnston--BACTEC) to determine the optimal assay for use in our aminoglycoside dosing service. The serum concentration determinations obtained via the three assay methods were analyzed by linear regression analysis. Significant positive correlations were noted between the three assay techniques (p less than 0.005) during both sample collection phases. The coefficients of determination for EMIT vs BACTEC and RIA vs BACTEC were 0.73 and 0.83 during phase 1, respectively, and 0.65 and 0.68 during phase 2, respectively. The slope of the regression lines also varied markedly during the two phases; 0.49 and 0.42 for EMIT and for RIA vs BACTEC, respectively, during phase 1 compound with 1.12 and 0.77, respectively, during phase 2. The differences noted in these relationships during phase 1 and 2 may be related to the alteration of the pH of the control sera utilized in the BACTEC assay. In contrast, RIA vs EMIT regression analysis indicated that existence of a highly significant relationship (p less than 0.0005 and r2 . 0.90). The EMIT technique was the easiest and most accurate for determination of serum gentamicin concentrations, whereas the BACTEC method was judged unacceptable for clinical use

A rapid inoculation technique for assessing pathogenicity of Fusarium oxysporum f. sp. niveum and F. o. melonis on Cucurbits

Science.gov (United States)

Freeman, S.; Rodriguez, R.J.

1993-01-01

A continuous-dip inoculation technique for rapid assessment of pathogenicity of Fusarium oxysporum f. sp. niveum and F. o. melonis was developed. The method, adapted from a similar procedure for determining pathogenicity of Colletotrichum magna (causal agent of anthracnose of cucurbits), involves constant exposure of seedlings and cuttings (seedlings with root systems excised) of watermelon and muskmelon to conidial suspensions contained in small scintillation vials. Disease development in intact seedlings corresponded well to disease responses observed with the standard root-dip inoculation/pot assay. The continuous-dip inoculation technique resulted in rapid disease development, with 50% of watermelon cuttings dying after 4–6 days of exposure to F. o. niveum. A mortality of 30% also was observed in watermelon cuttings exposed to conidia of F. o. melonis, as opposed to only a 0–2.5% mortality in seedlings with intact roots. Disease response was similar with muskmelon seedlings and cuttings continuously dip-inoculated with F. o. melonis isolates. However, no disease symptoms were observed in muskmelon seedlings or cuttings inoculated with F. o. niveum. Four nonpathogenic isolates of F. oxysporum did not cause disease symptoms in either watermelon or muskmelon cuttings and seedlings when assayed by this technique. The proposed method enables a rapid screening of pathogenicity and requires less time, labor, and greenhouse space than the standard root-dip inoculation/pot assay. The reliability of the continuous-dip inoculation technique is limited, however, to exposure of intact seedlings at a concentration of 1 × 106conidia per milliliter; the method is not accurate at this range for excised seedlings.

Rapid multiple immunoenzyme assay of mycotoxins.

Science.gov (United States)

Urusov, Alexandr E; Zherdev, Anatoly V; Petrakova, Alina V; Sadykhov, Elchin G; Koroleva, Olga V; Dzantiev, Boris B

2015-01-27

Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin-polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay.

Science.gov (United States)

François, Patrice; Bonetti, Eve-Julie; Fankhauser, Carolina; Baud, Damien; Cherkaoui, Abdessalam; Schrenzel, Jacques; Harbarth, Stephan

2017-09-01

Escherichia coli sequence type 131 is increasingly described in severe hospital infections. We developed a rapid real-time allelic discrimination assay for the rapid identification of E. coli ST131 isolates. This rapid assay represents an affordable alternative to sequence-based strategies before completing characterization of potentially highly virulent isolates of E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel approach for high precision rapid potentiometric titrations: application to hydrazine assay.

Science.gov (United States)

Sahoo, P; Malathi, N; Ananthanarayanan, R; Praveen, K; Murali, N

2011-11-01

We propose a high precision rapid personal computer (PC) based potentiometric titration technique using a specially designed mini-cell to carry out redox titrations for assay of chemicals in quality control laboratories attached to industrial, R&D, and nuclear establishments. Using this technique a few microlitre of sample (50-100 μl) in a total volume of ~2 ml solution can be titrated and the waste generated after titration is extremely low comparing to that obtained from the conventional titration technique. The entire titration including online data acquisition followed by immediate offline analysis of data to get information about concentration of unknown sample is completed within a couple of minutes (about 2 min). This facility has been created using a new class of sensors, viz., pulsating sensors developed in-house. The basic concept in designing such instrument and the salient features of the titration device are presented in this paper. The performance of the titration facility was examined by conducting some of the high resolution redox titrations using dilute solutions--hydrazine against KIO(3) in HCl medium, Fe(II) against Ce(IV) and uranium using Davies-Gray method. The precision of titrations using this innovative approach lies between 0.048% and 1.0% relative standard deviation in different redox titrations. With the evolution of this rapid PC based titrator it was possible to develop a simple but high precision potentiometric titration technique for quick determination of hydrazine in nuclear fuel dissolver solution in the context of reprocessing of spent nuclear fuel in fast breeder reactors. © 2011 American Institute of Physics

Development of a rapid, simple assay of plasma total carotenoids

Science.gov (United States)

2012-01-01

Background Plasma total carotenoids can be used as an indicator of risk of chronic disease. Laboratory analysis of individual carotenoids by high performance liquid chromatography (HPLC) is time consuming, expensive, and not amenable to use beyond a research laboratory. The aim of this research is to establish a rapid, simple, and inexpensive spectrophotometric assay of plasma total carotenoids that has a very strong correlation with HPLC carotenoid profile analysis. Results Plasma total carotenoids from 29 volunteers ranged in concentration from 1.2 to 7.4 μM, as analyzed by HPLC. A linear correlation was found between the absorbance at 448 nm of an alcohol / heptane extract of the plasma and plasma total carotenoids analyzed by HPLC, with a Pearson correlation coefficient of 0.989. The average coefficient of variation for the spectrophotometric assay was 6.5% for the plasma samples. The limit of detection was about 0.3 μM and was linear up to about 34 μM without dilution. Correlations between the integrals of the absorption spectra in the range of carotenoid absorption and total plasma carotenoid concentration gave similar results to the absorbance correlation. Spectrophotometric assay results also agreed with the calculated expected absorbance based on published extinction coefficients for the individual carotenoids, with a Pearson correlation coefficient of 0.988. Conclusion The spectrophotometric assay of total carotenoids strongly correlated with HPLC analysis of carotenoids of the same plasma samples and expected absorbance values based on extinction coefficients. This rapid, simple, inexpensive assay, when coupled with the carotenoid health index, may be useful for nutrition intervention studies, population cohort studies, and public health interventions. PMID:23006902

[Prediction of the side-cut product yield of atmospheric/vacuum distillation unit by NIR crude oil rapid assay].

Science.gov (United States)

Wang, Yan-Bin; Hu, Yu-Zhong; Li, Wen-Le; Zhang, Wei-Song; Zhou, Feng; Luo, Zhi

2014-10-01

In the present paper, based on the fast evaluation technique of near infrared, a method to predict the yield of atmos- pheric and vacuum line was developed, combined with H/CAMS software. Firstly, the near-infrared (NIR) spectroscopy method for rapidly determining the true boiling point of crude oil was developed. With commercially available crude oil spectroscopy da- tabase and experiments test from Guangxi Petrochemical Company, calibration model was established and a topological method was used as the calibration. The model can be employed to predict the true boiling point of crude oil. Secondly, the true boiling point based on NIR rapid assay was converted to the side-cut product yield of atmospheric/vacuum distillation unit by H/CAMS software. The predicted yield and the actual yield of distillation product for naphtha, diesel, wax and residual oil were compared in a 7-month period. The result showed that the NIR rapid crude assay can predict the side-cut product yield accurately. The near infrared analytic method for predicting yield has the advantages of fast analysis, reliable results, and being easy to online operate, and it can provide elementary data for refinery planning optimization and crude oil blending.

Rapid Multiple Immunoenzyme Assay of Mycotoxins

Directory of Open Access Journals (Sweden)

Alexandr E. Urusov

2015-01-01

Full Text Available Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin–polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

A fluorescence-based rapid screening assay for cytotoxic compounds

International Nuclear Information System (INIS)

Montoya, Jessica; Varela-Ramirez, Armando; Estrada, Abril; Martinez, Luis E.; Garza, Kristine; Aguilera, Renato J.

2004-01-01

A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death

Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay.

Science.gov (United States)

Kang, Sung-Il; Her, Moon; Kim, Ji-Yeon; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Jung, Suk Chan

2015-06-01

A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/μl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field. Copyright © 2015 Elsevier Ltd. All rights reserved.

Non destructive assay (NDA) techniques

International Nuclear Information System (INIS)

Mafra Guidicini, Olga; Llacer, Carlos D.; Rojo, Marcelo

2001-01-01

In the IAEA Safeguards System the basic verification method used is nuclear material accountancy, with containment and surveillance as important complementary measures. If nuclear material accountancy is to be effective, IAEA inspectors have to make independent measurements to verify declared material quantities. Most of the equipment available to the inspectors is designed to measure gamma rays and/or neutrons emitted by various nuclear materials. Equipment is also available to measure the gross weight of an item containing nuclear material. These types of measurement techniques are generally grouped under the title of nondestructive assay (NDA). The paper describes the NDA techniques and instruments used to verify the total amount of nuclear material held at a nuclear facility. (author)

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

International Nuclear Information System (INIS)

Takamiya, Mari; Sakurai, Masaaki; Teranishi, Fumie; Ikeda, Tomoko; Kamiyama, Tsutomu; Asai, Akira

2016-01-01

A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed a RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive 14 C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6. - Highlights: • A novel assay for elongation of very-long-chain fatty acids 6 (Elovl6) is proposed. • RapidFire mass spectrometry (RF-MS) assay is useful to select real screening hits. • RF-MS assay is proved to be beneficial because of

Non destructive assay techniques applied to nuclear materials

International Nuclear Information System (INIS)

Gavron, A.

2001-01-01

Nondestructive assay is a suite of techniques that has matured and become precise, easily implementable, and remotely usable. These techniques provide elaborate safeguards of nuclear material by providing the necessary information for materials accounting. NDA techniques are ubiquitous, reliable, essentially tamper proof, and simple to use. They make the world a safer place to live in, and they make nuclear energy viable. (author)

A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

Directory of Open Access Journals (Sweden)

Laura C Bonney

2017-10-01

Full Text Available Crimean-Congo Haemorrhagic fever Virus (CCHFV is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia.An isothermal recombinase polymerase amplification (RPA assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan.The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

Brief Report: Impact of Early Antiretroviral Therapy on the Performance of HIV Rapid Tests and HIV Incidence Assays.

Science.gov (United States)

Fogel, Jessica M; Piwowar-Manning, Estelle; Debevec, Barbara; Walsky, Tamara; Schlusser, Katherine; Laeyendecker, Oliver; Wilson, Ethan A; McCauley, Marybeth; Gamble, Theresa; Tegha, Gerald; Soko, Dean; Kumwenda, Johnstone; Hosseinipour, Mina C; Chen, Ying Q; Cohen, Myron S; Eshleman, Susan H

2017-08-01

Antiretroviral therapy (ART) can downregulate antibody responses to HIV infection. We evaluated the impact of early vs. delayed ART on the performance of HIV diagnostic and incidence assays. Samples were obtained from 207 participants in the HPTN 052 trial, who were stably suppressed on ART for ≥4 years [Malawi sites; pre-ART CD4 cell count 350-550 cells/mm (early ART arm, N = 180) or ART arm, N = 27)]. Samples were tested with 2 HIV rapid tests and 2 HIV incidence assays; selected samples were also tested with two fourth-generation immunoassays and a Western blot (WB) assay. A pre-ART sample was analyzed if the follow-up sample had a false-negative or weakly-reactive rapid test result, or had an incidence assay result indicative of recent infection (false-recent result). Ten (4.8%) samples had a nonreactive or weakly-reactive rapid test result (7/180 early ART arm, 3/27 delayed ART arm, P = 0.13); one sample had nonreactive fourth-generation assay results and 3 had indeterminate WBs. Forty (18.9%) samples had a false-recent incidence assay result; 16 (7.8%) had false-recent results with both incidence assays. Baseline samples had stronger rapid test and WB bands, higher fourth-generation assay signal-to-cutoff values, and fewer HIV incidence assay results indicative of recent infection. False-negative/weakly-reactive HIV rapid tests and false-recent HIV incidence assay results were observed in virally-suppressed individuals, regardless of pre-ART CD4 cell count. Downregulation of the antibody response to HIV infection in the setting of ART may impact population-level surveys of HIV prevalence and incidence.

Mini-column assay for rapid detection of malachite green in fish.

Science.gov (United States)

Shalaby, Ali R; Emam, Wafaa H; Anwar, Mervat M

2017-07-01

A simple, rapid and economical mini-column method for detecting malachite green (MG) residue in fish was developed. The method used a column with 2mm ID that was tightly packed with silica gel followed by alumina. Detection of MG was performed by viewing the developed mini-column at visible light by naked eye; where MG was seen as compact green band at the confluence of the silica gel layer with alumina layer. The limit of detection of the assay was 2ng which conform the minimum required performance limit (MRPL). Evaluation utility of the method indicated that all blank and spiked samples at levels below MRPL were assessed as accepted. The intensity of the green band increased whenever MG level in the extract increased; indicated that suggested mini-column technique could be used for semi-quantitative determination of MG in fish samples. The method can be used to select the questionable samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients

Directory of Open Access Journals (Sweden)

Chao Shan

2017-03-01

Full Text Available The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV diagnosis that can attain the “gold standard” of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus.

Science.gov (United States)

Bista, Bipin Raj; Ishwad, Chandra; Wadowsky, Robert M; Manna, Pradip; Randhawa, Parmjeet Singh; Gupta, Gaurav; Adhikari, Meena; Tyagi, Rakhi; Gasper, Gina; Vats, Abhay

2007-05-01

Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for "point of care" screening of BKV.

Neutron Resonance Transmission Analysis (NRTA): A Nondestructive Assay Technique for the Next Generation Safeguards Initiative’s Plutonium Assay Challenge

Energy Technology Data Exchange (ETDEWEB)

J. W. Sterbentz; D. L. Chichester

2010-12-01

This is an end-of-year report for a project funded by the National Nuclear Security Administration's Office of Nuclear Safeguards (NA-241). The goal of this project is to investigate the feasibility of using Neutron Resonance Transmission Analysis (NRTA) to assay plutonium in commercial light-water-reactor spent fuel. This project is part of a larger research effort within the Next-Generation Safeguards Initiative (NGSI) to evaluate methods for assaying plutonium in spent fuel, the Plutonium Assay Challenge. The first-year goals for this project were modest and included: 1) developing a zero-order MCNP model for the NRTA technique, simulating data results presented in the literature, 2) completing a preliminary set of studies investigating important design and performance characteristics for the NRTA measurement technique, and 3) documentation of this work in an end of the year report (this report). Research teams at Los Alamos National Laboratory (LANL), Lawrence Berkeley National Laboratory (LBNL), Pacific Northwest National Laboratory (PNNL), and at several universities are also working to investigate plutonium assay methods for spent-fuel safeguards. While the NRTA technique is well proven in the scientific literature for assaying individual spent fuel pins, it is a newcomer to the current NGSI efforts studying Pu assay method techniques having just started in March 2010; several analytical techniques have been under investigation within this program for two to three years or more. This report summarizes a nine month period of work.

Rapid detection of EBOLA VP40 in microchip immunofiltration assay

Science.gov (United States)

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

2015-05-01

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

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

Science.gov (United States)

Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

2015-01-01

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

The development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of abalone herpesvirus DNA.

Science.gov (United States)

Chen, M H; Kuo, S T; Renault, T; Chang, P H

2014-02-01

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized to 63°C and 60min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

Science.gov (United States)

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

2017-01-01

In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

Rapid intraoperative parathyroid hormone assay--more than just a comfort measure.

LENUS (Irish Health Repository)

Hanif, F

2012-02-03

BACKGROUND: Minimally invasive radio-guided parathyroidectomy (MIRP) has been embraced as an acceptable therapeutic approach to primary hyperparathyroidism. Preoperative sestamibi scanning has facilitated this technique. Here we evaluate the addition of a rapid intraoperative parathyroid hormone (iPTH) assay for patients undergoing MIRP. METHODS: A series of 51 patients underwent sestamibi localization of parathyroid glands followed by MIRP for primary hyperparathyroidism. Using peripheral venous samples, iPTH levels were measured prior to gland excision, as well as post-excision at 5, 10, and 15 minutes, taking a 50% reduction in iPTH level as indicative of complete excision. Next, changes in serum iPTH were compared with preoperative and postoperative changes in serum calcium, as well as levels of intraoperative ex-vivo radiation counts taken by hand-held gamma probe. RESULTS: In this series, a drop of greater than 50% in iPTH levels was observed in 94% of patients (n=48). Moreover, a significant drop in iPTH occurred within 10 minutes of excision in the majority (n=42) of cases (P<0.004). Changes in iPTH were comparable with the therapeutic reduction in calcium levels, as well as with the change in intraoperative ex-vivo gamma counts. CONCLUSIONS: This study demonstrates that the addition of an iPTH assay to MIRP provides a quick and reliable intraoperative diagnostic modality in confirming correct adenoma removal. Moreover, it precludes the requirement of frozen section.

Validation Study of Rapid Assays of Bioburden, Endotoxins and Other Contamination.

Science.gov (United States)

Shintani, Hideharu

2016-01-01

Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility. 　For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent. 　The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic

Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

Science.gov (United States)

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

2014-03-01

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. 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. 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 low-resource settings.

Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

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Camille Escadafal

2014-03-01

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

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus▿

Science.gov (United States)

Bista, Bipin Raj; Ishwad, Chandra; Wadowsky, Robert M.; Manna, Pradip; Randhawa, Parmjeet Singh; Gupta, Gaurav; Adhikari, Meena; Tyagi, Rakhi; Gasper, Gina; Vats, Abhay

2007-01-01

Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for “point of care” screening of BKV. PMID:17314224

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

Science.gov (United States)

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

NARCIS (Netherlands)

Groen, J; Van Dijk, G; Niesters, H G; Van Der Meijden, W I; Osterhaus, A D

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull

Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

Directory of Open Access Journals (Sweden)

Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*

2012-05-01

Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence.

Directory of Open Access Journals (Sweden)

Zachary J Farino

Full Text Available Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment.

Usefulness of a rapid immunometric assay for intraoperative parathyroid hormone measurements

Directory of Open Access Journals (Sweden)

M.N. Ohe

2003-06-01

Full Text Available Intraoperative parathyroid hormone (IO-PTH measurements have been proposed to improve operative success rates in primary, secondary and tertiary hyperparathyroidism (PHP, SHP and THP. Thirty-one patients requiring parathyroidectomy were evaluated retrospectively from June 2000 to January 2002. Sixteen had PHP, 7 SHP and 8 THP. Serum samples were taken at times 0 (before resection, 10, 20 and 30 min after resection of each abnormal parathyroid gland. Samples from 28 patients were frozen at -70ºC for subsequent tests, whereas samples from three patients were tested while surgery was being performed. IO-PTH was measured using the Elecsys immunochemiluminometric assay (Roche, Mannheim, Germany. The time necessary to perform the assay was 9 min. All samples had a second measurement taken by a conventional immunofluorimetric method. We considered as cured patients who presented normocalcemia in PHP and THP, and normal levels of PTH in SHP one month after surgery and who remained in this condition throughout the follow-up of 1 to 20 months. When rapid PTH assay was compared with a routine immunofluorimetric assay, excellent correlation was observed (r = 0.959, P < 0.0001. IO-PTH measurement showed a rapid average decline of 78.8% in PTH 10 min after adenoma resection in PHP and all patients were cured. SHP patients had an average IO-PTH decrease of 89% 30 min after total parathyroidectomy and cure was observed in 85.7%. THP showed an average IO-PTH decrease of 91.9%, and cure was obtained in 87.5% of patients. IO-PTH can be a useful tool that might improve the rate of successful treatment of PHP, SHP and THP.

Techniques for laser processing, assay, and examination of spent fuel

International Nuclear Information System (INIS)

Gray, J.H.; Mitchell, R.C.; Rogell, M.L.

1981-11-01

Fuel examination studies were performed which have application to interim spent fuel storage. These studies were in three areas, i.e., laser drilling and rewelding demonstration, nondestructive assay techniques survey, and fuel examination techniques survey

Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

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Kawahara Ryuji

2008-09-01

Full Text Available Abstract Background Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH and TDH-related hemolysin (TRH are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP assay for the sensitive and rapid detection of Vibrio parahaemolyticus. Results The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction. The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli▿

Science.gov (United States)

Hill, Joshua; Beriwal, Shilpa; Chandra, Ishwad; Paul, Vinod K.; Kapil, Aarti; Singh, Tripti; Wadowsky, Robert M.; Singh, Vinita; Goyal, Ankur; Jahnukainen, Timo; Johnson, James R.; Tarr, Phillip I.; Vats, Abhay

2008-01-01

We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform. PMID:18550738

Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli.

Science.gov (United States)

Hill, Joshua; Beriwal, Shilpa; Chandra, Ishwad; Paul, Vinod K; Kapil, Aarti; Singh, Tripti; Wadowsky, Robert M; Singh, Vinita; Goyal, Ankur; Jahnukainen, Timo; Johnson, James R; Tarr, Phillip I; Vats, Abhay

2008-08-01

We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.

Image-based ELISA on an activated polypropylene microtest plate--a spectrophotometer-free low cost assay technique.

Science.gov (United States)

Parween, Shahila; Nahar, Pradip

2013-10-15

In this communication, we report ELISA technique on an activated polypropylene microtest plate (APPµTP) as an illustrative example of a low cost diagnostic assay. Activated test zone in APPµTP binds a capture biomolecule through covalent linkage thereby, eliminating non-specific binding often prevalent in absorption based techniques. Efficacy of APPµTP is demonstrated by detecting human immunoglobulin G (IgG), human immunoglobulin E (IgE) and Aspergillus fumigatus antibody in patient's sera. Detection is done by taking the image of the assay solution by a desktop scanner and analyzing the color of the image. Human IgE quantification by color saturation in the image-based assay shows excellent correlation with absorbance-based assay (Pearson correlation coefficient, r=0.992). Significance of the relationship is seen from its p value which is 4.087e-11. Performance of APPµTP is also checked with respect to microtiter plate and paper-based ELISA. APPµTP can quantify an analyte as precisely as in microtiter plate with insignificant non-specific binding, a necessary prerequisite for ELISA assay. In contrast, paper-ELISA shows high non-specific binding in control sera (false positive). Finally, we have carried out ELISA steps on APPµTP by ultrasound waves on a sonicator bath and the results show that even in 8 min, it can convincingly differentiate a test sample from a control sample. In short, spectrophotometer-free image-based miniaturized ELISA on APPµTP is precise, reliable, rapid, and sensitive and could be a good substitute for conventional immunoassay procedures widely used in clinical and research laboratories. Copyright © 2013 Elsevier B.V. All rights reserved.

A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

Science.gov (United States)

Dave, Gayatri Ashwinkumar

2017-01-01

Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other

Passive nondestructive assay of nuclear materials

International Nuclear Information System (INIS)

Reilly, D.; Ensslin, N.; Smith, H. Jr.; Kreiner, S.

1991-03-01

The term nondestructive assay (NDA) is applied to a series of measurement techniques for nuclear fuel materials. The techniques measure radiation induced or emitted spontaneously from the nuclear material; the measurements are nondestructive in that they do not alter the physical or chemical state of the nuclear material. NDA techniques are characterized as passive or active depending on whether they measure radiation from the spontaneous decay of the nuclear material or radiation induced by an external source. This book emphasizes passive NDA techniques, although certain active techniques like gamma-ray absorption densitometry and x-ray fluorescence are discussed here because of their intimate relation to passive assay techniques. The principal NDA techniques are classified as gamma-ray assay, neutron assay, and calorimetry. Gamma-ray assay techniques are treated in Chapters 1--10. Neutron assay techniques are the subject of Chapters 11--17. Chapters 11--13 cover the origin of neutrons, neutron interactions, and neutron detectors. Chapters 14--17 cover the theory and applications of total and coincidence neutron counting. Chapter 18 deals with the assay of irradiated nuclear fuel, which uses both gamma-ray and neutron assay techniques. Chapter 19 covers perimeter monitoring, which uses gamma-ray and neutron detectors of high sensitivity to check that no unauthorized nuclear material crosses a facility boundary. The subject of Chapter 20 is attribute and semiquantitative measurements. The goal of these measurements is a rapid verification of the contents of nuclear material containers to assist physical inventory verifications. Waste and holdup measurements are also treated in this chapter. Chapters 21 and 22 cover calorimetry theory and application, and Chapter 23 is a brief application guide to illustrate which techniques can be used to solve certain measurement problems

Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

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Ahmed Abd El Wahed

Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

Science.gov (United States)

Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

2008-11-01

A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

Science.gov (United States)

Zozaya-Valdés, Enrique; Porter, Jessica L; Coventry, John; Fyfe, Janet A M; Carter, Glen P; Gonçalves da Silva, Anders; Schultz, Mark B; Seemann, Torsten; Johnson, Paul D R; Stewardson, Andrew J; Bastian, Ivan; Roberts, Sally A; Howden, Benjamin P; Williamson, Deborah A; Stinear, Timothy P

2017-06-01

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera . Copyright © 2017 American Society for Microbiology.

Colloidal gold-based immunochromatographic strip assay for the rapid detection of three natural estrogens in milk.

Science.gov (United States)

Wang, Zhongxing; Guo, Lingling; Liu, Liqiang; Kuang, Hua; Xu, Chuanlai

2018-09-01

In this study, we developed highly sensitive and specific monoclonal antibodies (mAbs) against estrone (E 1 ), 17β-estradiol (17β-E 2 ), and estriol (E 3 ). The half-maximal inhibitory concentration values of anti-E 1 , anti-17β-E 2 , and anti-E 3 mAbs were 0.46, 0.36, and 0.39 ng/mL, respectively, based on competitive enzyme-linked immunosorbent assay (ic-ELISA) results. A rapid colloidal gold-based immunoassay strip assay was developed for the determination of E 1, 17β-E 2 , and E 3 residues in milk samples. The assay had a visual cut-off value of 5 ng/mL, and required 10 min to assess with the naked eye. The results obtained from the immunochromatographic strip assay were consistent with those obtained from ic-ELISA and gas chromatography-mass spectrometry. The immunochromatographic strip assay is useful and rapid for the detection of E 1 , 17β-E 2 , and E 3 in milk. Copyright © 2018 Elsevier Ltd. All rights reserved.

A rapid colorimetric assay for mold spore germination using XTT tetrazolium salt

Science.gov (United States)

Carol A. Clausen; Vina W. Yang

2011-01-01

Current laboratory test methods to measure efficacy of new mold inhibitors are time consuming, some require specialized test equipment and ratings are subjective. Rapid, simple quantitative assays to measure the efficacy of mold inhibitors are needed. A quantitative, colorimetric microassay was developed using XTT tetrazolium salt to metabolically assess mold spore...

Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples.

Science.gov (United States)

Demerdash, Zeinab; Mohamed, Salwa; Hendawy, Mohamed; Rabia, Ibrahim; Attia, Mohy; Shaker, Zeinab; Diab, Tarek M

2013-02-01

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

A FIFO based neutron arrival time collection technique for assay of plutonium

International Nuclear Information System (INIS)

Parthasarathy, R.; Saisubalakshmi, D.; Venkatasubramani, C.R.

2004-01-01

The system assays plutonium by counting the time correlated neutrons emitted by the spontaneous fissions of the even-even Pu isotopes in the presence of random neutron background, originating principally from (a,n) reactions in the material. The correlation technique discussed in this paper utilizes twofold neutron coincidence counting but the system is proposed to be enhanced for neutron multiplicity counting. A microcontroller based data acquisition system has been developed using a couple of fast FIFO 2kX9 bit memory ICs and a 16 bit counter for identifying time-correlated neutrons. Since the neutron pulses are arriving at a rapid rate, the incoming pulses are buffered in the FIFO and then transferred to PC by the microcontroller through the parallel port. The correlation analysis based on this time arrival information is done in the PC off-line. (author)

Performance and diagnostic usefulness of commercially available enzyme linked immunosorbent assay and rapid kits for detection of HIV, HBV and HCV in India

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Maity Susmita

2012-11-01

Full Text Available Abstract Background HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. Results A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.; ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd. gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd., Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd. did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100% except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.. The kit efficiency didn’t vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p  Conclusions ELISA is a good screening assay for markers of HIV, HBV and HCV infections. Rapid tests are useful for

Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis

NARCIS (Netherlands)

Smits, Henk L.; Abdoel, Theresia H.; Solera, Javier; Clavijo, Encarnacion; Diaz, Ramon

2003-01-01

To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG

Laboratory evaluation of a simple and rapid latex agglutination assay for the serodiagnosis of typhoid fever

NARCIS (Netherlands)

Abdoel, Theresia H.; Pastoor, Rob; Smits, Henk L.; Hatta, Mochammad

2007-01-01

A latex agglutination assay for the serodiagnosis of typhoid fever was evaluated on samples collected from patients with clinical suspicion of typhoid fever in South Sulawesi, Indonesia, where the disease is endemic. The latex assay is very easy to use, gives a rapid result and may be used as a

Rapid in situ assessment for predicting soil quality using an algae-soaked disc seeding assay.

Science.gov (United States)

Nam, Sun-Hwa; Moon, Jongmin; Kim, Shin Woong; Kim, Hakyeong; Jeong, Seung-Woo; An, Youn-Joo

2017-11-16

The soil quality of remediated land is altered and this land consequently exerts unexpected biological effects on terrestrial organisms. Therefore, field evaluation of such land should be conducted using biological indicators. Algae are a promising new biological indicator since they are a food source for organisms in higher soil trophic levels and easily sampled from the soil. Field evaluation of soil characteristics is preferred to be testing in laboratory conditions because many biological effects cannot be duplicated during laboratory evaluations. Herein, we describe a convenient and rapid algae-soaked disc seeding assay for assessing soil quality in the field based on soil algae. The collection of algae is easy and rapid and the method predicts the short-term quality of contaminated, remediated, and amended farm and paddy soils. The algae-soaked disc seeding assay is yet to be extensively evaluated, and the method cannot be applied to loamy sand soil in in situ evaluations. The algae-soaked disc seeding assay is recommended for prediction of soil quality in in situ evaluations because it reflects all variations in the environment. The algae-soaked disc seeding assay will help to develop management strategies for in situ evaluation.

Rapid and highly informative diagnostic assay for H5N1 influenza viruses.

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Nader Pourmand

Full Text Available A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts.

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

Science.gov (United States)

Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

2016-01-01

ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

Science.gov (United States)

Yang, Xue; Hameed, Uzma; Zhang, Ai-Fang; Zang, Hao-Yu; Gu, Chun-Yan; Chen, Yu; Xu, Yi-Liu

2017-01-01

Pilidiella granati, a causal agent of twig blight and crown rot of pomegranate, is an emerging threat that may cause severe risk to the pomegranate industry in the future. Development of a rapid assay for the timely and accurate detection of P. granati will be helpful in the active surveillance and management of the disease caused by this pathogen. In this study, a nested PCR method was established for the detection of P. granati. Comparative analysis of genetic diversity within 5.8S rDNA internal transcribed spacer (ITS) sequences of P. granati and 21 other selected fungal species was performed to design species-specific primers (S1 and S2). This primer pair successfully amplified a 450 bp product exclusively from the genomic DNA of P. granati. The developed method can detect 10 pg genomic DNA of the pathogen in about 6 h. This technique was successfully applied to detect the natural infection of P. granati in the pomegranate fruit. The designed protocol is rapid and precise with a high degree of sensitivity. PMID:28106107

Soil Baiting, Rapid PCR Assay and Quantitative Real Time PCR to Diagnose Late Blight of Potato in Quarantine Programs

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Touseef Hussain

2018-05-01

Full Text Available Phytophthora infestans (mont de Bary is a pathogen of great concern across the globe, and accurate detection is an important component in responding to the outbreaks of potential disease. Although the molecular diagnostic protocol used in regulatory programs has been evaluated but till date methods implying direct comparison has rarely used. In this study, a known area soil samples from potato fields where light blight appear every year (both A1 and A2 mating type was assayed by soil bait method, PCR assay detection and quantification of the inoculums. Suspected disease symptoms appeared on bait tubers were further confirmed by rapid PCR, inoculums were quantified through Real Time PCR, which confirms presence of P. infestans. These diagnostic methods can be highly correlated with one another. Potato tuber baiting increased the sensitivity of the assay compared with direct extraction of DNA from tuber and soil samples. Our study determines diagnostic sensitivity and specificity of the assays to determine the performance of each method. Overall, molecular techniques based on different types of PCR amplification and Real-time PCR can lead to high throughput, faster and more accurate detection method which can be used in quarantine programmes in potato industry and diagnostic laboratory.

Development of loop-mediated isothermal amplification (LAMP assay for rapid and sensitive identification of ostrich meat.

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Amir Abdulmawjood

Full Text Available Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes.

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

Science.gov (United States)

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

2016-01-01

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 104 CFU mL−1 or 105 CFU mL−1 for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R2) of 0.916–0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water. PMID:26884128

Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay.

Science.gov (United States)

Ai, L; Li, C; Elsheikha, H M; Hong, S J; Chen, J X; Chen, S H; Li, X; Cai, X Q; Chen, M X; Zhu, X Q

2010-12-15

The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of Fasciola hepatica and Fasciola gigantica. The LAMP assay is inexpensive, easy to perform and shows rapid reaction, wherein the amplification can be obtained in 45 min under isothermal conditions of 61 °C or 62 °C by employing a set of four species-specific primer mixtures and results can be checked through naked-eye visualization. The optimal assay conditions with no cross-reaction with other closely related trematodes (Clonorchis sinensis, Opisthorchis viverrini, Orientobilharzia turkestanicum and Schistosoma japonicum) as well as within the two Fasciola species were established. The assay was validated by examining F. gigantica DNA in the intermediate host snails and in faecal samples. The results indicated that the LAMP assay is approximately 10(4) times more sensitive than the conventional specific PCR assays. These findings indicate that this Fasciola species-specific LAMP assay may have a potential clinical application for detection and differentiation of Fasciola species, especially in endemic countries. Copyright © 2010 Elsevier B.V. All rights reserved.

Shell Vial culture Assay for the rapid diagnosis of Japanese encephalitis, West Nile and Dengue-2 viral encephalitis

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Badrinath S

2006-01-01

Full Text Available Abstract Background Encephalitis caused by flaviviruses, Japanese encephalitis virus (JEV and West Nile virus (WNV is responsible for significant morbidity and mortality in many endemic countries. Dengue-2 (Den-2 virus is a recent addition to the list of encephalitogenic viruses, after its Central Nervous System (CNS invasion capability has been established. There is a wide array of laboratory tools that have helped us not only in the diagnosis of these conditions but also in understanding their pathogenesis and pathology. However, there are no reports of Shell Vial Culture (SVC, a centrifuge enhanced tissue culture assay that has revolutionized viral culturing in terms of rapidity and sensitivity being optimized for these flaviviral encephalitic conditions. The present study is an attempt to standardize and evaluate the usefulness of SVC for the laboratory diagnosis of JE, WN and Den-2 encephalitis cases and to compare it with Indirect Immunofluorescence (IIF technique that detects cell associated virus antigen. Analysis of the various clinical parameters with respect to viral etiology has also been carried out. Results Pediatric patients constituted the major group involved in the study (92%. Etiological diagnosis of viral encephalitis could be established in twenty nine (58% patients. JE encephalitis was the commonest with 19 (39% cases being positive followed by, WN (9 cases-18% and Den-2 (one case. IIF test could detect antigens of JE, WN and Den-2 viruses in 16(32%, 7(14% and 1 case respectively. Shell vial culture assay picked up all cases that were positive by IIF test. In addition, SVC assay could detect 3 and 2 more cases of JE and WN encephalitis respectively, that were negative by the IIF test. Conclusion Shell vial culture is a rapid and efficient tool for the etiological diagnosis of JE, WN and Den-2 encephalitis cases. Early, prompt collection, transport and processing of the CSF samples, would make SVC a better method for the

A rapid and user-friendly assay to detect the Neutrophil gelatinase-associated lipocalin (NGAL) using up-converting nanoparticles.

Science.gov (United States)

Lei, Lijiang; Zhu, Jin; Xia, Gangqiang; Feng, Hui; Zhang, Hongman; Han, Yuwang

2017-01-01

NGAL is a promising novel biomarker for acute kidney injury (AKI) and chronic kidney disease (CKD). More rapid and user-friendly methods are needed for the timely monitoring of NGAL in human urine and serum. UCP technology-based lateral flow assay (UPT-LFA) was developed for rapid, user-friendly and quantitative detection of the NGAL in human serum specimens and urine specimens. Under optimal conditions, the UPT-LFA displayed a rapid response to NGAL with a LOD of 7.68ng/mL and detection range from 7.68 to 1000ng/mL. The UPT-LFA method was compared with commercial immunoturbidimetry (103 urine specimens), and by ELISA (26 serum specimens), respectively. The results demonstrated that the UPT-LFA was consistent with immunoturbidimetry assay, reporting a 97.92% of positive and 92.73% of negative coincidence rates, respectively. Meanwhile, the concordance rate between UPT-LFA and ELISA, as shown by correlative regression analysis, was also high (R 2 =0.95). The whole assay can be completed within 30min compared to 4h consuming with ELISA. The research implies that, the UPT-LFA provides a potential to be used in point of care testing (POCT) to define early acute kidney injury with advantages of user-friendly and rapid testing, promising this new assay a bright future. Copyright © 2016 Elsevier B.V. All rights reserved.

Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

Science.gov (United States)

Yamashoji, Shiro; Yoshikawa, Naoko; Kirihara, Masayuki; Tsuneyoshi, Toshihiro

2013-06-15

The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, ethanol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for 4h. Menadione-catalysed H2O2 production by living mammalian cells exposed to each extract was determined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in order of inhibitory effect on H2O2 production by intact cells. The well known natural toxins such as Fusarium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and brevetoxin could be detected by the above chemiluminescent assay. Copyright © 2012 Elsevier Ltd. All rights reserved.

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

Science.gov (United States)

Benitez, Alvaro J; Winchell, Jonas M

2016-04-01

We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis of Acanthamoeba keratitis

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Abhishek Mewara

2017-01-01

Full Text Available Background: The clinical features of Acanthamoeba keratitis (AK are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP with microscopy, non-nutrient agar (NNA culture and polymerase chain reaction (PCR in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis, 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%, and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%. The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings

Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay.

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Rosemary S Turingan

Full Text Available Nucleic acid amplification tests (NAATs are recommended by the CDC for detection of Chlamydia trachomatis (Ct urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4. The remaining 19 blinded samples were correctly identified as LGV clade 1 (3, ocular clade 3 (4, or as negatives (12. To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out, the assay has significant potential as a rapid POC diagnostic for Ct infections.

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

Science.gov (United States)

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

2017-08-01

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

Loop-mediated isothermal amplification assay for rapid and sensitive detection of sheep pox and goat pox viruses in clinical samples.

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Venkatesan, G; Balamurugan, V; Bhanuprakash, V; Singh, R K; Pandey, A B

2016-06-01

A Loop-mediated isothermal amplification (LAMP) assay targeting the highly conserved DNA polymerase gene of capripox virus genome was developed and evaluated for rapid detection of sheep pox and goat pox viruses. The optimized LAMP assay is found specific and sensitive for amplification of target DNA with a diagnostic sensitivity and specificity of 96.6% and 100% respectively compared to quantitative PCR. The detection rate of LAMP, PCR and Q-PCR assays is found to be 81.5%, 67% and 83% respectively. This LAMP assay has the potential for rapid clinical diagnosis and surveillance of sheep pox and goat pox in field diagnostic laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of four novel isothermal amplification assays towards simple and rapid genotyping of chloroquine resistant Plasmodium falciparum.

Science.gov (United States)

Chahar, Madhvi; Anvikar, Anup; Dixit, Rajnikant; Valecha, Neena

2018-07-01

Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and field deployable technique for nucleic acid amplification under isothermal conditions. In this study, we have developed and optimized four different visualization methods of loop-mediated isothermal amplification (LAMP) assay to detect Pfcrt K76T mutants of P. falciparum and compared their important features for one-pot in-field applications. Even though all the four tested LAMP methods could successfully detect K76T mutants of P. falciparum, however considering the time, safety, sensitivity, cost and simplicity, the malachite green and HNB based methods were found more efficient. Among four different visual dyes uses to detect LAMP products accurately, hydroxynaphthol blue and malachite green could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk. Our results indicated that the LAMP offers an interesting novel and convenient best method for the rapid, sensitive, cost-effective, and fairly user friendly tool for detection of K76T mutants of P. falciparum and therefore presents an alternative to PCR-based assays. Based on our comparative analysis, better field based LAMP visualization method can be chosen easily for the monitoring of other important drug targets (Kelch13 propeller region). Copyright © 2018 Elsevier Inc. All rights reserved.

Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

DEFF Research Database (Denmark)

Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

2002-01-01

) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect r = 0.99) over...... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro....... In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

Development of a nematode offspring counting assay for rapid and simple soil toxicity assessment.

Science.gov (United States)

Kim, Shin Woong; Moon, Jongmin; Jeong, Seung-Woo; An, Youn-Joo

2018-05-01

Since the introduction of standardized nematode toxicity assays by the American Society for Testing and Materials (ASTM) and International Organization for Standardization (ISO), many studies have reported their use. Given that the currently used standardized nematode toxicity assays have certain limitations, in this study, we examined the use of a novel nematode offspring counting assay for evaluating soil ecotoxicity based on a previous soil-agar isolation method used to recover live adult nematodes. In this new assay, adult Caenorhabditis elegans were exposed to soil using a standardized toxicity assay procedure, and the resulting offspring in test soils attracted by a microbial food source in agar plates were counted. This method differs from previously used assays in terms of its endpoint, namely, the number of nematode offspring. The applicability of the bioassay was demonstrated using metal-spiked soils, which revealed metal concentration-dependent responses, and with 36 field soil samples characterized by different physicochemical properties and containing various metals. Principal component analysis revealed that texture fraction (clay, sand, and silt) and electrical conductivity values were the main factors influencing the nematode offspring counting assay, and these findings warrant further investigation. The nematode offspring counting assay is a rapid and simple process that can provide multi-directional toxicity assessment when used in conjunction with other standard methods. Copyright © 2018 Elsevier Ltd. All rights reserved.

Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay.

Science.gov (United States)

Linares, María; Viera, Sara; Crespo, Benigno; Franco, Virginia; Gómez-Lorenzo, María G; Jiménez-Díaz, María Belén; Angulo-Barturen, Íñigo; Sanz, Laura María; Gamo, Francisco-Javier

2015-11-05

The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium-high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'. The quantification of new infections to determine parasite viability offers important

Rapid high-throughput assay to assess scavenging capacity index using DPPH.

Science.gov (United States)

Abderrahim, Fatima; Arribas, Silvia M; Gonzalez, M Carmen; Condezo-Hoyos, Luis

2013-11-15

A new microplate-adapted DPPH rapid assay was developed to assess the antioxidant capacity of pure compounds and foods. The assay was carried out in buffered medium (methanol: 10mmol/l Tris buffer pH 7.5, 1:1 v/v) and reaction was completed at 10min. The scavenging capacity index (SCI), a theoretical antioxidant parameter directly related to the antioxidant capacity of samples, was calculated. SCI for pure compounds: gallic acid (6.76±0.08), quercetin (7.89±0.24), catechin (6.05±0.23), trolox (2.32±0.03), ascorbic acid (2.52±0.15) and gluthatione (1.08±0.08) and foods (μmol DPPH scavenged/100ml): tropical juice (655.62±12.18), mediterraneo juice (702.87±11.13), apple juice (212.52±17.22), pomegranate juice (319.83±9.45), red grape nectar (1093.05±18.69), Don Simon orange juice (632.94±17.22) and date syrup (15992.34±250.7) were comparable to those in previous reports using the classic DPPH assay. The relative standard deviation (RSD) for the SCI on the same and different days was less than 8.12% in all cases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda.

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Freddie Bwanga

Full Text Available The most common method for detection of drug resistant (DR TB in resource-limited settings (RLSs is indirect susceptibility testing on Lowenstein-Jensen medium (LJ which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA and Microscopic Observation Drug Susceptibility (MODS for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95% with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.

Manufacturing implant supported auricular prostheses by rapid prototyping techniques.

Science.gov (United States)

Karatas, Meltem Ozdemir; Cifter, Ebru Demet; Ozenen, Didem Ozdemir; Balik, Ali; Tuncer, Erman Bulent

2011-08-01

Maxillofacial prostheses are usually fabricated on the models obtained following the impression procedures. Disadvantages of conventional impression techniques used in production of facial prosthesis are deformation of soft tissues caused by impression material and disturbance of the patient due to. Additionally production of prosthesis by conventional methods takes longer time. Recently, rapid prototyping techniques have been developed for extraoral prosthesis in order to reduce these disadvantages of conventional methods. Rapid prototyping technique has the potential to simplify the procedure and decrease the laboratory work required. It eliminates the need for measurement impression procedures and preparation of wax model to be performed by prosthodontists themselves In the near future this technology will become a standard for fabricating maxillofacial prostheses.

Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

Directory of Open Access Journals (Sweden)

A K Singh

2013-01-01

Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

Colloidal gold probe based rapid immunochromatographic strip assay for cortisol

International Nuclear Information System (INIS)

Nara, Seema; Tripathi, Vinay; Singh, Harpal; Shrivastav, Tulsidas G.

2010-01-01

A rapid and semi-quantitative immunochromatographic strip (ICS) test for cortisol analysis in serum was developed. The test strip was based on a competitive assay format. Colloidal gold nanoparticles were synthesized and coupled with cortisol-3-carboxymethyloxime-adipic acid dihydrazide-bovine serum albumin (F-3-CMO-ADH-BSA) antigen to directly compete with cortisol in human serum samples. F-3-CMO-ADH-BSA-gold label and uncoupled colloidal gold nanoparticles were appropriately characterized using UV-vis spectroscopy, transmission electron microscopy and atomic force microscopy. Anticortisol antibody raised against F-3-CMO-BSA immunogen in New Zealand white rabbits was coated on the NC membrane as test line. Anti-BSA antibody was used as control line. The lower detection limit of the ICS test was 30 ng mL -1 with visual detection and was completed in 10 min. About 30 human serum samples were also analyzed by the developed strip test and their range of cortisol concentration was established. The developed ICS test is rapid, economic and user friendly.

Colloidal gold probe based rapid immunochromatographic strip assay for cortisol

Energy Technology Data Exchange (ETDEWEB)

Nara, Seema, E-mail: seemanara@mnnit.ac.in [Department of Applied Mechanics (Biotechnology), Motilal Nehru National Institute of Technology, Allahabad 211004 (India); Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Tripathi, Vinay [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Singh, Harpal [Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Shrivastav, Tulsidas G. [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India)

2010-12-03

A rapid and semi-quantitative immunochromatographic strip (ICS) test for cortisol analysis in serum was developed. The test strip was based on a competitive assay format. Colloidal gold nanoparticles were synthesized and coupled with cortisol-3-carboxymethyloxime-adipic acid dihydrazide-bovine serum albumin (F-3-CMO-ADH-BSA) antigen to directly compete with cortisol in human serum samples. F-3-CMO-ADH-BSA-gold label and uncoupled colloidal gold nanoparticles were appropriately characterized using UV-vis spectroscopy, transmission electron microscopy and atomic force microscopy. Anticortisol antibody raised against F-3-CMO-BSA immunogen in New Zealand white rabbits was coated on the NC membrane as test line. Anti-BSA antibody was used as control line. The lower detection limit of the ICS test was 30 ng mL{sup -1} with visual detection and was completed in 10 min. About 30 human serum samples were also analyzed by the developed strip test and their range of cortisol concentration was established. The developed ICS test is rapid, economic and user friendly.

Development of versatile universal reagent immunoradiometric assay technique

International Nuclear Information System (INIS)

Hazra, D.K.

1982-10-01

Immunoradiometric assays, which make use of labelled antibodies, potentially offer better sensitivity and specificity than do radioimmunoassays, which use labelled antigens. In addition, they can in principle be performed in a particularly convenient scheme wherein the same labelled reagent may be used for many different analytes - thus serving as a ''universal'' labelled reagent. Thus if the specific antibody for every analyte is raised in rabbits, and an anti-rabbit antibody is labelled, the latter may be added after the specific antibody to quantify the amount of specific antibody bound to analyte and thereby the amount of analyte present. The potential greater sensitivity and specificity of the immunoradiometric procedure, coupled with the potential convenience of the ''universal'' labelled reagent, might allow such immunoradiometric techniques to be used effectively in the study of communicable diseases in developing countries. Development of these procedures was the subject of this investigation. Many components of these procedures had to be explored and provisionally optimized, including coating of assay tubes with ''extraction'' antibody, immunological purification of antibodies, labelling of antibodies, and intermediate steps toward these goals. Applications were thereupon tested using those provisionally optimized components. The ''universal'' labelled reagent, a donkey anti-rabbit antiserum, was successfully used in the assay of TSH; however, cross reactions of the reagent with non-rabbit immunoglobulins and other materials present seriously limited the sensitivity of the method. Using conventional immunoradiometric procedures, circulating TB and amoebic antibodies could be detected in patients suffering from these diseases. Similarly, circulating antigens in the same patients could also be detected, but not with sufficient sensitivity and specificity to provide a reliable analytical system. Numerous improvements will be required before these techniques

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

Science.gov (United States)

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.

Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

Science.gov (United States)

Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

2016-02-01

Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

DEFF Research Database (Denmark)

Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

2002-01-01

A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD......) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect ... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...

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

Science.gov (United States)

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

2011-08-01

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

Development of in-vitro radiometric assay for the rapid assessment of chloroquine resistant plasmodium vivax

International Nuclear Information System (INIS)

Myint Oo; Myo Khin; Nwe Nwe Oo

1997-01-01

Previously, resistance of malaria parasite to chloroquine has been restricted only to Plasmodium falciparum. Recently, there have been many reports of chloroquine-resistant Plasmodium vivax. One of the mechanisms of chloroquine resistance is the decreased uptake of chloroquine or rapid efflux of the drug from the food vacuole of the parasite. In this study, we have measured the rapid efflux of IH-chloroquine in fifty blood samples from patients with P Vivax infection. All 50 patients were hospitalised for 28 days for the standard treatment with chloroquine. It was found that seven patients who did not respond to the standard regimen of chloroquine have parasites with rapid effluxes of IH-chloroquine. Since rapid effluxes of IH-chloroquine in the resistant parasites showed strong correlation with in vivo 28 days clinical trial, this assay could be used as rapid assessment of chloroquine resistance in patients with P vivax infection

Development of in-vitro radiometric assay for the rapid assessment of chloroquine resistant plasmodium vivax

Energy Technology Data Exchange (ETDEWEB)

Oo, Myint; Khin, Myo; Oo, Nwe Nwe [Department of Medical Research, Yangon (Myanmar)

1997-12-01

Previously, resistance of malaria parasite to chloroquine has been restricted only to Plasmodium falciparum. Recently, there have been many reports of chloroquine-resistant Plasmodium vivax. One of the mechanisms of chloroquine resistance is the decreased uptake of chloroquine or rapid efflux of the drug from the food vacuole of the parasite. In this study, we have measured the rapid efflux of IH-chloroquine in fifty blood samples from patients with P Vivax infection. All 50 patients were hospitalised for 28 days for the standard treatment with chloroquine. It was found that seven patients who did not respond to the standard regimen of chloroquine have parasites with rapid effluxes of IH-chloroquine. Since rapid effluxes of IH-chloroquine in the resistant parasites showed strong correlation with in vivo 28 days clinical trial, this assay could be used as rapid assessment of chloroquine resistance in patients with P vivax infection.

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

Energy Technology Data Exchange (ETDEWEB)

Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

1983-10-01

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of (3H)thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of (3H)thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

International Nuclear Information System (INIS)

Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

1983-01-01

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of [3H]thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of [3H]thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay

Rapid Estimation of Tocopherol Content in Linseed and Sunflower Oils-Reactivity and Assay

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Tjaša Prevc

2015-08-01

Full Text Available The reactivity of tocopherols with 2,2-diphenyl-1-picrylhydrazyl (DPPH was studied in model systems in order to establish a method for quantifying vitamin E in plant oils. The method was optimized with respect to solvent composition of the assay medium, which has a large influence on the course of reaction of tocopherols with DPPH. The rate of reaction of α-tocopherol with DPPH is higher than that of γ-tocopherol in both protic and aprotic solvents. In ethyl acetate, routinely applied for the analysis of antioxidant potential (AOP of plant oils, reactions of tocopherols with DPPH are slower and concentration of tocopherols in the assay has a large influence on their molar reactivity. In 2-propanol, however, two electrons are exchanged for both α- and γ-tocopherols, independent of their concentration. 2-propanol is not toxic and is fully compatible with polypropylene labware. The chromatographically determined content of tocopherols and their molar reactivity in the DPPH assay reveal that only tocopherols contribute to the AOP of sunflower oil, whereas the contribution of tocopherols to the AOP of linseed oil is 75%. The DPPH assay in 2-propanol can be applied for rapid and cheap estimation of vitamin E content in plant oils where tocopherols are major antioxidants.

Rapid Estimation of Tocopherol Content in Linseed and Sunflower Oils-Reactivity and Assay.

Science.gov (United States)

Prevc, Tjaša; Levart, Alenka; Cigić, Irena Kralj; Salobir, Janez; Ulrih, Nataša Poklar; Cigić, Blaž

2015-08-13

The reactivity of tocopherols with 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied in model systems in order to establish a method for quantifying vitamin E in plant oils. The method was optimized with respect to solvent composition of the assay medium, which has a large influence on the course of reaction of tocopherols with DPPH. The rate of reaction of α-tocopherol with DPPH is higher than that of γ-tocopherol in both protic and aprotic solvents. In ethyl acetate, routinely applied for the analysis of antioxidant potential (AOP) of plant oils, reactions of tocopherols with DPPH are slower and concentration of tocopherols in the assay has a large influence on their molar reactivity. In 2-propanol, however, two electrons are exchanged for both α- and γ-tocopherols, independent of their concentration. 2-propanol is not toxic and is fully compatible with polypropylene labware. The chromatographically determined content of tocopherols and their molar reactivity in the DPPH assay reveal that only tocopherols contribute to the AOP of sunflower oil, whereas the contribution of tocopherols to the AOP of linseed oil is 75%. The DPPH assay in 2-propanol can be applied for rapid and cheap estimation of vitamin E content in plant oils where tocopherols are major antioxidants.

Contrast bolus technique with rapid CT scanning

International Nuclear Information System (INIS)

Arnold, H.; Kuehne, D.; Rohr, W.; Heller, M.

1981-01-01

Twenty-three patients complying with the clinical criteria for brain death were studied by contrast-enhanced CT. In all but one, the great intracranial vessels escaped visualization; accordingly, angiography demonstrated cerebral circulatory arrest. In the remaining case, faint enhancement of the circle of Willis corresponded to angiographic demonstration of the proximal segments of cerebral arteris. Neither in normal brain nor in dead brain did slow CT scanning disclose any postcontrast increase in parenchymal attenuation. An improved technique is proposed to demonstrate the transit of the contrast bolus by rapid CT with image splitting. If cerebral blood flow is preserved, the grey and white matter will enhance significantly following administration of contrast medium. Vice versa, the absence of enhancement confirms brain death, even in instances in which the great cerebral vessels are obscured by hemorrhage or other extensive lesions. Two additional cases of brain death were evaluated by rapid CT scanning. As to brain death, the technique obviates the need for angiography or radionuclide angiography, usually applied in prospective organ donors, because its informative content is superior to that of either method. The CT technique described affords a reliable and safe diagnosis of brain death, and can be interpreted easily. (orig.)

Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

Science.gov (United States)

Meng, Juncai; Lai, Ming-Tain; Munshi, Vandna; Grobler, Jay; McCauley, John; Zuck, Paul; Johnson, Eric N; Uebele, Victor N; Hermes, Jeffrey D; Adam, Gregory C

2015-06-01

HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors. © 2015 Society for Laboratory Automation and Screening.

Rapid detection of Cyprinid herpesvirus-3 (CyHV-3) using a gold nanoparticle-based hybridization assay.

Science.gov (United States)

Saleh, Mona; El-Matbouli, Mansour

2015-06-01

Cyprinid herpesvirus-3 (CyHV-3) is a highly infectious pathogen that causes fatal disease in common and koi carp Cyprinus carpio L. CyHV-3 detection is usually based on virus propagation or amplification of the viral DNA using the PCR or LAMP techniques. However, due to the limited susceptibility of cells used for propagation, it is not always possible to successfully isolate CyHV-3 even from tissue samples that have high virus titres. All previously described detection methods including PCR-based assays are time consuming, laborious and require specialized equipment. To overcome these limitations, gold nanoparticles (AuNPs) have been explored for direct and sensitive detection of DNA. In this study, a label-free colorimetric nanodiagnostic method for direct detection of unamplified CyHV-3 DNA using gold nanoparticles is introduced. Under appropriate conditions, DNA probes hybridize with their complementary target sequences in the sample DNA, which results in aggregation of the gold nanoparticles and a concomitant colour change from red to blue, whereas test samples with non complementary DNA sequences remain red. In this study, gold nanoparticles were used to develop and evaluate a specific and sensitive hybridization assay for direct and rapid detection of the highly infectious pathogen termed Cyprinid herpesvirus-3. Copyright © 2015 Elsevier B.V. All rights reserved.

Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

International Nuclear Information System (INIS)

Hovi, T.; Roivainen, M.

1989-01-01

We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with 51 Cr, [ 3 H]leucine, or, preferentially, with [ 3 H]uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30 degree C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

Directory of Open Access Journals (Sweden)

Xingmei Xie

Full Text Available Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR. Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY, five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377, one X/Y-common STR (X22, and two autosomal STRs (D13S305 and D21S11. Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay.

Directory of Open Access Journals (Sweden)

Gregory M Laird

Full Text Available HIV-1 persists in infected individuals in a stable pool of resting CD4(+ T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4(+ T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4(+ T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

Science.gov (United States)

Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

2017-08-01

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Comparison of Enzymatic Assay and Multiple Tube Fermentation Technique in the Assessment of Microbial Quality of the Karoon River

Directory of Open Access Journals (Sweden)

Mahnaz Nikaeen

2010-09-01

Full Text Available Microbiological monitoring of surface waters designated for use as drinking water is essential by water utilities for the design and operation of drinking water treatment plants. Enzymatic assays have been applied as a rapid alternative approach to assess the microbiological quality of freshwater. In this study, the LMX broth (LMX as an enzymatic assay was compared with the standard method of multiple tube fermentation technique (MTF for the microbial monitoring of the Karoon River. Enumeration of total coliforms and E. coli averaged 9928 and 6684 MPN/ 100 ml by the LMX and 7564 and 6546 MPN/ 100 ml for the MTF, respectively. This difference was statistically significant for TC but the overall analysis revealed no difference between E. coli recoveries on LMX and MTF. In conclusion, LMX can be used for the enumeration of coliforms and E. coli in surface waters as it is less lobar-intensive, yields faster result, and simultaneously detects both total coliforms and E. coli.

Evaluation of rapid immuno chromatographic assay kit using monoclonal mpt64 antibodies for identification of mycobacterium tuberculosis complex

International Nuclear Information System (INIS)

Satti, L.; Ikram, A.; Malik, N.

2010-01-01

To evaluate the performance of rapid immuno chromatographic kit MPT64 Ag for the identification of Mycobacterium tuberculosis complex from various Mycobacterium tuberculosis culture positive specimens. Department of Microbiology, Armed Forces Institute of Pathology Rawalpindi, from August 2008 through March 2009. Eighty four Mycobacterium tuberculosis positive cultures on I BACTEC 460 and MGIT 960, one ATCC 25177 MTB strain, three institutional control MTB strains, two institutional control MOTT strains and 20 different bacterial isolates were tested. Tests were performed according to the instructional manual. Out of total 84 tested samples, MPT64 showed positive result in 80 cultures. Only four positive cultures did not display any band on MPT64 kit. These four strains were reconfirmed as Mycobacterium tuberculosis by PCR method. MOTT control strains and all the 20 bacterial isolates were negative for band. The sensitivity and specificity of ICT assay in our study was 95.2% and 100% respectively. Rapid MPT64 Kit is a good diagnostic tool to differentiate between Mycobacterium tuberculosis complex and MOTT with 100% specificity. The technique is simple and can provide prompt information to the clinicians to initiate early and appropriate antituberculosis therapy. (author)

A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.

Science.gov (United States)

Wang, Dan; Xie, Jiangbi; Zhu, Xiaocui; Li, Jinqiu; Zhao, Dongqin; Zhao, Meiping

2014-05-15

In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes. Copyright © 2013 Elsevier B.V. All rights reserved.

A Rapid and Simple Real-Time PCR Assay for Detecting Foodborne Pathogenic Bacteria in Human Feces.

Science.gov (United States)

Hanabara, Yutaro; Ueda, Yutaka

2016-11-22

A rapid, simple method for detecting foodborne pathogenic bacteria in human feces is greatly needed. Here, we examined the efficacy of a method that employs a combination of a commercial PCR master mix, which is insensitive to PCR inhibitors, and a DNA extraction method which used sodium dodecyl benzene sulfonate (SDBS), and Tween 20 to counteract the inhibitory effects of SDBS on the PCR assay. This method could detect the target genes (stx1 and stx2 of enterohemorrhagic Escherichia coli, invA of Salmonella Enteritidis, tdh of Vibrio parahaemolyticus, gyrA of Campylobacter jejuni, ceuE of Campylobacter coli, SEA of Staphylococcus aureus, ces of Bacillus cereus, and cpe of Clostridium perfringens) in a fecal suspension containing 1.0 × 10 1 to 1.0 × 10 3 CFU/ml. Furthermore, the assay was neither inhibited nor influenced by individual differences among the fecal samples of 10 subjects or fecal concentration (40-160 mg/ml in the fecal suspension). When we attempted to detect the genes of pathogenic bacteria in 4 actual clinical cases, we found that this method was more sensitive than standard culture method. These results showed that this assay is a rapid, simple detection method for foodborne pathogenic bacteria in human feces.

Evaluation of a simple and rapid dipstick assay for the diagnosis of typhoid fever in Indonesia

NARCIS (Netherlands)

Gasem, M. Hussein; Smits, Henk L.; Goris, Marga G. A.; Dolmans, Wil M. V.

2002-01-01

To support the clinical diagnosis of typhoid fever in Indonesia, where most hospitals and health centres have no facilities for culture, a rapid dipstick assay for the detection of Salmonella typhi-specific IgM antibodies was evaluated on serum samples from 127 patients clinically suspected of

Development of an immunochromatographic assay for the rapid detection of bromoxynil in water

International Nuclear Information System (INIS)

Zhu Jiang; Chen Wenchao; Lu Yitong; Cheng Guohua

2008-01-01

A rapid immunochromatographic one-step strip test was developed to specifically determine bromoxynil in surface and drinking water by competitive inhibition with the nano colloidal gold-conjugated monoclonal antibody (mAb). Bromoxynil standard samples of 0.01-10 mg L -1 in water were tested by this method and the visual limit was 0.06 mg L -1 . The assay only required 5 min and one-step by dispensing a drop of sample solution onto a strip. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA. Therefore, the one-step strip test is very useful as a screening method for qualitative detection of bromoxynil in water. - One-step strip test is a rapid method for qualitative detection of bromoxynil residues in water

A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

Science.gov (United States)

Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

1975-01-01

Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of Burkholderia pseudomallei and Burkholderia mallei

OpenAIRE

U'Ren, Jana M.; Van Ert, Matthew N.; Schupp, James M.; Easterday, W. Ryan; Simonson, Tatum S.; Okinaka, Richard T.; Pearson, Talima; Keim, Paul

2005-01-01

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.

Performance Values for Non-Destructive Assay (NDA) Technique Applied to Wastes: Evaluation by the ESARDA NDA Working Group

International Nuclear Information System (INIS)

Rackham, Jamie; Weber, Anne-Laure; Chard, Patrick

2012-01-01

The first evaluation of NDA performance values was undertaken by the ESARDA Working Group for Standards and Non Destructive Assay Techniques and was published in 1993. Almost ten years later in 2002 the Working Group reviewed those values and reported on improvements in performance values and new measurement techniques that had emerged since the original assessment. The 2002 evaluation of NDA performance values did not include waste measurements (although these had been incorporated into the 1993 exercise), because although the same measurement techniques are generally applied, the performance is significantly different compared to the assay of conventional Safeguarded special nuclear material. It was therefore considered more appropriate to perform a separate evaluation of performance values for waste assay. Waste assay is becoming increasingly important within the Safeguards community, particularly since the implementation of the Additional Protocol, which calls for declaration of plutonium and HEU bearing waste in addition to information on existing declared material or facilities. Improvements in the measurement performance in recent years, in particular the accuracy, mean that special nuclear materials can now be accounted for in wastes with greater certainty. This paper presents an evaluation of performance values for the NDA techniques in common usage for the assay of waste containing special nuclear material. The main topics covered by the document are: 1- Techniques for plutonium bearing solid wastes 2- Techniques for uranium bearing solid wastes 3 - Techniques for assay of fissile material in spent fuel wastes. Originally it was intended to include performance values for measurements of uranium and plutonium in liquid wastes; however, as no performance data for liquid waste measurements was obtained it was decided to exclude liquid wastes from this report. This issue of the performance values for waste assay has been evaluated and discussed by the ESARDA

Performance Values for Non-Destructive Assay (NDA) Technique Applied to Wastes: Evaluation by the ESARDA NDA Working Group

Energy Technology Data Exchange (ETDEWEB)

Rackham, Jamie [Babcock International Group, Sellafield, Seascale, Cumbria, (United Kingdom); Weber, Anne-Laure [Institut de Radioprotection et de Surete Nucleaire Fontenay-Aux-Roses (France); Chard, Patrick [Canberra, Forss Business and Technology park, Thurso, Caithness (United Kingdom)

2012-12-15

The first evaluation of NDA performance values was undertaken by the ESARDA Working Group for Standards and Non Destructive Assay Techniques and was published in 1993. Almost ten years later in 2002 the Working Group reviewed those values and reported on improvements in performance values and new measurement techniques that had emerged since the original assessment. The 2002 evaluation of NDA performance values did not include waste measurements (although these had been incorporated into the 1993 exercise), because although the same measurement techniques are generally applied, the performance is significantly different compared to the assay of conventional Safeguarded special nuclear material. It was therefore considered more appropriate to perform a separate evaluation of performance values for waste assay. Waste assay is becoming increasingly important within the Safeguards community, particularly since the implementation of the Additional Protocol, which calls for declaration of plutonium and HEU bearing waste in addition to information on existing declared material or facilities. Improvements in the measurement performance in recent years, in particular the accuracy, mean that special nuclear materials can now be accounted for in wastes with greater certainty. This paper presents an evaluation of performance values for the NDA techniques in common usage for the assay of waste containing special nuclear material. The main topics covered by the document are: 1- Techniques for plutonium bearing solid wastes 2- Techniques for uranium bearing solid wastes 3 - Techniques for assay of fissile material in spent fuel wastes. Originally it was intended to include performance values for measurements of uranium and plutonium in liquid wastes; however, as no performance data for liquid waste measurements was obtained it was decided to exclude liquid wastes from this report. This issue of the performance values for waste assay has been evaluated and discussed by the ESARDA

Development of a VHH-Based Erythropoietin Quantification Assay

DEFF Research Database (Denmark)

Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon

2015-01-01

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against...... human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification...

Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

Directory of Open Access Journals (Sweden)

Rosemary S Turingan

Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

A novel bench-scale column assay to investigate site-specific nitrification biokinetics in biological rapid sand filters

DEFF Research Database (Denmark)

Tatari, Karolina; Smets, Barth F.; Albrechtsen, Hans-Jørgen

2013-01-01

A bench-scale assay was developed to obtain site-specific nitrification biokinetic information from biological rapid sand filters employed in groundwater treatment. The experimental set-up uses granular material subsampled from a full-scale filter, packed in a column, and operated with controlled...

Nested PCR Assay for Eight Pathogens: A Rapid Tool for Diagnosis of Bacterial Meningitis.

Science.gov (United States)

Bhagchandani, Sharda P; Kubade, Sushant; Nikhare, Priyanka P; Manke, Sonali; Chandak, Nitin H; Kabra, Dinesh; Baheti, Neeraj N; Agrawal, Vijay S; Sarda, Pankaj; Mahajan, Parikshit; Ganjre, Ashish; Purohit, Hemant J; Singh, Lokendra; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

2016-02-01

Bacterial meningitis is a dreadful infectious disease with a high mortality and morbidity if remained undiagnosed. Traditional diagnostic methods for bacterial meningitis pose a challenge in accurate identification of pathogen, making prognosis difficult. The present study is therefore aimed to design and evaluate a specific and sensitive nested 16S rDNA genus-based polymerase chain reaction (PCR) assay using clinical cerebrospinal fluid (CSF) for rapid diagnosis of eight pathogens causing the disease. The present work was dedicated to development of an in-house genus specific 16S rDNA nested PCR covering pathogens of eight genera responsible for causing bacterial meningitis using newly designed as well as literature based primers for respective genus. A total 150 suspected meningitis CSF obtained from the patients admitted to Central India Institute of Medical Sciences (CIIMS), India during the period from August 2011 to May 2014, were used to evaluate clinical sensitivity and clinical specificity of optimized PCR assays. The analytical sensitivity and specificity of our newly designed genus-specific 16S rDNA PCR were found to be ≥92%. With such a high sensitivity and specificity, our in-house nested PCR was able to give 100% sensitivity in clinically confirmed positive cases and 100% specificity in clinically confirmed negative cases indicating its applicability in clinical diagnosis. Our in-house nested PCR system therefore can diagnose the accurate pathogen causing bacterial meningitis and therefore be useful in selecting a specific treatment line to minimize morbidity. Results are obtained within 24 h and high sensitivity makes this nested PCR assay a rapid and accurate diagnostic tool compared to traditional culture-based methods.

Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers.

Science.gov (United States)

Farcas, Gabriella A; Soeller, Rainer; Zhong, Kathleen; Zahirieh, Alireza; Kain, Kevin C

2006-03-01

Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations. We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)-resistance determinants in returned travelers who are febrile. A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR-restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly. The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation. This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

Science.gov (United States)

Redmile-Gordon, M A; Armenise, E; White, R P; Hirsch, P R; Goulding, K W T

2013-12-01

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

Manufacturing Implant Supported Auricular Prostheses by Rapid Prototyping Techniques

OpenAIRE

Karatas, Meltem Ozdemir; Cifter, Ebru Demet; Ozenen, Didem Ozdemir; Balik, Ali; Tuncer, Erman Bulent

2011-01-01

Maxillofacial prostheses are usually fabricated on the models obtained following the impression procedures. Disadvantages of conventional impression techniques used in production of facial prosthesis are deformation of soft tissues caused by impression material and disturbance of the patient due to. Additionally production of prosthesis by conventional methods takes longer time. Recently, rapid prototyping techniques have been developed for extraoral prosthesis in order to reduce these disadv...

Development of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea.

Science.gov (United States)

Ghosh, Raju; Nagavardhini, Avuthu; Sengupta, Anindita; Sharma, Mamta

2015-02-11

Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt is a devastating pathogen of chickpea. In chickpea, various soil borne pathogens produce (s) similar symptoms, therefore cannot be distinguished easily at field level. There is real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeting the elongation factor 1 alpha gene sequence for visual detection of Foc. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue (HNB) was added before amplification, samples with Foc DNA developed a characteristic sky blue colour but those without DNA or with the DNA of six other plant pathogenic fungi did not. Results obtained with LAMP and HNB were confirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for Foc was 10 fg of genomic DNA per reaction, while that of conventional PCR was 100 pg. In conclusion, it was found that a LAMP assay combined with HNB is simple, rapid, sensitive, and specific. The LAMP assay does not require specialized equipment, hence can be used in the field for the rapid detection of Foc. This is the first report of the use of LAMP assay for the detection of Foc. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of Foc, with the potential to be standardized as a detection method for Foc in endemic areas and will be very useful for monitoring the disease complex in the field further suggesting the management strategies.

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

Science.gov (United States)

Verma, Sandeep; Singh, Ruchi; Sharma, Vanila; Bumb, Ram Avtar; Negi, Narendra Singh; Ramesh, V; Salotra, Poonam

2017-03-23

Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

Science.gov (United States)

Ambagala, A; Fisher, M; Goolia, M; Nfon, C; Furukawa-Stoffer, T; Ortega Polo, R; Lung, O

2017-10-01

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT ™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco ™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. © 2016 Her Majesty the Queen in Right of Canada.

Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

Science.gov (United States)

Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

2016-01-01

Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane.

Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay

Directory of Open Access Journals (Sweden)

Dinggang eZhou

2016-03-01

Full Text Available Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was ten-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100% by LAMP and 97/100 cases (97% by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable and cost-effective for detection of the bar specific transgenic sugarcane.

Preliminary report of the comparison of multiple non-destructive assay techniques on LANL Plutonium Facility waste drums

International Nuclear Information System (INIS)

Bonner, C.; Schanfein, M.; Estep, R.

1999-01-01

Prior to disposal, nuclear waste must be accurately characterized to identify and quantify the radioactive content. The DOE Complex faces the daunting task of measuring nuclear material with both a wide range of masses and matrices. Similarly daunting can be the selection of a non-destructive assay (NDA) technique(s) to efficiently perform the quantitative assay over the entire waste population. In fulfilling its role of a DOE Defense Programs nuclear User Facility/Technology Development Center, the Los Alamos National Laboratory Plutonium Facility recently tested three commercially built and owned, mobile nondestructive assay (NDA) systems with special nuclear materials (SNM). Two independent commercial companies financed the testing of their three mobile NDA systems at the site. Contained within a single trailer is Canberra Industries segmented gamma scanner/waste assay system (SGS/WAS) and neutron waste drum assay system (WDAS). The third system is a BNFL Instruments Inc. (formerly known as Pajarito Scientific Corporation) differential die-away imaging passive/active neutron (IPAN) counter. In an effort to increase the value of this comparison, additional NDA techniques at LANL were also used to measure these same drums. These are comprised of three tomographic gamma scanners (one mobile unit and two stationary) and one developmental differential die-away system. Although not certified standards, the authors hope that such a comparison will provide valuable data for those considering these different NDA techniques to measure their waste as well as the developers of the techniques

Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

DEFF Research Database (Denmark)

Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

2002-01-01

6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...... axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer......A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD...

Statistical precision of delayed-neutron nondestructive assay techniques

International Nuclear Information System (INIS)

Bayne, C.K.; McNeany, S.R.

1979-02-01

A theoretical analysis of the statistical precision of delayed-neutron nondestructive assay instruments is presented. Such instruments measure the fissile content of nuclear fuel samples by neutron irradiation and delayed-neutron detection. The precision of these techniques is limited by the statistical nature of the nuclear decay process, but the precision can be optimized by proper selection of system operating parameters. Our method is a three-part analysis. We first present differential--difference equations describing the fundamental physics of the measurements. We then derive and present complete analytical solutions to these equations. Final equations governing the expected number and variance of delayed-neutron counts were computer programmed to calculate the relative statistical precision of specific system operating parameters. Our results show that Poisson statistics do not govern the number of counts accumulated in multiple irradiation-count cycles and that, in general, maximum count precision does not correspond with maximum count as first expected. Covariance between the counts of individual cycles must be considered in determining the optimum number of irradiation-count cycles and the optimum irradiation-to-count time ratio. For the assay system in use at ORNL, covariance effects are small, but for systems with short irradiation-to-count transition times, covariance effects force the optimum number of irradiation-count cycles to be half those giving maximum count. We conclude that the equations governing the expected value and variance of delayed-neutron counts have been derived in closed form. These have been computerized and can be used to select optimum operating parameters for delayed-neutron assay devices

Evaluation of the microscopic observational drug susceptibility assay for rapid and efficient diagnosis of multi-drug resistant tuberculosis

Directory of Open Access Journals (Sweden)

R P Lazarus

2012-01-01

Full Text Available Purpose: Tuberculosis (TB is endemic in India and the burden of multi-drug-resistant tuberculosis (MDR-TB is high. Early detection of MDR-TB is of primary importance in controlling the spread of TB. The microscopic observational drug susceptibility (MODS assay has been described as a cost-effective and rapid method by which mycobacterial culture and the drug susceptibility test (DST can be done at the same time. Materials and Methods: A total of 302 consecutive sputum samples that were received in an accredited mycobacteriology laboratory for conventional culture and DST were evaluated by the MODS assay. Results: In comparison with conventional culture on Lowenstein Jensen (LJ media, the MODS assay showed a sensitivity of 94.12% and a specificity of 89.39% and its concordance with the DST by the proportion method on LJ media to isoniazid and rifampicin was 90.8% and 91.5%, respectively. The turnaround time for results by MODS was 9 days compared to 21 days by culture on LJ media and an additional 42 days for DST by the 1% proportion method. The cost of performing a single MODS assay was Rs. 250/-, compared to Rs. 950/- for culture and 1st line DST on LJ. Conclusion: MODS was found to be a sensitive and rapid alternative method for performing culture and DST to identify MDR-TB in resource poor settings.

Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

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Pravas Ranjan Sahoo

2016-05-01

Full Text Available India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

Science.gov (United States)

Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

2016-05-01

India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

Further Evaluation of the Neutron Resonance Transmission Analysis (NRTA) Technique for Assaying Plutonium in Spent Fuel

Energy Technology Data Exchange (ETDEWEB)

J. W. Sterbentz; D. L. Chichester

2011-09-01

This is an end-of-year report (Fiscal Year (FY) 2011) for the second year of effort on a project funded by the National Nuclear Security Administration's Office of Nuclear Safeguards (NA-241). The goal of this project is to investigate the feasibility of using Neutron Resonance Transmission Analysis (NRTA) to assay plutonium in commercial light-water-reactor spent fuel. This project is part of a larger research effort within the Next-Generation Safeguards Initiative (NGSI) to evaluate methods for assaying plutonium in spent fuel, the Plutonium Assay Challenge. The second-year goals for this project included: (1) assessing the neutron source strength needed for the NRTA technique, (2) estimating count times, (3) assessing the effect of temperature on the transmitted signal, (4) estimating plutonium content in a spent fuel assembly, (5) providing a preliminary assessment of the neutron detectors, and (6) documenting this work in an end of the year report (this report). Research teams at Los Alamos National Laboratory (LANL), Lawrence Berkeley National Laboratory (LBNL), Pacific Northwest National Laboratory (PNNL), and at several universities are also working to investigate plutonium assay methods for spent-fuel safeguards. While the NRTA technique is well proven in the scientific literature for assaying individual spent fuel pins, it is a newcomer to the current NGSI efforts studying Pu assay method techniques having just started in March 2010; several analytical techniques have been under investigation within this program for two to three years or more. This report summarizes work performed over a nine month period from January-September 2011 and is to be considered a follow-on or add-on report to our previous published summary report from December 2010 (INL/EXT-10-20620).

A novel rapid genotyping technique for Collie eye anomaly: SYBR Green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on Flinders Technology Associates filter paper.

Science.gov (United States)

Chang, Hye-Sook; Mizukami, Keijiro; Yabuki, Akira; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Arai, Toshiro; Yamato, Osamu

2010-09-01

Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

Improved laboratory assays of Pu and U for SRP purification and finishing processes

International Nuclear Information System (INIS)

Holland, M.K.; Dorsett, R.S.

1986-01-01

Significant improvements have been made in routine assay techniques for uranium and plutonium as part of an effort to improve accountability at the Savannah River Plant (SRP). Emphasis was placed on input/output accountability points and key physical inventory tanks associated with purification and finishing processes. Improvements were made in existing assay methods; new methods were implemented; and the application of these methods was greatly expanded. Prior to assays, samples were validated via density measurements. Digital density meters precise to four, five, and six decimal places were used to meet specific requirements. Improved plutonium assay techniques are now in routine use: controlled-potential coulometry, ion-exchange coulometry, and Pu(III) diode-array spectrophotometry. A new state-of-the-art coulometer was fabricated and used to ensure maximum accuracy in verifying standards and in measuring plutonium in product streams. The diode-array spectrophotometer for Pu(III) measurements was modified with fiber optics to facilitate remote measurements; rapid, precise measurements made the technique ideally suited for high-throughput assays. For uranium assays, the isotope-dilution mass spectrometric (IDMS) method was converted to a gravimetric basis. The IDMS method and the existing Davies-Gray titration (gravimetric basis) have met accountability requirements for uranium. More recently, a Pu(VI) diode-array spectrophotometric method was used on a test basis to measure plutonium in shielded-cell input accountability samples. In addition, tests to measure uranium via diode-array spectrophotometry were initiated. This rapid, precise method will replace IDMS for certain key sample points

The effect of varying incubation times for hypotonic treatment of lymphocytes in dicentric assay technique

International Nuclear Information System (INIS)

Noraisyah Yusof; Noriah Jamal; Rahimah Abdul Rahim; Juliana Mahamad Napiah

2010-01-01

The International Atomic Energy Agency (IAEA) has recommended that incubation time for the hypotonic treatment of lymphocytes in dicentric assay technique to be between 15 to 20 minutes. Incubation time will effect the hypotonic treatment of lymphocytes and thus, the breakage of cytoplasmic membrane. The objective of this study is to examine the effect of varying incubation times for hypotonic treatment of lymphocytes in dicentric assay technique. In this study, we choose to use our standard protocol for dicentric assay technique. However, for the hypotonic treatment of lymphocytes, the incubation times were varied from 10, 15, 20, 25 and 30 minutes respectively. Lymphocytes were then fixed and stained with Giemsa. The cells were then analyzed for clear background that indicates good metaphases. We found that incubation time of 30 minutes gives the best metaphase images. This incubation time is longer than what has been recommended by the IAEA. This maybe explained by the fact that our country's climate is of higher humidity compared with the European countries. (author)

DNA Comet Assay. A simple screening technique for identification of some irradiated foods

International Nuclear Information System (INIS)

Khan, A.A.; Khan, H.M.

2008-01-01

DNA Comet Assay method was carried out to detect irradiation treatment of some foods like meat, spices, beans and lentils. The fresh meat of cow and duck were irradiated up to radiation doses of 3 kGy, the spices (cardamoms and cumin black) were irradiated to radiation doses of 5, 10, 15 and 20 kGy while the beans (black beans and white beans) and lentils (red and green lentils) were irradiated to 0.5 and 1 kGy. All the foods were then analyzed for radiation treatment using simple microgel electrophoresis of single cells or nuclei (DNA Comet Assay). Sedimentation, lysis and staining times were adjusted to get optimized conditions for correct and easy analysis of each food. Using these optimized conditions, it was found out that radiation damaged DNA showed comets in case of irradiated food samples, whereas in non-treated food samples, round or conical spots of stained DNA were visible. Shape, length and intensity of these comets were also radiation dose dependent. Screening of unirradiated and irradiated samples by Comet Assay was successful in the case of all the foods under consideration under the optimized conditions of assay. Therefore, for different kinds of irradiated foods studied in the present study, the DNA Comet Assay can be used as a rapid, simple and inexpensive screening test. (author)

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

Science.gov (United States)

Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

2013-01-01

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

Directory of Open Access Journals (Sweden)

Xin Zhang

Full Text Available Fusarium oxysporum f. sp. cubense (Foc, the causal agent of Fusarium wilt (Panama disease, is one of the most devastating diseases of banana (Musa spp.. The Foc tropical race 4 (TR4 is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05. Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

Science.gov (United States)

Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

2015-01-01

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

Directory of Open Access Journals (Sweden)

Anna Grazia Recchia

Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

Nondestructive techniques for assaying fuel debris in piping at Three Mile Island Unit 2

International Nuclear Information System (INIS)

Vinjamuri, K.; McIsaac, C.V.; Beller, L.S.; Isaacson, L.; Mandler, J.W.; Hobbins, R.R. Jr.

1981-11-01

Four major categories of nondestructive techniques - ultrasonic, passive gamma ray, infrared detection, and remote video examination - have been determined to be feasible for assaying fuel debris in the primary coolant system of the Three Mile Island Unit 2 (TMI-2) Reactor. Passive gamma ray detection is the most suitable technique for the TMI-2 piping; however, further development of this technique is needed for specific application to TMI-2

Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples.

Science.gov (United States)

Leach, L; Zhu, Y; Chaturvedi, S

2018-02-01

Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 ( ITS 2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities. Copyright © 2018 Leach et al.

Rapid assay of resveratrol in red wine by paper spray tandem mass spectrometry and isotope dilution.

Science.gov (United States)

Di Donna, Leonardo; Taverna, Domenico; Indelicato, Serena; Napoli, Anna; Sindona, Giovanni; Mazzotti, Fabio

2017-08-15

A rapid analytical approach for the assay of resveratrol in red wines, based on Paper Spray Mass Spectrometry (PS-MS) and Multiple Reaction Monitoring (MRM) is described. The assay involves the use of the stable isotope dilution method. The analytical parameters calculated analyzing fortified samples confirm the reliability of the proposed approach, with accuracy values about 100%, and LOD and LOQ values calculated at 0.5 and 0.8μg/mL, respectively. Furthermore, both the recovery, which was quantitative for the analyte, and the reproducibility (RSD%), checked on different days on the same wine, always below 7%, highlighted the consistency of the methodology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Science.gov (United States)

Coban, Ahmet Yilmaz; Uzun, Meltem

2013-01-01

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results. PMID:24402143

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Directory of Open Access Journals (Sweden)

Ahmet Yilmaz Coban

2013-12-01

Full Text Available Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA in detecting isoniazid (INH and rifampicin (RIF resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV, negative predictive value (NPV and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.

Loop-mediated isothermal amplification assay for rapid detection of Streptococcus agalactiae (group B streptococcus) in vaginal swabs - a proof of concept study.

Science.gov (United States)

McKenna, James Patrick; Cox, Ciara; Fairley, Derek John; Burke, Rachael; Shields, Michael D; Watt, Alison; Coyle, Peter Valentine

2017-03-01

Neonatal sepsis caused by Streptococcus agalactiae [group B streptococcus (GBS)] is a life-threatening condition, which is preventable if colonized mothers are identified and given antibiotic prophylaxis during labour. Conventional culture is time consuming and unreliable, and many available non-culture diagnostics are too complex to implement routinely at point of care. Loop-mediated isothermal amplification (LAMP) is a method that, enables the rapid and specific detection of target nucleic acid sequences in clinical materials without the requirement for extensive sample preparation. A prototype LAMP assay targeting GBS sip gene is described. The assay was 100 % specific for GBS, with a limit of detection of 14 genome copies per reaction. The clinical utility of the LAMP assay for rapid direct molecular detection of GBS was determined by testing a total of 157 vaginal swabs with minimal sample processing using a rapid lysis solution. Compared to a reference quantitative real-time PCR assay, the direct LAMP protocol had a sensitivity and specificity of 95.4 and 100 %, respectively, with positive and negative predictive values of 100 and 98.3 %, respectively. Positive and negative likelihood ratios were infinity and 0.05, respectively. The direct LAMP method required a mean time of 45 min from the receipt of a swab to generation of a confirmed result, compared to 2 h 30 min for the reference quantitative real-time PCR test. The direct LAMP protocol described is easy to perform, facilitating rapid and accurate detection of GBS in vaginal swabs. This test has a potential for use at point of care.

Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

Science.gov (United States)

Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi

2017-08-12

Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

A DNA Barcode-Based RPA Assay (BAR-RPA) for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao).

Science.gov (United States)

Tian, Enwei; Liu, Qianqian; Ye, Haoting; Li, Fang; Chao, Zhi

2017-12-18

Background: Wuzhimaotao (the dry root of Ficus hirta ) is used as both medicine and food ingredient by the locals in areas around Nanling Mountains of China. Due to its very similar external morphologies with Duanchangcao (the root of Gelsemium elegans , which contains gelsemine that is extremely neurotoxic) and the associated growth of these two plants, incidents of food poisoning and even death frequently occur, resulting from the misuse of Duanchangcao as Wuzhimaotao. The aim of this study is to develop a fast, even, on-spot approach to identification of Wuzhimaotao. Methods: We used DNA barcode-based recombinase polymerase amplification (BAR-RPA) with species-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA of F. hirta. BAR-RPA reaction time and temperature were optimized and the specificity and sensitivity of BAR-RPA species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of F. hirta and allowed for rapid amplification (within 15 min) of the ITS region under a constant and mild temperature range of 37-42 °C without using thermocyclers. Conclusions: The BAR-RPA assay with a fast DNA extraction protocol provides a simple, energy-saving, and rapid method for identification of Wuzhimaotao in both laboratory and field settings.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

Science.gov (United States)

Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

2015-03-01

Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs.

Rapid prototyping: An innovative technique in dentistry

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Shakeba Quadri

2017-01-01

Full Text Available Emergence of advanced digital technology has opened up new perspectives for design and production in the field of dentistry. Rapid prototyping (RP is a technique to quickly and automatically construct a three-dimensional (3D model of a part or product using 3D printers or stereolithography machines. RP has various dental applications, such as fabrication of implant surgical guides, zirconia prosthesis and molds for metal castings, maxillofacial prosthesis and frameworks for fixed and removable partial dentures, wax patterns for the dental prosthesis and complete denture. Rapid prototyping presents fascinating opportunities, but the process is difficult as it demands a high level of artistic skill, which means that the dental technicians should be able to work with the models obtained after impression to form a mirror image and achieve good esthetics. This review aims to focus on various RP methods and its application in dentistry.

A rapid microtiter plate serum bactericidal assay method for determining serum complement-mediated killing of Mannheimia haemolytica.

Science.gov (United States)

Ayalew, Sahlu; Confer, Anthony W; Shrestha, Binu; Payton, Mark E

2012-05-01

In this study, we describe a rapid microtiter serum bactericidal assay (RMSBA) that can be used to measure the functionality of immune sera. It quantifies bactericidal activity of immune sera in the presence of complement against a homologous bacterium, M. haemolytica in this case. There is high correlation between data from RMSBA and standard complement-mediated bacterial killing assay (r=0.756; p<0.0001). The RMSBA activity of sera can be generated in less than 5 h instead of overnight incubation. RMSBA costs substantially less in terms of time, labor, and resources and is highly reproducible. Copyright © 2012 Elsevier B.V. All rights reserved.

In vitro and in vivo assay of radio-induced damage in Escherichia Coli, DNA labelled on thymidilic fragment

International Nuclear Information System (INIS)

Bonicel, A.

1977-01-01

A technique of rapid assay for a particular and very important damage, N-formamido (DNA), is described. Using this technique, the importance of radio-induced DNA damage can be evaluated before the repair enzymatic system takes place [fr

Single-cell microgel electrophoresis: an in vitro assay of radiosensitivity

International Nuclear Information System (INIS)

Deeley, J.O.T.; Moore, J.L.

1993-01-01

The results obtained by a microgel electrophoresis are comparable to conventional gel electrophoresis and elution techniques (Singh et al, 1989), DNA precipitation, alkali unwinding and cell clonogenicity assays (Olive et al, 1990). Since single cells are assessed, microgel electrophoresis is particularly appropriate for end-points such as the intercell variation in response. The simplicity, low cost and rapidity of microgel electrophoresis compared with other assays makes it particularly attractive for assessing the effects on DNA of radiation and other genotoxic agents on the general population. (Author)

Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

Directory of Open Access Journals (Sweden)

Wan Zhixiang

2005-07-01

Full Text Available Abstract Background Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR-based assays and enzyme-linked immunosorbent assays (ELISA are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Methods Chemically modified glass slides were used as solid supports (named chip, on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. Results We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05 existed between the results determined by our assay and ELISA respectively. Conclusion

Inspection with non destructive assay techniques of the aluminium coating of the TRIGA Mark III reactor vat

International Nuclear Information System (INIS)

Reyes A, A.I.; Gonzalez M, A.; Castaneda J, G.; Rivera M, H.; Sandoval G, I.

2001-01-01

In June 2000, the Reactor Department assigned to the Scientific Research Direction of the National Institute of Nuclear Research requested to the Non-destructive Assays Laboratory (LEND), assigned to the Materials Science Management, the inspection and measurement of thickness of the aluminium coating (liner) of the TRIGA Mark III reactor vat with non-destructive assay techniques, due to that the aluminium coating is exposed mainly to undergo slimming on its back side due to corrosion phenomena. Activity that was able to be carried out from april until august 2001. It is worth pointing out that this type of inspection with these techniques was realized by first time. The non-destructive assays (NDA) are techniques which use indirect physical methods for inspecting the sanitation of components in process or in service, for detect lack of continuity or defects which affect their quality or usefulness. The application of those do not alter the physical, chemical, mechanical or dimensional properties of the part subject of inspection. The results of the application of the ultrasound inspection techniques, industrial radiography and penetrating liquids are presented. (Author)

A histogram-based technique for rapid vector extraction from PIV photographs

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Humphreys, William M., Jr.

1991-01-01

A new analysis technique, performed totally in the image plane, is proposed which rapidly extracts all available vectors from individual interrogation regions on PIV photographs. The technique avoids the need for using Fourier transforms with the associated computational burden. The data acquisition and analysis procedure is described, and results of a preliminary simulation study to evaluate the accuracy of the technique are presented. Recently obtained PIV photographs are analyzed.

Evaluation of a new T2 Magnetic Resonance assay for rapid detection of emergent fungal pathogen Candida auris on clinical skin swab samples.

Science.gov (United States)

Sexton, D Joseph; Bentz, Meghan L; Welsh, Rory M; Litvintseva, Anastasia P

2018-06-25

Candida auris is a multidrug-resistant pathogenic yeast whose recent emergence is of increasing public-health concern. C. auris can colonize multiple body sites, including patients' skin, and survive for weeks in the healthcare environment, facilitating patient-to-patient transmission and fueling healthcare-associated outbreaks. Rapid and accurate detection of C. auris colonization is essential for timely implementation of infection control measures and prevent transmission. Currently, axilla/groin composite swabs, used to assess colonization status, are processed using a culture-based method that is sensitive and specific but requires 14 days. This delay limits the opportunity to respond and highlights the need for a faster alternative. The culture-independent T2 Magnetic Resonance (T2MR) system is a rapid diagnostic platform shown to detect target pathogens of interest from unprocessed blood samples in T2 assay was evaluated for screening of the skin surveillance samples. Inclusivity and limit of detection of the T2 C. auris assay were assessed with spiked samples in a representative skin flora background. The T2 C. auris assay recognized isolates from each of the 4 known clades of C. auris and consistently detected cells at 5 CFU/mL. Finally, 89 clinical axilla/groin swab samples were processed with the T2 C. auris assay. The culture-based diagnostic assay was used as a gold standard to determine performance statistics including sensitivity (0.89) and specificity (0.98). Overall, the T2 C. auris assay performed well as a rapid diagnostic and could help expedite the detection of C. auris in patient skin swabs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Benzodiazepine Synthesis and Rapid Toxicity Assay

Science.gov (United States)

Fletcher, James T.; Boriraj, Grit

2010-01-01

A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

Development of Techniques for Spent Fuel Assay - Differential Dieaway Final Report

International Nuclear Information System (INIS)

Swinhoe, Martyn Thomas; Goodsell, Alison; Ianakiev, Kiril Dimitrov; Iliev, Metodi; Desimone, David J.; Rael, Carlos D.; Henzl, Vladimir; Polk, Paul John

2016-01-01

This report summarizes the work done under a DNDO R&D funded project on the development of the differential dieaway method to measure plutonium in spent fuel. There are large amounts of plutonium that are contained in spent fuel assemblies, and currently there is no way to make quantitative non-destructive assay. This has led NA24 under the Next Generation Safeguards Initiative (NGSI) to establish a multi-year program to investigate, develop and implement measurement techniques for spent fuel. The techniques which are being experimentally tested by the existing NGSI project do not include any pulsed neutron active techniques. The present work covers the active neutron differential dieaway technique and has advanced the state of knowledge of this technique as well as produced a design for a practical active neutron interrogation instrument for spent fuel. Monte Carlo results from the NGSI effort show that much higher accuracy (1-2%) for the Pu content in spent fuel assemblies can be obtained with active neutron interrogation techniques than passive techniques, and this would allow their use for nuclear material accountancy independently of any information from the operator. The main purpose of this work was to develop an active neutron interrogation technique for spent nuclear fuel.

Immunochromatographic strip assay for the rapid and sensitive detection of Salmonella Typhimurium in artificially contaminated tomato samples.

Science.gov (United States)

Shukla, Shruti; Leem, Hyerim; Lee, Jong-Suk; Kim, Myunghee

2014-06-01

This study was designed to confirm the applicability of a liposome-based immunochromatographic assay for the rapid detection of Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) in artificially contaminated tomato samples. To determine the detection limit and pre-enrichment incubation time (10, 12, and 18 h pre-enrichment in 1% buffered peptone water), the tests were performed with different cell numbers of Salmonella Typhimurium (3 × 10(0), 3 × 10(1), 3 × 10(2), and 3 × 10(3) CFU·mL(-1)) inoculated into 25 g of crushed tomato samples. The assay was able to detect as few as 30 Salmonella Typhimurium cells per 25 g of tomato samples (1.2 cells·g(-1)) after 12 h pre-enrichment incubation. Moreover, when the developed assay was compared with traditional morphological and biochemical culture-based methods as well as colloidal gold nanoparticle-based commercial test strips, the developed assay yielded positive results for the detection of Salmonella Typhimurium within a shorter period time. These findings confirm that the developed assay may have practical application for the sensitive detection of Salmonella Typhimurium in various food samples, including raw vegetables, with a relatively low detection limit and shorter analysis time.

Interactions between heavy metals and photosynthetic materials studied by optical techniques.

Science.gov (United States)

Ventrella, Andrea; Catucci, Lucia; Piletska, Elena; Piletsky, Sergey; Agostiano, Angela

2009-11-01

In this work studies on rapid inhibitory interactions between heavy metals and photosynthetic materials at different organization levels were carried out by optical assay techniques, investigating the possibility of applications in the heavy metal detection field. Spinach chloroplasts, thylakoids and Photosystem II proteins were employed as biotools in combination with colorimetric assays based on dichlorophenol indophenole (DCIP) photoreduction and on fluorescence emission techniques. It was found that copper and mercury demonstrated a strong and rapid photosynthetic activity inhibition, that varied from proteins to membranes, while other metals like nickel, cobalt and manganese produced only slight inhibition effects on all tested photosynthetic materials. By emission measurements, only copper was found to rapidly influence the photosynthetic material signals. These findings give interesting information about the rapid effects of heavy metals on isolated photosynthetic samples, and are in addition to the literature data concerning the effects of growth in heavy metal enriched media.

Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex

Science.gov (United States)

Wachiralurpan, Sirirat; Sriyapai, Thayat; Areekit, Supatra; Sriyapai, Pichapak; Augkarawaritsawong, Suphitcha; Santiwatanakul, Somchai; Chansiri, Kosum

2018-04-01

ABSTRACT Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity and accuracy were 100%, 90.20% and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

Development of an ATP assay for rapid onboard testing to detect living microorganisms in ballast water

Science.gov (United States)

Hyun, Bonggil; Cha, Hyung-Gon; Lee, Nayoung; Yum, Seungshic; Baek, Seung Ho; Shin, Kyoungsoon

2018-03-01

Ballast water is a principal pathway for the introduction of pathogens and non-indigenous species to ports worldwide. The International Maritime Organization (IMO) and the United States Coast Guard (USCG) have adopted ballast water management regulations that require, e.g., the installation of shipboard ballast water management systems (BWMS). Rapid and simple analytical methods are needed to monitor whether ballast water disinfection ensures compliance with the discharge standards. In this study laboratory and full scale land-based testing was used to investigate the suitability of an adenosine triphosphate (ATP) assay for quantifying living organisms (≥ 10 and land-based testing the ATP assay also showed a good correlation with the presence of living natural plankton cells in control samples, but the ATP concentration (137 pg mL- 1) was much lower than the ATP guideline. The low ATP concentration in natural plankton cells may reflect a decline in their biological activity because of extended exposure to dark conditions. Although our results need further validation, the ATP assay is a suitable tool for monitoring compliance of ballast water treatment.

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

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Mindy I Davis

Full Text Available Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538, was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Rapid Immuno-Chromatographic Assay for the Detection of Antibodies to HIV Compare with Elisa among Voluntary and Replacement Blood Donor of Mymensingh Medical College Hospital.

Science.gov (United States)

Chakrabarty, P; Rudra, S; Hossain, M A; Begum, S A; Mirza, T T; Rudra, M

2015-04-01

Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from voluntary and replacement blood donors & HIV-infected patients (positive samples from BSMMU, Dhaka). Five rapid HIV assays: Determine™ HIV-1/2 (Inverness Medical), SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), First Response HIV Card 1-2.0 (PMC Medical India Pvt Ltd.), HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc) and Uni-Gold™ HIV-1/2 (Biotech) were evaluated between 1st February to 30th June, 2013 using 400 whole blood samples from voluntary and replacement blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics). Only 01 sample including ten positive samples from BSMMU were confirmed HIV-1 antibody positive, while 399 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold™ was 100% (95% CI; 99.1-100) while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2-99.9) and 97.7% (95% CI; 95.7-98.9) respectively, which increased to 100% (95% CI; 99.1-100) on repeat testing. The initial specificity of the Uni-Gold™ assay was 100% (95% CI; 99.6-100) while specificities were 99.6% (95% CI; 99-99.9), 99.4% (95% CI; 98.8-99.7), 99.6% (95% CI; 99-99.9) and 99.8% (95% CI; 99.3-99.9) for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was concordantly false positive in Uni-Gold™, Determine and SD Bioline assays. An alternative confirmatory HIV testing strategy based on initial testing on either SD Bioline or Determine assays followed by testing of reactive

Multidrug-resistant tuberculosis: Rapid molecular detection with MTBDRplus® assay in clinical samples

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Rita Macedo

2009-05-01

Full Text Available Nowadays, the greatest concern of tuberculosis control programmes is the appearance of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Rapid determination of drug resistance in clinical samples, with Mycobacterium tuberculosis complex (MTC, is the prerequisite for initiating effective chemotherapy, ensuring successful treatment of the patient and preventing further spread of drugresistant isolates.The aim of our study was to determine the sensitivity of the new MTBDRplus® assay in comparison to culture, identification and classic DST, directly from smear-positive clinical specimens.A total of 68 smear-positive sputum specimens were processed by both the classical mycobacteriological methods and the molecular assay, MTBDRplus®.MTBDRplus® assay allowed an accurate identification of MTC species by detection of the specific band in all samples, from which we also isolated and identified MTC strains by culture methods. In the samples from which we isolated susceptible strains (63.2%, wild type patterns were found using MTBDRplus® assay. The samples from which we isolated resistant strains (36.8% showed specific mutations associated with the correspondent resistant phenotype.Our study indicated that this assay allows rapid detection of resistance, always in agreement with classic methods. Resumo: Uma das principais problematicas no controlo da tuberculose e o aparecimento de casos de tuberculose multirresistente (TB-MR e tuberculose extensivamente resistente (TB-XDR. A deteccao precoce da resistencia a farmacos, directamente a partir de amostras respiratorias, e essencial para que se assegure o tratamento atempado, adequado e eficaz da tuberculose, bem como para prevenir a disseminacao destes casos de especial gravidade.O nosso objectivo foi avaliar a sensibilidade e comparar os resultados obtidos com um metodo de genetica molecular disponivel comercialmente – MTBDRplus® – e o isolamento

Prion strain discrimination based on rapid in vivo amplification and analysis by the cell panel assay.

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Yervand Eduard Karapetyan

Full Text Available Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.

Performance values for non destructive assay (NDA) techniques applied to safeguards: the 2002 evaluation by the ESARDA NDA Working Group

International Nuclear Information System (INIS)

Guardini, S.

2003-01-01

The first evaluation of NDA performance values undertaken by the ESARDA Working Group for Standards and Non Destructive Assay Techniques (WGNDA) was published in 1993. Almost 10 years later the Working Group decided to review those values, to report about improvements and to issue new performance values for techniques which were not applied in the early nineties, or were at that time only emerging. Non-Destructive Assay techniques have become more and more important in recent years, and they are used to a large extent in nuclear material accountancy and control both by operators and control authorities. As a consequence, the performance evaluation for NDA techniques is of particular relevance to safeguards authorities in optimising Safeguards operations and reducing costs. Performance values are important also for NMAC regulators, to define detection levels, limits for anomalies, goal quantities and to negotiate basic audit rules. This paper presents the latest evaluation of ESARDA Performance Values (EPVs) for the most common NDA techniques currently used for the assay of nuclear materials for Safeguards purposes. The main topics covered by the document are: techniques for plutonium bearing materials: PuO 2 and MOX; techniques for U-bearing materials; techniques for U and Pu in liquid form; techniques for spent fuel assay. This issue of the performance values is the result of specific international round robin exercises, field measurements and ad hoc experiments, evaluated and discussed in the ESARDA NDA Working Group. (author)

A comparison of high-throughput techniques for assaying circadian rhythms in plants.

Science.gov (United States)

Tindall, Andrew J; Waller, Jade; Greenwood, Mark; Gould, Peter D; Hartwell, James; Hall, Anthony

2015-01-01

Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions.

A DNA Barcode-Based RPA Assay (BAR-RPA for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao

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Enwei Tian

2017-12-01

Full Text Available Background: Wuzhimaotao (the dry root of Ficus hirta is used as both medicine and food ingredient by the locals in areas around Nanling Mountains of China. Due to its very similar external morphologies with Duanchangcao (the root of Gelsemium elegans, which contains gelsemine that is extremely neurotoxic and the associated growth of these two plants, incidents of food poisoning and even death frequently occur, resulting from the misuse of Duanchangcao as Wuzhimaotao. The aim of this study is to develop a fast, even, on-spot approach to identification of Wuzhimaotao. Methods: We used DNA barcode-based recombinase polymerase amplification (BAR-RPA with species–specific primers targeting the internal transcribed spacer (ITS region of the rDNA of F. hirta. BAR-RPA reaction time and temperature were optimized and the specificity and sensitivity of BAR-RPA species–specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of F. hirta and allowed for rapid amplification (within 15 min of the ITS region under a constant and mild temperature range of 37–42 °C without using thermocyclers. Conclusions: The BAR-RPA assay with a fast DNA extraction protocol provides a simple, energy-saving, and rapid method for identification of Wuzhimaotao in both laboratory and field settings.

Pulsating potentiometric titration technique for assay of dissolved oxygen in water at trace level.

Science.gov (United States)

Sahoo, P; Ananthanarayanan, R; Malathi, N; Rajiniganth, M P; Murali, N; Swaminathan, P

2010-06-11

A simple but high performance potentiometric titration technique using pulsating sensors has been developed for assay of dissolved oxygen (DO) in water samples down to 10.0 microg L(-1) levels. The technique involves Winkler titration chemistry, commonly used for determination of dissolved oxygen in water at mg L(-1) levels, with modification in methodology for accurate detection of end point even at 10.0 microg L(-1) levels DO present in the sample. An indigenously built sampling cum pretreatment vessel has been deployed for collection and chemical fixing of dissolved oxygen in water samples from flowing water line without exposure to air. A potentiometric titration facility using pulsating sensors developed in-house is used to carry out titration. The power of the titration technique has been realised in estimation of very dilute solution of iodine equivalent to 10 microg L(-1) O(2). Finally, several water samples containing dissolved oxygen from mg L(-1) to microg L(-1) levels were successfully analysed with excellent reproducibility using this new technique. The precision in measurement of DO in water at 10 microg L(-1) O(2) level is 0.14 (n=5), RSD: 1.4%. Probably for the first time a potentiometric titration technique has been successfully deployed for assay of dissolved oxygen in water samples at 10 microg L(-1) levels. Copyright 2010 Elsevier B.V. All rights reserved.

Pulsating potentiometric titration technique for assay of dissolved oxygen in water at trace level

International Nuclear Information System (INIS)

Sahoo, P.; Ananthanarayanan, R.; Malathi, N.; Rajiniganth, M.P.; Murali, N.; Swaminathan, P.

2010-01-01

A simple but high performance potentiometric titration technique using pulsating sensors has been developed for assay of dissolved oxygen (DO) in water samples down to 10.0 μg L -1 levels. The technique involves Winkler titration chemistry, commonly used for determination of dissolved oxygen in water at mg L -1 levels, with modification in methodology for accurate detection of end point even at 10.0 μg L -1 levels DO present in the sample. An indigenously built sampling cum pretreatment vessel has been deployed for collection and chemical fixing of dissolved oxygen in water samples from flowing water line without exposure to air. A potentiometric titration facility using pulsating sensors developed in-house is used to carry out titration. The power of the titration technique has been realised in estimation of very dilute solution of iodine equivalent to 10 μg L -1 O 2 . Finally, several water samples containing dissolved oxygen from mg L -1 to μg L -1 levels were successfully analysed with excellent reproducibility using this new technique. The precision in measurement of DO in water at 10 μg L -1 O 2 level is 0.14 (n = 5), RSD: 1.4%. Probably for the first time a potentiometric titration technique has been successfully deployed for assay of dissolved oxygen in water samples at 10 μg L -1 levels.

Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay

NARCIS (Netherlands)

Hansenova Manaskova, S.; Bikker, F.J.; Veerman, E.C.I.; van Belkum, A.; van Wamel, W.J.B.

2013-01-01

The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In

Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.

Science.gov (United States)

Zhou, Qian-Jin; Wang, Lei; Chen, Jiong; Wang, Rui-Na; Shi, Yu-Hong; Li, Chang-Hong; Zhang, De-Min; Yan, Xiao-Jun; Zhang, Yan-Jun

2014-09-01

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture. Copyright © 2014 Elsevier B.V. All rights reserved.

A review of rapid prototyping techniques for tissue engineering purposes

NARCIS (Netherlands)

Peltola, Sanna M.; Melchels, Ferry P. W.; Grijpma, Dirk W.; Kellomaki, Minna

2008-01-01

Rapid prototyping (RP) is a common name for several techniques, which read in data from computer-aided design (CAD) drawings and manufacture automatically three-dimensional objects layer-by-layer according to the virtual design. The utilization of RP in tissue engineering enables the production of

Evaluation of OXA-48 K-Se T: an immunochromatographic assay for rapid detection of OXA-48-producing Enterobacteriaceae.

Science.gov (United States)

Fernández, Javier; Fleites, Ana; Rodcio, María Rosario; Vazquez, Fernando

2016-05-01

The OXA-48 K-Se T, a new immunochromatographic assay for rapid detection of OXA-48-producing Enterobacteriaceae, has been evaluated in a Spanish Hospital during a 3-month period. A collection of 100 Enterobacteriaceae including 79 isolates producing OXA-48 has been tested. Sensitivity and specificity of 100% were obtained. Copyright © 2016 Elsevier Inc. All rights reserved.

Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay.

Science.gov (United States)

Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan; Qi, Shunxiang; Ma, Xuejun

2014-06-01

Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71

A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections.

Science.gov (United States)

Díaz de Arce, Heidy; Pérez, Lester J; Frías, Maria T; Rosell, Rosa; Tarradas, Joan; Núñez, José I; Ganges, Llilianne

2009-11-18

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5' non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89TCID(50). The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.

Simulation of the fissureless technique for thoracoscopic segmentectomy using rapid prototyping.

Science.gov (United States)

Akiba, Tadashi; Nakada, Takeo; Inagaki, Takuya

2015-01-01

The fissureless lobectomy or anterior fissureless technique is a novel surgical technique, which avoids dissection of the lung parenchyma over the pulmonary artery during lobectomy by open thoracotomy approach or direct vision thoracoscopic surgery. This technique is indicated for fused lobes. We present two cases where thoracoscopic pulmonary segmentectomy was performed using the fissureless technique simulated by three-dimensional (3D) pulmonary models. The 3D model and rapid prototyping provided an accurate anatomical understanding of the operative field in both cases. We believe that the construction of these models is useful for thoracoscopic and other complicated surgeries of the chest.

Simulation of the Fissureless Technique for Thoracoscopic Segmentectomy Using Rapid Prototyping

Science.gov (United States)

Nakada, Takeo; Inagaki, Takuya

2014-01-01

The fissureless lobectomy or anterior fissureless technique is a novel surgical technique, which avoids dissection of the lung parenchyma over the pulmonary artery during lobectomy by open thoracotomy approach or direct vision thoracoscopic surgery. This technique is indicated for fused lobes. We present two cases where thoracoscopic pulmonary segmentectomy was performed using the fissureless technique simulated by three-dimensional (3D) pulmonary models. The 3D model and rapid prototyping provided an accurate anatomical understanding of the operative field in both cases. We believe that the construction of these models is useful for thoracoscopic and other complicated surgeries of the chest. PMID:24633132

Performance of the new automated Abbott RealTime MTB assay for rapid detection of Mycobacterium tuberculosis complex in respiratory specimens.

Science.gov (United States)

Chen, J H K; She, K K K; Kwong, T-C; Wong, O-Y; Siu, G K H; Leung, C-C; Chang, K-C; Tam, C-M; Ho, P-L; Cheng, V C C; Yuen, K-Y; Yam, W-C

2015-09-01

The automated high-throughput Abbott RealTime MTB real-time PCR assay has been recently launched for Mycobacterium tuberculosis complex (MTBC) clinical diagnosis. This study would like to evaluate its performance. We first compared its diagnostic performance with the Roche Cobas TaqMan MTB assay on 214 clinical respiratory specimens. Prospective analysis of a total 520 specimens was then performed to further evaluate the Abbott assay. The Abbott assay showed a lower limit of detection at 22.5 AFB/ml, which was more sensitive than the Cobas assay (167.5 AFB/ml). The two assays demonstrated a significant difference in diagnostic performance (McNemar's test; P = 0.0034), in which the Abbott assay presented significantly higher area under curve (AUC) than the Cobas assay (1.000 vs 0.880; P = 0.0002). The Abbott assay demonstrated extremely low PCR inhibition on clinical respiratory specimens. The automated Abbott assay required only very short manual handling time (0.5 h), which could help to improve the laboratory management. In the prospective analysis, the overall estimates for sensitivity and specificity of the Abbott assay were both 100 % among smear-positive specimens, whereas the smear-negative specimens were 96.7 and 96.1 %, respectively. No cross-reactivity with non-tuberculosis mycobacterial species was observed. The superiority in sensitivity of the Abbott assay for detecting MTBC in smear-negative specimens could further minimize the risk in MTBC false-negative detection. The new Abbott RealTime MTB assay has good diagnostic performance which can be a useful diagnostic tool for rapid MTBC detection in clinical laboratories.

Potentiometric titration in a low volume of solution for rapid assay of uranium. Application to quantitative electro-reduction of uranium(VI)

International Nuclear Information System (INIS)

Sahoo, P.; Ananthanarayanan, R.; Murali, N.; Mallika, C.; Falix Lawrence; Kamachi Mudali, U.

2012-01-01

A simple, inexpensive PC based potentiometric titration technique for the assay of uranium using low volumes of sample aliquot (25-100 μL) along with all reagents (total volume of solution being less than 2.5 mL) is presented. The technique involves modification of the well known Davies and Gray Method recommended for assay of uranium(VI) in nuclear materials by introducing an innovative potentiometric titration device with a mini cell developed in-house. After appropriate chemical conditioning the titration is completed within a couple of minutes with display of online titration plot showing the progress of titration. The first derivative plot generated immediately after titration provides information of end point. The main advantage of using this technique is to carry out titration with minimum volumes of sample and reagents generating minimum volume of wastes after titration. The validity of the technique was evaluated using standard certified samples. This technique was applied for assay of uranium in a typical sample collected from fuel reprocessing laboratory. Further, the present technique was deployed in investigating the optimum conditions for efficient in situ production of U(IV). The precision in the estimation of uranium is highly satisfactory (RSD less than 1.0%). (author)

Experimental program for development and evaluation of nondestructive assay techniques for plutonium holdup

International Nuclear Information System (INIS)

Brumbach, S.B.

1977-05-01

An outline is presented for an experimental program to develop and evaluate nondestructive assay techniques applicable to holdup measurement in plutonium-containing fuel fabrication facilities. The current state-of-the-art in holdup measurements is reviewed. Various aspects of the fuel fabrication process and the fabrication facility are considered for their potential impact on holdup measurements. The measurement techniques considered are those using gamma-ray counting, neutron counting, and temperature measurement. The advantages and disadvantages of each technique are discussed. Potential difficulties in applying the techniques to holdup measurement are identified. Experiments are proposed to determine the effects of such problems as variation in sample thickness, in sample distribution, and in background radiation. These experiments are also directed toward identification of techniques most appropriate to various applications. Also proposed are experiments to quantify the uncertainties expected for each measurement

Protein subcellular localization assays using split fluorescent proteins

Science.gov (United States)

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

Identification of radiation treatment of foods using novel technique of 'DNA comet assay'

International Nuclear Information System (INIS)

Khan, A.A.; Khan, H.M.; Wasim, M.A.

2005-01-01

Treatment of food using ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests and to extend shelf life thereby contributing to safer and more plentiful food supply. Food control agencies throughout the world need some reliable, simple and rapid methods for the detection foods to ensure free choice of consumer and to enforce labeling. The DNA comet assay offers great potential as a rapid tool to screen irradiated and unirradiated samples of several kinds of foods. In the present study samples of fresh and frozen beef has investigated for the detection of irradiation treatment. The samples were subjected to radiation doses of 0,4.5 and 7 KGy and were stored in freezer before analysis. The cells were extracted into cold PBS solutions, embedded into agarose gel on microscope slides, lysed and eletrophoressed at a voltage of 2V/cm for 2 minutes. The fragmented DNA as a irradiation treatment was stretched in the gel producing the dose dependent comets. These comets were visible using a simple transmission microscope after silver staining. The controlled and irradiation samples of meat were clearly distinguishable on the basis of the stained patterns of DNA in from of round or conical intact cells for unirradiated samples or in from of comets for irradiated samples. It is therefore concluded that DNA comet Assay offers a potential to screen unirradiated and irradiated meat samples. (author)

Detection of radiation treatment of meat by novel techniques of DNA comet assay

International Nuclear Information System (INIS)

Khan, A.A.; Khan, H.M.

2002-01-01

Treatment of food to ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests and to extend shelf life; thereby contributing to safer and more plentiful food supply. Food control agencies throughout the world need some reliable, simple and rapid methods for detection of irradiated foods to ensure free choice of consumer and to enforce labeling. The DNA comet assay offers great potential as a rapid tool to screen irradiated and unirradiated samples of several kinds of foods. In the present study, frozen beef has been investigated for detection of irradiation treatment. The samples were subjected to radiation doses of 0,4,5 and 7.0 kGy and were stored in freezer before analysis. The cells were extracted into cold PBS solutions, embedded into the agarose gel on microscope slides, lysed and electrophoressed at a voltage of 2v/cm for 2 min. The fragmented DNA as a result of irradiation treatment was stretched in the gel producing the dose dependent comets. These comets were visible using a simple transmission microscope after silver staining. The controlled and irradiated samples of meat were clearly distinguishable on the basis of the stained patterns of DNA in form of round or conical intact cells for unirradiated samples or in form of comets for irradiated samples. It is therefore, concluded that 'DNA Comet Assay' offers a potential to screen unirradiated and irradiated meat samples. (author)

Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification.

Science.gov (United States)

Eguiara, Arrate; Holgado, Olaia; Beloqui, Izaskun; Abalde, Leire; Sanchez, Yolanda; Callol, Carles; Martin, Angel G

2011-11-01

The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.

H5N1-SeroDetect EIA and rapid test: a novel differential diagnostic assay for serodiagnosis of H5N1 infections and surveillance.

Science.gov (United States)

Khurana, Surender; Sasono, Pretty; Fox, Annette; Nguyen, Van Kinh; Le, Quynh Mai; Pham, Quang Thai; Nguyen, Tran Hien; Nguyen, Thanh Liem; Horby, Peter; Golding, Hana

2011-12-01

Continuing evolution of highly pathogenic (HP) H5N1 influenza viruses in wild birds with transmission to domestic poultry and humans poses a pandemic threat. There is an urgent need for a simple and rapid serological diagnostic assay which can differentiate between antibodies to seasonal and H5N1 strains and that could provide surveillance tools not dependent on virus isolation and nucleic acid technologies. Here we describe the establishment of H5N1 SeroDetect enzyme-linked immunosorbent assay (ELISA) and rapid test assays based on three peptides in HA2 (488-516), PB1-F2 (2-75), and M2e (2-24) that are highly conserved within H5N1 strains. These peptides were identified by antibody repertoire analyses of H5N1 influenza survivors in Vietnam using whole-genome-fragment phage display libraries (GFPDLs). To date, both platforms have demonstrated high levels of sensitivity and specificity in detecting H5N1 infections (clade 1 and clade 2.3.4) in Vietnamese patients as early as 7 days and up to several years postinfection. H5N1 virus-uninfected individuals in Vietnam and the United States, including subjects vaccinated with seasonal influenza vaccines or with confirmed seasonal virus infections, did not react in the H5N1-SeroDetect assays. Moreover, sera from individuals vaccinated with H5N1 subunit vaccine with moderate anti-H5N1 neutralizing antibody titers did not react positively in the H5N1-SeroDetect ELISA or rapid test assays. The simple H5N1-SeroDetect ELISA and rapid tests could provide an important tool for large-scale surveillance for potential exposure to HP H5N1 strains in both humans and birds.

Considerations for Task Analysis Methods and Rapid E-Learning Development Techniques

Directory of Open Access Journals (Sweden)

Dr. Ismail Ipek

2014-02-01

Full Text Available The purpose of this paper is to provide basic dimensions for rapid training development in e-learning courses in education and business. Principally, it starts with defining task analysis and how to select tasks for analysis and task analysis methods for instructional design. To do this, first, learning and instructional technologies as visions of the future were discussed. Second, the importance of task analysis methods in rapid e-learning was considered, with learning technologies as asynchronous and synchronous e-learning development. Finally, rapid instructional design concepts and e-learning design strategies were defined and clarified with examples, that is, all steps for effective task analysis and rapid training development techniques based on learning and instructional design approaches were discussed, such as m-learning and other delivery systems. As a result, the concept of task analysis, rapid e-learning development strategies and the essentials of online course design were discussed, alongside learner interface design features for learners and designers.

Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay.

Science.gov (United States)

Chen, Jingfang; Zhang, Rusheng; Ou, Xinhua; Yao, Dong; Huang, Zheng; Li, Linzhi; Sun, Biancheng

2017-06-01

A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found. Copyright © 2017 Elsevier Ltd. All rights reserved.

Automated immunoradiometric assay of thyrotrophin (TSH) in dried blood filter paper spots

Energy Technology Data Exchange (ETDEWEB)

John, R.; Woodhead, J.S. (Welsh National School of Medicine, Cardiff (UK))

1982-11-10

An immunoradiometric two-site assay for thyrotrophin (TSH) in dried blood filter paper spots is described. The assay is automated by means of the Kemtek 3000 automated immunoassay system. The technique uses a 6.0 mm disc punched from the dried blood samples collected as part of the screening programme for phenylketonuria. The method is sensitive and precise, and results correlate well with those obtained in TSH assays of serum samples. The procedure is rapid, results being available within 24 h of receipt of samples. Of 25204 specimens so far screened by this assay, 99.9% have TSH levels less than 15 mU/l. One false positive result has been obtained and six confirmed cases of neonatal hypothyroidism detected, giving a prevalence of 1 in 4200.

Development of Techniques for Spent Fuel Assay – Differential Dieaway Final Report

Energy Technology Data Exchange (ETDEWEB)

Swinhoe, Martyn Thomas [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Goodsell, Alison [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Ianakiev, Kiril Dimitrov [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Iliev, Metodi [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Desimone, David J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Rael, Carlos D. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Henzl, Vladimir [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Polk, Paul John [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2016-07-28

This report summarizes the work done under a DNDO R&D funded project on the development of the differential dieaway method to measure plutonium in spent fuel. There are large amounts of plutonium that are contained in spent fuel assemblies, and currently there is no way to make quantitative non-destructive assay. This has led NA24 under the Next Generation Safeguards Initiative (NGSI) to establish a multi-year program to investigate, develop and implement measurement techniques for spent fuel. The techniques which are being experimentally tested by the existing NGSI project do not include any pulsed neutron active techniques. The present work covers the active neutron differential dieaway technique and has advanced the state of knowledge of this technique as well as produced a design for a practical active neutron interrogation instrument for spent fuel. Monte Carlo results from the NGSI effort show that much higher accuracy (1-2%) for the Pu content in spent fuel assemblies can be obtained with active neutron interrogation techniques than passive techniques, and this would allow their use for nuclear material accountancy independently of any information from the operator. The main purpose of this work was to develop an active neutron interrogation technique for spent nuclear fuel.

Logging technique for assaying for uranium in rocks

International Nuclear Information System (INIS)

Givens, W.W.

1973-01-01

A uranium exploration technique is described for determining the uranium content of a formation traversed by borehole. A delayed fission neutron assay log is obtained by irradiating the formation with repetitive bursts of fast neutrons and detecting delayed neutrons resulting from neutron fission of uranium at time intervals between the fast neutron bursts and after dissipation of the neutrons originating in the bursts. In addition, a response log is obtained by irradiating the formation with a source of fast neutrons whereby the neutrons from this source are moderated in the formation to lower energy levels and are subject to absorption. Secondary radiation attendant to these lower energy neutrons is recorded in order to obtain a log representative of the response of the formation to moderation and absorption of the neutrons. The two logs thus obtained are correlated in order to determine a corrected value of uranium content of the formation. (author)

Uranium solution mining cost estimating technique: means for rapid comparative analysis of deposits

International Nuclear Information System (INIS)

Anon.

1978-01-01

Twelve graphs provide a technique for determining relative cost ranges for uranium solution mining projects. The use of the technique can provide a consistent framework for rapid comparative analysis of various properties of mining situations. The technique is also useful to determine the sensitivities of cost figures to incremental changes in mining factors or deposit characteristics

Radioreceptor opioid assay

International Nuclear Information System (INIS)

Miller, R.J.; Chang, K.-J.

1981-01-01

A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles

Directory of Open Access Journals (Sweden)

Qi H

2011-11-01

Full Text Available Hui Qi1, Zhen Zhong1, Han-Xin Zhou1, Chun-Yan Deng1, Hai Zhu2, Jin-Feng Li2, Xi-Li Wang2, Fu-Rong Li1,31Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People's Hospital, Jinan University, 2Shenzhen Bioeasy Biotechnologies Co, Ltd, 3Shenzhen Institute of Gerontology, Shenzhen, People's Republic of ChinaBackground: Escherichia coli O157:H7 (E. coli O157:H7 is an important pathogenic bacterium that threatens human health. A rapid, simple, highly sensitive, and specific method for the detection of E. coli O157:H7 is necessary.Methods: In the present study, immunomagnetic nanoparticles (IMPs were prepared with nanopure iron as the core, coated with E. coli O157:H7 polyclonal antibodies. These IMPs were used in combination with immunochromatographic assay (ICA and used to establish highly sensitive and rapid kits (IMPs+ICA to detect E. coli O157:H7. The kits were then used to detect E. coli O157:H7 in 150 food samples and were compared with conventional ICA to evaluate their efficacy.Results: The average diameter of IMPs was 56 nm and the amount of adsorbed antibodies was 106.0 µg/mg. The sensitivity of ICA and IMPs+ICA was 105 colony-forming units/mL and 103 CFUs/mL, respectively, for purified E. coli O157:H7 solution. The sensitivity of IMPs+ICA was increased by two orders, and its specificity was similar to ICA.Conclusion: The kits have the potential to offer important social and economic benefits in the screening, monitoring, and control of food safety.Keywords: colloidal gold, immunomagnetic nanoparticles, Escherichia coli O157:H7, immunochromatographic assay

Random assay in radioimmunoassay: Feasibility and application compared with batch assay

Energy Technology Data Exchange (ETDEWEB)

Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

2016-12-15

The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature

International Nuclear Information System (INIS)

Dawson, R.M.

1986-01-01

Benzodiazepine receptors of rat cerebellum were assayed with [ 3 H]-labeled flunitrazepam at 37 0 C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37 0 C until just prior to filtration

A rapid and cell-free assay to test the activity of lynch syndrome-associated MSH2 and MSH6 missense variants

DEFF Research Database (Denmark)

Drost, Mark; Zonneveld, José B M; van Hees, Sandrine

2012-01-01

amino acid alterations. The pathogenicity of these variants of uncertain significance (VUS) is difficult to assess, precluding diagnosis of carriers and their relatives. Here we present a rapid cell-free assay to investigate MMR activity of MSH2 or MSH6 VUS. We used this assay to analyze a series of MSH......2 and MSH6 VUS, selected from the Leiden Open Variation Database. Whereas a significant fraction of the MSH2 VUS has lost MMR activity, suggesting pathogenicity, the large majority of the MSH6 VUS appears MMR proficient. We anticipate that this assay will be an important tool in the development...... of a comprehensive and widely applicable diagnostic procedure for LS-associated VUS....

Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

Directory of Open Access Journals (Sweden)

Adeline Bidault

2015-12-01

Full Text Available The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD in the Manila clam Venerupis (=Ruditapes philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW or extrapallial fluids (EF samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

Performance and diagnostic usefulness of commercially available enzyme linked immunosorbent assay and rapid kits for detection of HIV, HBV and HCV in India.

Science.gov (United States)

Maity, Susmita; Nandi, Srijita; Biswas, Subrata; Sadhukhan, Salil Kumar; Saha, Malay Kumar

2012-11-26

HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.); ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd.) gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd.), Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd.) did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100%) except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.). The kit efficiency didn't vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p bank. For availability of quality commercial diagnostic assays, evaluation of kit may be helpful.

Use of refractometry and colorimetry as field methods to rapidly assess antimalarial drug quality.

Science.gov (United States)

Green, Michael D; Nettey, Henry; Villalva Rojas, Ofelia; Pamanivong, Chansapha; Khounsaknalath, Lamphet; Grande Ortiz, Miguel; Newton, Paul N; Fernández, Facundo M; Vongsack, Latsamy; Manolin, Ot

2007-01-04

The proliferation of counterfeit and poor-quality drugs is a major public health problem; especially in developing countries lacking adequate resources to effectively monitor their prevalence. Simple and affordable field methods provide a practical means of rapidly monitoring drug quality in circumstances where more advanced techniques are not available. Therefore, we have evaluated refractometry, colorimetry and a technique combining both processes as simple and accurate field assays to rapidly test the quality of the commonly available antimalarial drugs; artesunate, chloroquine, quinine, and sulfadoxine. Method bias, sensitivity, specificity and accuracy relative to high-performance liquid chromatographic (HPLC) analysis of drugs collected in the Lao PDR were assessed for each technique. The HPLC method for each drug was evaluated in terms of assay variability and accuracy. The accuracy of the combined method ranged from 0.96 to 1.00 for artesunate tablets, chloroquine injectables, quinine capsules, and sulfadoxine tablets while the accuracy was 0.78 for enterically coated chloroquine tablets. These techniques provide a generally accurate, yet simple and affordable means to assess drug quality in resource-poor settings.

New Application of the Comet Assay

Science.gov (United States)

Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

2011-01-01

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

Rapid, low-cost fluorescent assay of β-lactamase-derived antibiotic resistance and related antibiotic susceptibility

Science.gov (United States)

Erdem, S. Sibel; Khan, Shazia; Palanisami, Akilan; Hasan, Tayyaba

2014-10-01

Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of β-lactamase resistance (the most common form of AR) that uses a modified β-lactam structure decorated with two fluorophores quenched due to their close proximity. When the β-lactam core is cleaved by β-lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the β-lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of β-lactamase resistance, both for epidemiological purposes as well as individualized patient care.

Nondestructive assay uncertainties - present status and future possibilities

International Nuclear Information System (INIS)

Parker, J.L.; Ensslin, N.

1989-01-01

Nondestructive assay (NDA) techniques play an important role in nuclear safeguards by providing rapid, noninvasive measurements of many nuclear materials. The development of these techniques has proceeded in parallel with the increasing need for near-real-time accountability and the increasing use of automation and robotics to save time and labor in all aspects of nuclear materials processing. Gamma-ray counting, neutron counting, and calorimetry are the most common NDA techniques. This paper discusses the random and systematic measurement uncertainties associated with the first two. It is worth nothing that the random and systematic error components encountered in NDA often have a very different origin from those encountered in destructive analysis; the random component in NDA, for example, is dominated by counting statistics

Impact of a Rapid Herpes Simplex Virus PCR Assay on Duration of Acyclovir Therapy.

Science.gov (United States)

Van, Tam T; Mongkolrattanothai, Kanokporn; Arevalo, Melissa; Lustestica, Maryann; Dien Bard, Jennifer

2017-05-01

Herpes simplex virus (HSV) infections of the central nervous system (CNS) are associated with significant morbidity and mortality rates in children. This study assessed the impact of a direct HSV (dHSV) PCR assay on the time to result reporting and the duration of acyclovir therapy for children with signs and symptoms of meningitis and encephalitis. A total of 363 patients with HSV PCR results from cerebrospinal fluid (CSF) samples were included in this retrospective analysis, divided into preimplementation and postimplementation groups. For the preimplementation group, CSF testing was performed using a laboratory-developed real-time PCR assay; for the postimplementation group, CSF samples were tested using a direct sample-to-answer assay. All CSF samples were negative for HSV. Over 60% of patients from both groups were prescribed acyclovir. The average HSV PCR test turnaround time for the postimplementation group was reduced by 14.5 h (23.6 h versus 9.1 h; P < 0.001). Furthermore, 79 patients (43.6%) in the postimplementation group had dHSV PCR results reported <4 h after specimen collection. The mean time from specimen collection to acyclovir discontinuation was 17.1 h shorter in the postimplementation group (31.1 h versus 14 h; P < 0.001). The median duration of acyclovir therapy was also significantly reduced in the postimplementation group (29.2 h versus 14.3 h; P = 0.01). Our investigation suggests that implementation of rapid HSV PCR testing can decrease turnaround times and the duration of unnecessary acyclovir therapy. Copyright © 2017 American Society for Microbiology.

Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos

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Mulero Victoriano

2008-10-01

Full Text Available Abstract Background The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism. Results Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the in vivo response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase and inducible (NF-κB-dependent promoters. The flexibility of this assay is demonstrated by induction of the NF-κB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown. Conclusion This assay has several advantages compared with the classical in vitro (cell lines and in vivo (transgenic mice approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.

Nondestructive assay of special nuclear material for uranium fuel-fabrication facilities

International Nuclear Information System (INIS)

Smith, H.A. Jr.; Schillebeeckx, P.

1997-01-01

A high-quality materials accounting system and effective international inspections in uranium fuel-fabrication facilities depend heavily upon accurate nondestructive assay measurements of the facility's nuclear materials. While item accounting can monitor a large portion of the facility inventory (fuel rods, assemblies, storage items), the contents of all such items and mass values for all bulk materials must be based on quantitative measurements. Weight measurements, combined with destructive analysis of process samples, can provide highly accurate quantitative information on well-characterized and uniform product materials. However, to cover the full range of process materials and to provide timely accountancy data on hard-to-measure items and rapid verification of previous measurements, radiation-based nondestructive assay (NDA) techniques play an important role. NDA for uranium fuel fabrication facilities relies on passive gamma spectroscopy for enrichment and U isotope mass values of medium-to-low-density samples and holdup deposits; it relies on active neutron techniques for U-235 mass values of high-density and heterogeneous samples. This paper will describe the basic radiation-based nondestructive assay techniques used to perform these measurements. The authors will also discuss the NDA measurement applications for international inspections of European fuel-fabrication facilities

Safeguards and Non-destructive Assay

International Nuclear Information System (INIS)

Carchon, R.; Bruggeman, M.

2001-01-01

SCK-CEN's programme on safeguards and non-destructive assay includes: (1) various activities to assure nuclear materials accountancy; (2) contributes to the implementation of Integrated Safeguards measures in Belgium and to assist the IAEA through the Belgian Support Programme; (3) renders services to internal and external customers in the field of safeguards; (4) improves passive neutron coincidence counting techniques for waste assay and safeguards verification measurements by R and D on correlation algorithms implemented via software or dedicated hardware; (5) improves gamma assay techniques for waste assay by implementing advanced scanning techniques and different correlation algorithms; and (6) develops numerical calibration techniques. Major achievements in these areas in 2000 are reported

Micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

Energy Technology Data Exchange (ETDEWEB)

Layton, G T [Royal Infirmary, Manchester (UK)

1980-10-01

Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10/sup -3/ units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus.

Techniques for rapid determination of effects of synergy between radionuclides and pollutants

International Nuclear Information System (INIS)

Saas, A.; Grauby, A.

1975-01-01

The authors present a number of chromatographic techniques for rapid determination of synergy between radionuclides and various compounds in water. The first technique consists in studying how the chemical equilibrium of iodine varies in the presence of various organic and mineral compounds. The second makes it possible to define the effects of synergy within a given hydrographic basin. A third technique deals with the effects of synergy in ground water in the presence of various types of irrigation water. Finally, to complete this set of techniques, the authors define the mobility potential of a radionuclide in a given aqueous effluent

Role of genotype® mycobacterium common mycobacteria/additional species assay for rapid differentiation between Mycobacterium tuberculosis complex and different species of non-tuberculous mycobacteria

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Amresh Kumar Singh

2013-01-01

Full Text Available Background: Mycobacterium tuberculosis complex (MTBC and non-tuberculous mycobacteria (NTM may or may not have same clinical presentations, but the treatment regimens are always different. Laboratory differentiation between MTBC and NTM by routine methods are time consuming and cumbersome to perform. We have evaluated the role of GenoType® Mycobacterium common mycobacteria/additional species (CM/AS assay for differentiation between MTBC and different species of NTM in clinical isolates from tuberculosis (TB cases. Materials and Methods: A total of 1080 clinical specimens were collected from January 2010 to June 2012. Diagnosis was performed by Ziehl-Neelsen staining followed by culture in BacT/ALERT 3D system (bioMerieux, France. A total of 219 culture positive clinical isolates (BacT/ALERT® MP cultures were selected for differentiation by p-nitrobenzoic acid (PNB sensitivity test as and BIO-LINE SD Ag MPT64 TB test considering as the gold standard test. Final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType® Mycobacterium CM/AS assay (Hain Lifescience, Nehren, Germany. Results: Out of 219 BacT/ALERT® MP culture positive isolates tested by PNB as 153 MTBC (69.9% and by GenoType® Mycobacterium CM/AS assay as 159 (72.6% MTBC and remaining 60 (27.4% were considered as NTM species. The GenoType® Mycobacterium CM/AS assay was proved 99.3% sensitive and 98.3% specific for rapid differentiation of MTBC and NTM. The most common NTM species were; Mycobacterium fortuitum 20 (33.3% among rapid growing mycobacteria and Mycobacterium intracellulare 11 (18.3% among slow growing mycobacteria. Conclusion: The GenoType® Mycobacterium assay makes rapid and accurate identification of NTM species as compared with different phenotypic and molecular diagnostic tool and helps in management of infections caused by different mycobacteria.

A field-practical assay for rapid detection of chlorophenols

Energy Technology Data Exchange (ETDEWEB)

Washburn, K.S.; Phillips, T.D. [Texas A and M Univ., College Station, TX (United States). Dept. of Veterinary Anatomy and Public Health

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).

Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India

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Anand Kumar Maurya

2013-01-01

Full Text Available Background: The problem of multi-drug resistance tuberculosis (MDR-TB is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST of MDR-TB cases in Northern India. Materials and Methods: A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Results: Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%, 52 (94.5% and 17 (30.9% strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05. The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3% in S531L, 52 (94.5% in S315T1 and 11 (20% in C15T regions respectively (P < 0.05. Conclusions: Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

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

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Sascha D Braun

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

Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria.

Science.gov (United States)

Zhang, D F; Zhang, Q Q; Li, A H

2014-11-01

Species of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (mPCR) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus-specific primers instead of species-specific ones, the current mPCR covered much more target bacterial species compared with previously reported species-specific mPCR methods. The specificity of the four putative genus-specific primers was validated experimentally while used exclusively (uniplex PCR) or combined (mPCR) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: Aeromonas (875 bp), Vibrio (524 bp), Edwardsiella (302 bp) and Streptococcus (197 bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The mPCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The mPCR detection limits for each target bacterial genera were 50 colony-forming units (CFU) in pure culture and 100 CFU in fish tissue samples. In conclusion, the mPCR assay was proven to be a powerful alternative to the conventional culture-based method, given its rapid, specific, sensitive and reliable detection of target pathogens. The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus-specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The mPCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species-specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This m

Rapid small intestinal permeability assay based on riboflavin and lactulose detected by bis-boronic acid appended benzyl viologens.

Science.gov (United States)

Resendez, Angel; Abdul Halim, Md; Landhage, Caroline M; Hellström, Per M; Singaram, Bakthan; Webb, Dominic-Luc

2015-01-15

Although organoboronic acids are efficient high-throughput sugar sensors, they have not been pursued for gut permeability studies. A modification of the lactulose/mannitol assay is described by which small intestinal permeability is assessed at the time of urine collection using a lactulose/riboflavin ratio. Volunteers ingested 50mg riboflavin and either 5 g mannitol or 10 g lactulose. Urine was collected for 6 hrs. Riboflavin was assayed by autofluorescence. Riboflavin was removed by C18 solid phase extraction. Lactulose and mannitol were then assayed using 1,1'-bis(2-boronobenzyl)-4,4'-bipyridinium (4,4'oBBV) coupled to the fluorophore HPTS. The temporal profile over 6 hrs for riboflavin paralleled mannitol. Riboflavin recovery in urine was 11.1 ± 1.9 % (mean ± SEM, n=7), similar to mannitol. There was selective binding of 4,4'oBBV to lactulose, likely involving cooperativity between the fructose and galactose moieties. Lower limits of detection and quantification were 90 and 364 μM. The lactulose assay was insensitive to other permeability probes (e.g., sucrose, sucralose) while tolerating glucose or lactose. This assay can be adapted to automated systems. Stability of 4,4'oBBV exceeds 4 years. Riboflavin measured by autofluorescence combined with lactulose measured with 4,4'oBBV represents a useful new chemistry for rapid measurement of intestinal permeability with excellent stability, cost and throughput benefits. Copyright © 2014. Published by Elsevier B.V.

A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

Science.gov (United States)

Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

2012-01-01

ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

Science.gov (United States)

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

Science.gov (United States)

Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

2016-08-01

Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid assay of intrinsic radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes

International Nuclear Information System (INIS)

Ozsahin, Mahmut; Ozsahin, Huelya; Yuquan, Shi; Larsson, Boerje; Wuergler, Friedrich E.; Crompton, Nigel E. A.

1997-01-01

Purpose: An assay for radiosensitivity has numerous applications in the clinic. Avoidance of acute responses, prediction of normal tissue toxicity, and individualization of patient radiotherapy are included among these. We have developed a rapid assay (about 24 h) able to predict intrinsic radiosensitivity of CD4 and CD8 T-lymphocytes based on radiation-induced apoptosis. Methods and Materials: Fresh blood samples (1-2 ml in heparinized tubes) were irradiated with 0-, 2-, and 8-Gy X rays at a dose rate of approximately 3 Gy/min. Following irradiation, the cells were collected and prepared for flow-cytometric analysis and cell sorting. In conjunction with the CellQuest software available with the FACSVantage cell sorter (Becton-Dickinson), two T-lymphocyte types were analyzed on the basis of their cell-specific antigens (CD4 and CD8), and DNA was stained with DAPI. Following the separation of these cell types, radiation-induced cell death was assessed. Cytotoxicity was characterized by gradual degradation of internucleosomal DNA which results in a sub-G1 peak on the DNA histogram, and by the associated loss of surface antigens causing an intermediate positive peak in the antibody histogram. Using the assay, we investigated the interdonor variation in a cohort of 45 healthy adult blood donors and 5 children [one had immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF), and one had ataxia telangiectasia (AT)]. Intradonor variation was assessed with 10 different experiments from a single donor. Results: CD4 and CD8 T-lymphocyte radiosensitivities were correlated (r 0.63 and 0.65 for 2 and 8 Gy, respectively) in 45 adult donors. Both for CD4 and CD8 cells, 2 and 8 Gy irradiation responses showed a good correlation (r 0.77 for both). Interdonor variation was significantly higher than intradonor variation (p < 0.0005) for all CD4 and CD8 data. We observed a decrease in the antigen fluorescence of dying cells, a phenomenon referred to as antigen

A novel technique for presurgical nasoalveolar molding using computer-aided reverse engineering and rapid prototyping.

Science.gov (United States)

Yu, Quan; Gong, Xin; Wang, Guo-Min; Yu, Zhe-Yuan; Qian, Yu-Fen; Shen, Gang

2011-01-01

To establish a new method of presurgical nasoalveolar molding (NAM) using computer-aided reverse engineering and rapid prototyping technique in infants with unilateral cleft lip and palate (UCLP). Five infants (2 males and 3 females with mean age of 1.2 w) with complete UCLP were recruited. All patients were subjected to NAM before the cleft lip repair. The upper denture casts were recorded using a three-dimensional laser scanner within 2 weeks after birth in UCLP infants. A digital model was constructed and analyzed to simulate the NAM procedure with reverse engineering software. The digital geometrical data were exported to print the solid model with rapid prototyping system. The whole set of appliances was fabricated based on these solid models. Laser scanning and digital model construction simplified the NAM procedure and estimated the treatment objective. The appliances were fabricated based on the rapid prototyping technique, and for each patient, the complete set of appliances could be obtained at one time. By the end of presurgical NAM treatment, the cleft was narrowed, and the malformation of nasoalveolar segments was aligned normally. We have developed a novel technique of presurgical NAM based on a computer-aided design. The accurate digital denture model of UCLP infants could be obtained with laser scanning. The treatment design and appliance fabrication could be simplified with a computer-aided reverse engineering and rapid prototyping technique.

New Strategies for Powder Compaction in Powder-based Rapid Prototyping Techniques

NARCIS (Netherlands)

Budding, A.; Vaneker, Thomas H.J.

2013-01-01

In powder-based rapid prototyping techniques, powder compaction is used to create thin layers of fine powder that are locally bonded. By stacking these layers of locally bonded material, an object is made. The compaction of thin layers of powder mater ials is of interest for a wide range of

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

Science.gov (United States)

Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

2014-06-01

Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

An automated immunoradiometric assay of thyrotrophin (TSH) in dried blood filter paper spots

International Nuclear Information System (INIS)

John, R.; Woodhead, J.S.

1982-01-01

An immunoradiometric two-site assay for thyrotrophin (TSH) in dried blood filter paper spots is described. The assay is automated by means of the Kemtek 3000 automated immunoassay system. The technique uses a 6.0 mm disc punched from the dried blood samples collected as part of the screening programme for phenylketonuria. The method is sensitive and precise, and results correlate well with those obtained in TSH assays of serum samples. The procedure is rapid, results being available within 24 h of receipt of samples. Of 25204 specimens so far screened by this assay, 99.9% have TSH levels less than 15 mU/l. One false positive result has been obtained and six confirmed cases of neonatal hypothyroidism detected, giving a prevalence of 1 in 4200. (Auth.)

Development of Colloidal Gold-Based Immunochromatographic Assay for Rapid Detection of Goose Parvovirus

Directory of Open Access Journals (Sweden)

Xianglong Yu

2018-05-01

Full Text Available Goose parvovirus (GPV remains as a worldwide problem in goose industry. For this reason, it is necessary to develop a new diagnostic approach that is easier and faster than conventional tests. A rapid immunochromatographic assay based on antibody colloidal gold nanoparticles specific to GPV was developed for the detection of GPV in goose allantoic fluid and supernatant of tissue homogenate. The monoclonal antibodies (Mab was produced by immunizing the BALB/c mice with purified GPV suspension, and the polyclonal antibody (pAb was produced by immunizing the rabbits with recombinant VP3 protein. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with Mab against GPV. The optimal concentrations of the coating antibody and capture antibody were determined to be 1.6 mg/ml and 9 μg/ml. With visual observation, the lower limit was found to be around 1.2 μg/ml. Common diseases of goose were tested to evaluate the specificity of the immune colloidal gold (ICG strip, and no cross-reaction was observed. The clinical detection was examined by carrying out the ICG strip test with 92 samples and comparing the results of these tests with those obtained via agar diffusion test and polymerase chain reaction (PCR test. Therefore, the ICG strip test was a sufficiently sensitive and accurate detection method for a rapid screening of GPV.

Immunocapture loop-mediated isothermal amplification assays for the detection of canine parvovirus.

Science.gov (United States)

Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

2017-11-01

A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10 -1 , 10 -1 , and 10 -1 TCID 50 /mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV. Copyright © 2017 Elsevier B.V. All rights reserved.

Real‑time, fast neutron detection for stimulated safeguards assay

International Nuclear Information System (INIS)

Joyce, Malcolm J.; Adamczyk, Justyna; Plenteda, Romano; Aspinall, Michael D.; Cave, Francis D.

2015-01-01

The advent of low‑hazard organic liquid scintillation detectors and real‑time pulse‑shape discrimination (PSD) processing has suggested a variety of modalities by which fast neutrons, as opposed to neutrons moderated prior to detection, can be used directly to benefit safeguards needs. In this paper we describe a development of a fast‑neutron based safeguards assay system designed for the assessment of 235 U content in fresh fuel. The system benefits from real‑time pulse‑shape discrimination processing and auto‑calibration of the detector system parameters to ensure a rapid and effective set‑up protocol. These requirements are essential in optimising the speed and limit of detection of the fast neutron technique, whilst minimising the intervention needed to perform the assay.

Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation

NARCIS (Netherlands)

Dittmer, W.U.; Kievit, de P.; Prins, M.W.J.; Vissers, J.L.M.; Mersch, M.E.C.; Martens, M.F.W.C.

2008-01-01

A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions

Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals

DEFF Research Database (Denmark)

Vinggaard, Anne; Jørgensen, E.C.B.; Larsen, John Christian

1999-01-01

Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrog......Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential...... antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were...... calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands...

A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

International Nuclear Information System (INIS)

Layton, G.T.

1980-01-01

Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10 -3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk: A Modern Laboratory Project

Science.gov (United States)

Inda, Luis A.; Razquín, Pedro; Lampreave, Fermín; Alava, María A.; Calvo, Miguel

1998-12-01

Specificity, sensitivity, and experimental simplicity make the immunoenzymatic assay suitable for a variety of laboratories dedicated to diverse activities such as research, quality control in food analysis, or clinical biochemistry. In these assays, the antibody that specifically recognizes the antigen is covalently attached to an enzyme. Once the antigen-antibody immunocomplex is formed, the enzymatic reaction gives a colored product that allows the detection of the initial antigen. The aim of this work was the design of a new laboratory project appropriate for use in courses of biochemistry, immunochemistry, or analytical chemistry. The assay described here detects the presence of cow milk in milk of other species. The main application is the detection of cow milk in sheep milk and cheese. Specific proteins, immunoglobulins (IgG) of the fraudulent bovine milk, are specifically recognized and retained by antibodies immobilized on a membrane. The binding of a second antibody covalently attached to horseradish peroxidase (HRP) allows the development of a visible signal. Thus, students can rapidly detect milk adulterations using a specific, sensitive, and safe experimental approach. The experiment allows students to apply their theoretical knowledge, resulting in a stimulating experience of solving a real problem during a 4-hour laboratory period.

Rapid Detection of Staphylococcus aureus and Methicillin-Resistant S. aureus in Atopic Dermatitis by Using the BD Max StaphSR Assay.

Science.gov (United States)

Lee, Mi Kyung; Park, Kui Young; Jin, Taewon; Kim, Ju Hee; Seo, Seong Jun

2017-07-01

Eczematous lesions of atopic dermatitis (AD) patients are known to be a source of Staphylococcus aureus (SA) transmission and might be a reservoir for community-associated methicillin-resistant SA (MRSA). The BD Max StaphSR (BD-SR) is a fully automated, multiplex real-time PCR assay for the direct detection and differentiation of SA and MRSA from nasal swab samples. We evaluated the detection rates of SA and MRSA from skin lesions of outpatients with AD using the BD-SR assay, and determined the usefulness of the BD-SR assay. A total of 244 skin swab samples (skin lesions of 213 outpatients with AD and normal skin of 31 healthy controls) were tested directly by using the BD-SR assay. Of the 213 samples from patients with AD, 69 (32.4%) were positive for SA, 6 (8.7%) of which were positive for MRSA. Only 1 (3.2%) of 31 samples from healthy controls was positive for SA. The BD-SR assay is effective for the rapid detection of SA and MRSA from skin swab samples, which can provide important information for managing patients with AD and preventing the spread of MRSA. © The Korean Society for Laboratory Medicine.

A locked nucleic acid (LNA-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

Directory of Open Access Journals (Sweden)

Lingxiang Zhu

Full Text Available Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA-based quantitative real-time PCR (LNA-qPCR method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4% were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

Impacts of Extraction Methods in the Rapid Determination of Atrazine Residues in Foods using Supercritical Fluid Chromatography and Enzyme-Linked Immunosorbent Assay: Microwave Solvent vs. Supercritical Fluid Extractions

Directory of Open Access Journals (Sweden)

Mohamed H. El-Saeid

2005-01-01

Full Text Available It is an accepted fact that many food products that we eat today have the possibility of being contaminated by various chemicals used from planting to processing. These chemicals have been shown to cause illnesses for which some concerned government agencies have instituted regulatory mechanisms to minimize the risks and the effects on humans. It is for these concerns that reliable and accurate rapid determination techniques are needed to effect proper regulatory standards for the protection of people's nutritional health. This paper, therefore, reports the comparative evaluation of the extraction methods in the determination of atrazine (commonly used in agricultural as a herbicide residues in foods using supercritical fluid chromatography (SFC and enzyme-linked immunosorbent assay (ELISA techniques. Supercritical fluid extraction (SFE and microwave solvent extraction (MSE methods were used to test samples of frozen vegetables, fruit juice, and jam from local food markets in Houston. Results showed a high recovery percentage of atrazine residues using supercritical fluid coupled with ELISA and SFC than with MSE. Comparatively, however, atrazine was detected 90.9 and 54.5% using SFC and ELISA techniques, respectively. ELISA technique was, however, less time consuming, lower in cost, and more sensitive with low detection limit of atrazine residues than SFC technique.

A novel and rapid microbiological assay for ciprofloxacin hydrochloride

Directory of Open Access Journals (Sweden)

Edith Cristina Laignier Cazedey

2013-10-01

Full Text Available The present work reports a simple, fast and sensitive microbiological assay applying the turbidimetric method for the determination of ciprofloxacin hydrochloride (CIPRO HCl in ophthalmic solutions. The validation method yielded good results and included excellent linearity, precision, accuracy and specificity. The bioassay is based on the inhibitory effect of CIPRO HCl upon the strain of Staphylococcus epidermidis ATCC 12228 used as the test microorganism. The results were treated statistically by analysis of variance (ANOVA and were found to be linear (r=0.9994, in the range of 14.0–56.0 µg/mL, precise (intraday RSD%=2.06; interday RSD%=2.30 and accurate (recovery=99.71%. The turbidimetric assay was compared to the UV spectrophotometric and HPLC methods for the same drug. The turbidimetric bioassay described on this paper for determination of ciprofloxacin hydrochloride in ophthalmic solution is an alternative to the physicochemical methods disclosed in the literature and can be used in quality control routine. Keywords: Antibiotics, Fluoroquinolones, Ciprofloxacin hydrochloride, Quality control, Microbiological assay, Turbidimetric method

Rapid assay of plutonium in soils by passive L x-ray counting

International Nuclear Information System (INIS)

Gehrke, R.J.; Putnam, M.H.; Goodwin, S.G.; Kynaston, R.L.

1992-05-01

A technique has been developed to rapidly measure the presence of plutonium in soils, filters, smears, and glass waste forms by measuring the uranium L-shell x-ray emissions associated with the decay of plutonium. In addition, the technique can simultaneously acquire spectra of samples and automatically analyze them for the amount of americium, and gamma-ray emitting activation and fission products present. The samples are counted with a large area, thin-window, n-type Ge spectrometer which is equally efficient for the detection of low energy x-rays (>10 key), as well as high-energy gamma rays (>1 MeV). A 8192-channel analyzer is used to acquire the entire photon spectrum at one time. A dual-energy, time-tagged pulser, that is injected into the test input of the preamplifier to monitor the energy scale, detector resolution, and pulse pile-up will be installed in FY-92. The L x-ray portion of each spectrum is analyzed by a linear least-squares spectral fitting technique originally developed for the analysis of spectra from NaI(Tl) detectors. The gamma-ray portion of each spectrum is analyzed by a standard Ge gamma-ray analysis package. Detection limits (also referred to as lower limits of detection) for plutonium in contaminated soils that have been achieved by this technique are reported

GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

Science.gov (United States)

Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

2015-06-01

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

Real-time RPA assay for rapid detection and differentiation of wild-type pseudorabies and gE-deleted vaccine viruses.

Science.gov (United States)

Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Pang, Xiaoyu; Yuan, Wanzhe

2018-02-15

The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 10 2 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R 2 value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas. Copyright © 2017 Elsevier Inc. All rights reserved.

Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

Energy Technology Data Exchange (ETDEWEB)

Hondroulis, Evangelia; Liu Chang; Li Chenzhong, E-mail: licz@fiu.edu [Nanobioengineering/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, 10555 West Flagler Street, Miami, FL 33174 (United States)

2010-08-06

A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

Science.gov (United States)

Hondroulis, Evangelia; Liu, Chang; Li, Chen-Zhong

2010-08-01

A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

International Nuclear Information System (INIS)

Hondroulis, Evangelia; Liu Chang; Li Chenzhong

2010-01-01

A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

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

International Nuclear Information System (INIS)

Villavicencio, A.L.C.H.; Delincee, H.

1998-01-01

Complete text of publication follows. 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 ionizing radiation causes DNA fragmentation, the analysis of DNA damage might be promising. In fact, DNA fragmentation measured in single cells by agarose gel electrophoresis - DNA Comet Assay - has shown to offer great potential as a rapid tool to detect whether a wide variety of foodstuffs has been radiation processed. However, more work is needed to exploit the full potential of this promising technique. In this paper, the DNA Comet Assay was used to identify exotic meat (boar, jacare and capybara), irradiated with 60 Co gamma-rays. The applied radiation doses were 0, 1.5, 3.0 and 4.5 kGy. Analysis of the DNA migration enable a rapid identification of the radiation treatment

Automation of the dicentric chromosome assay and related assays

International Nuclear Information System (INIS)

Balajee, Adayabalam S.; Dainiak, Nicholas

2016-01-01

Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

Two-monoclonal-antibody sandwich-type assay for thyrotropin, with use of an avidin-biotin separation technique

International Nuclear Information System (INIS)

Odell, W.D.; Griffin, J.; Zahradnik, R.

1986-01-01

We have developed a sensitive, specific, noncompetitive, sandwich-type radioimmunoassay for human thyrotropin (hTSH), which can be performed in 30 min. The assay involves two monoclonal antibodies, selected for high affinity and specificity and also for reaction against antigenic sites on hTSH that are distal from each other. One of these antibodies is labeled with 125 I; the other is conjugated covalently to biotin. Polystyrene beads were also conjugated covalently to biotin. After conjugation, the beads were incubated with avidin. These beads represent a rapid, simple method for separating hTSH-bound antibody from free antibody. The biotin-antibody-hTSH- 125 I-labeled antibody complexes bind to the beads and hTSH concentration is directly related to counts per minute. This assay can detect hTSH at a concentration of 0.06 milli-unit/L in serum

Accuracy of a dual path platform (DPP assay for the rapid point-of-care diagnosis of human leptospirosis.

Directory of Open Access Journals (Sweden)

Scott A Nabity

Full Text Available Diagnosis of leptospirosis by the gold standard serologic assay, the microscopic agglutination test (MAT, requires paired sera and is not widely available. We developed a rapid assay using immunodominant Leptospira immunoglobulin-like (Lig proteins in a Dual Path Platform (DPP. This study aimed to evaluate the assay's diagnostic performance in the setting of urban transmission.We determined test sensitivity using 446 acute and convalescent sera from MAT-confirmed case-patients with severe or mild leptospirosis in Brazil. We assessed test specificity using 677 sera from the following groups: healthy residents of a Brazilian slum with endemic transmission, febrile outpatients from the same slum, healthy blood donors, and patients with dengue, hepatitis A, and syphilis. Three operators independently interpreted visual results without knowing specimen status.The overall sensitivity for paired sera was 100% and 73% for severe and mild disease, respectively. In the acute phase, the assay achieved a sensitivity of 85% and 64% for severe and mild leptospirosis, respectively. Within seven days of illness onset, the assay achieved a sensitivity of 77% for severe disease and 60% for mild leptospirosis. Sensitivity of the DPP assay was similar to that for IgM-ELISA and increased with both duration of symptoms (chi-square regression P = 0.002 and agglutinating titer (Spearman ρ = 0.24, P<0.001. Specificity was ≥93% for dengue, hepatitis A, syphilis, febrile outpatients, and blood donors, while it was 86% for healthy slum residents. Inter-operator agreement ranged from very good to excellent (kappa: 0.82-0.94 and test-to-test reproducibility was also high (kappa: 0.89.The DPP assay performed acceptably well for diagnosis of severe acute clinical leptospirosis and can be easily implemented in hospitals and health posts where leptospirosis is a major public health problem. However, test accuracy may need improvement for mild disease and early stage

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

DEFF Research Database (Denmark)

Hum, S.; Quinn, K.; Brunner, J.

1997-01-01

methods were attributed to methodological differences used in various laboratories. Conclusion Our results indicate that misidentification of C fetus in routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive......Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies. Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme...... by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping...

Integration of electrochemistry in micro-total analysis systems for biochemical assays: recent developments.

Science.gov (United States)

Xu, Xiaoli; Zhang, Song; Chen, Hui; Kong, Jilie

2009-11-15

Micro-total analysis systems (microTAS) integrate different analytical operations like sample preparation, separation and detection into a single microfabricated device. With the outstanding advantages of low cost, satisfactory analytical efficiency and flexibility in design, highly integrated and miniaturized devices from the concept of microTAS have gained widespread applications, especially in biochemical assays. Electrochemistry is shown to be quite compatible with microanalytical systems for biochemical assays, because of its attractive merits such as simplicity, rapidity, high sensitivity, reduced power consumption, and sample/reagent economy. This review presents recent developments in the integration of electrochemistry in microdevices for biochemical assays. Ingenious microelectrode design and fabrication methods, and versatility of electrochemical techniques are involved. Practical applications of such integrated microsystem in biochemical assays are focused on in situ analysis, point-of-care testing and portable devices. Electrochemical techniques are apparently suited to microsystems, since easy microfabrication of electrochemical elements and a high degree of integration with multi-analytical functions can be achieved at low cost. Such integrated microsystems will play an increasingly important role for analysis of small volume biochemical samples. Work is in progress toward new microdevice design and applications.

A rapid method for infectivity titration of Andes hantavirus using flow cytometry.

Science.gov (United States)

Barriga, Gonzalo P; Martínez-Valdebenito, Constanza; Galeno, Héctor; Ferrés, Marcela; Lozach, Pierre-Yves; Tischler, Nicole D

2013-11-01

The focus assay is currently the most commonly used technique for hantavirus titer determination. This method requires an incubation time of between 5 and 11 days to allow the appearance of foci after several rounds of viral infection. The following work presents a rapid Andes virus (ANDV) titration assay, based on viral nucleocapsid protein (N) detection in infected cells by flow cytometry. To this end, an anti-N monoclonal antibody was used that was developed and characterized previously. ANDV N could be detected as early as 6 h post-infection, while viral release was not observed until 24-48 h post-infection. Given that ANDV detection was performed during its first round of infection, a time reduction for titer determination was possible and provided results in only two days. The viral titer was calculated from the percentage of N positive cells and agreed with focus assay titers. Furthermore, the assay was applied to quantify the inhibition of ANDV cell entry by patient sera and by preventing endosome acidification. This novel hantavirus titration assay is a highly quantitative and sensitive tool that facilitates infectivity titration of virus stocks, rapid screening for antiviral drugs, and may be further used to detect and quantify infectious virus in human samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid assay for cell age response to radiation by electronic volume flow cell sorting

International Nuclear Information System (INIS)

Freyer, J.P.; Wilder, M.E.; Raju, M.R.

1987-01-01

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. Sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [ 3 H]thymidine autoradiography of the sorted cell populations, it is demonstrated that resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. Variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. Advantages of this method include: (1) no treatment of the cells is required, thus, this method is noncytotoxic; (2) no cell cycle progression is needed to obtain different cell age compartments; (3) the cell population can be held in complete growth medium at any desired temperature during sorting; (4) a complete radiation age - response assay can be plated in 2 h. Applications of this method are discussed, along with some technical limitations. (author)

Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

Energy Technology Data Exchange (ETDEWEB)

Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

2007-06-26

A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

simple and rapid spectrophotometric assay of levocetirizine

African Journals Online (AJOL)

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Simple, rapid, selective and fairly sensitive method is described for the ... Determination of small amounts of LCTZ in pharmaceutical preparations is important for .... sodium hydroxide and extraction of HCl-free-amine into chloroform followed ...

A one-step, real-time PCR assay for rapid detection of rhinovirus.

Science.gov (United States)

Do, Duc H; Laus, Stella; Leber, Amy; Marcon, Mario J; Jordan, Jeanne A; Martin, Judith M; Wadowsky, Robert M

2010-01-01

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID(50) (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.

Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

Directory of Open Access Journals (Sweden)

Feiwu Li

2014-08-01

Full Text Available The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

Science.gov (United States)

Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

2014-08-27

The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

Determining Plutonium Mass in Spent Fuel with Nondestructive Assay Techniques -- Preliminary Modeling Results Emphasizing Integration among Techniques

International Nuclear Information System (INIS)

Tobin, S.J.; Fensin, M.L.; Ludewigt, B.A.; Menlove, H.O.; Quiter, B.J.; Sandoval, N.P.; Swinhoe, M.T.; Thompson, S.J.

2009-01-01

There are a variety of motivations for quantifying Pu in spent (used) fuel assemblies by means of nondestructive assay (NDA) including the following: strengthen the capabilities of the International Atomic Energy Agencies to safeguards nuclear facilities, quantifying shipper/receiver difference, determining the input accountability value at reprocessing facilities and providing quantitative input to burnup credit determination for repositories. For the purpose of determining the Pu mass in spent fuel assemblies, twelve NDA techniques were identified that provide information about the composition of an assembly. A key point motivating the present research path is the realization that none of these techniques, in isolation, is capable of both (1) quantifying the elemental Pu mass of an assembly and (2) detecting the diversion of a significant number of pins. As such, the focus of this work is determining how to best integrate 2 or 3 techniques into a system that can quantify elemental Pu and to assess how well this system can detect material diversion. Furthermore, it is important economically to down-select among the various techniques before advancing to the experimental phase. In order to achieve this dual goal of integration and down-selection, a Monte Carlo library of PWR assemblies was created and is described in another paper at Global 2009 (Fensin et al.). The research presented here emphasizes integration among techniques. An overview of a five year research plan starting in 2009 is given. Preliminary modeling results for the Monte Carlo assembly library are presented for 3 NDA techniques: Delayed Neutrons, Differential Die-Away, and Nuclear Resonance Fluorescence. As part of the focus on integration, the concept of 'Pu isotopic correlation' is discussed and the role of cooling time determination.

Candida albicans Germ-Tube Antibody: Evaluation of a New Automatic Assay for Diagnosing Invasive Candidiasis in ICU Patients.

Science.gov (United States)

Parra-Sánchez, Manuel; Zakariya-Yousef Breval, Ismail; Castro Méndez, Carmen; García-Rey, Silvia; Loza Vazquez, Ana; Úbeda Iglesias, Alejandro; Macías Guerrero, Desiree; Romero Mejías, Ana; León Gil, Cristobal; Martín-Mazuelos, Estrella

2017-08-01

Testing for Candida albicans germ-tube antibody IFA IgG assay (CAGTA) is used to detect invasive candidiasis infection. However, most suitable assays lack automation and rapid single-sample testing. The CAGTA assay was adapted in an automatic monotest system (invasive candidiasis [CAGTA] VirClia ® IgG monotest (VirClia ® ), a chemiluminescence assay with ready-to-use reagents that provides a rapid objective result. CAGTA assay was compared with the monotest automatic VirClia ® assay in order to establish the diagnostic reliability, accuracy, and usefulness of this method. A prospective study with 361 samples from 179 non-neutropenic critically ill adults patients was conducted, including 21 patients with candidemia, 18 with intra-abdominal candidiasis, 84 with Candida spp. colonization, and 56 with culture-negative samples, as well as samples from ten healthy subjects. Overall agreement between the two assays (CAGTA and VirCLIA) was 85.3%. These assays were compared with the gold-standard method to determine the sensitivity, specificity as well as positive and negative predictive values. In patients with candidemia, values for CAGTA and VirCLIA assays were 76.2 versus 85.7%, 80.3 versus 75.8%, 55.2 versus 52.9%, and 91.4 versus 94.3%, respectively. The corresponding values in patients with intra-abdominal candidiasis were 61.1 versus 66.7%, 80.3 versus 75.8%, 45.8 versus 42.9%, and 88.3 versus 89.3%, respectively. No differences were found according to the species of Candida isolated in culture, except for Candida albicans and C. parapsilosis, for which VirClia ® was better than CAGTA. According to these results, the automated VirClia ® assay was a reliable, rapid, and very easy to perform technique as tool for the diagnosis invasive candidiasis.

A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.

Science.gov (United States)

Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

2010-06-17

Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity. Copyright 2010 Elsevier B.V. All rights reserved.

Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

Science.gov (United States)

Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

2006-01-01

A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592

Phosphodiesterase (PDE5) inhibition assay for rapid detection of erectile dysfunction drugs and analogs in sexual enhancement products.

Science.gov (United States)

Santillo, Michael F; Mapa, Mapa S T

2018-02-28

Products marketed as dietary supplements for sexual enhancement are frequently adulterated with phosphodiesterase-5 (PDE5) inhibitors, which are erectile dysfunction drugs or their analogs that can cause adverse health effects. Due to widespread adulteration, a rapid screening assay was developed to detect PDE5 inhibitors in adulterated products. The assay employs fluorescence detection and is based on measuring inhibition of PDE5 activity, the pharmacological mechanism shared among the adulterants. Initially, the assay reaction scheme was established and characterized, followed by analysis of 9 representative PDE5 inhibitors (IC 50 , 0.4-4.0 ng mL -1 ), demonstrating sensitive detection in matrix-free solutions. Next, dietary supplements serving as matrix blanks (n = 25) were analyzed to determine matrix interference and establish a threshold value; there were no false positives. Finally, matrix blanks were spiked with 9 individual PDE5 inhibitors, along with several mixtures. All 9 adulterants were successfully detected (≤ 5 % false negative rate; n = 20) at a concentration of 1.00 mg g -1 , which is over 5 times lower than concentrations commonly encountered in adulterated products. A major distinction of the PDE5 inhibition assay is the ability to detect adulterants without prior knowledge of their chemical structures, demonstrating a broad-based detection capability that can address a continuously evolving threat of new adulterants. The PDE5 inhibition assay can analyze over 40 samples simultaneously within 15 minutes and involves a single incubation step and simple data analysis, all of which are advantageous for combating the widespread adulteration of sex-enhancement products. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Nondestructive assay technology and automated ''real-time'' materials control

International Nuclear Information System (INIS)

Keepin, G.R.

1977-01-01

Significant advances in nondestructive assay techniques and instrumentation now enable rapid, accurate and direct in-plant measurement of nuclear material on a continuous or ''real-time'' basis as it progresses through a nuclear facility. A variety of passive and active assay instruments are required for the broad range of materials measurement problems encountered by safeguards inspectors and facility operators in various types of nuclear plants. Representative NDA techniques and instruments are presented and reviewed with special attention to their assay capabilities and areas of applicability in the nuclear fuel cycle. An advanced system of materials control - called ''DYMAC'', for Dynamic Materials Control - is presently under development by the U.S. Energy Research and Development Administration; the DYMAC program integrates new nondestructive assay instrumentation and modern data-processing methods, with the overall objective of demonstrating a workable, cost-effective system of stringent safeguards and materials control in various generic types of facilities found in the nuclear fuel cycle. Throughout the program, emphasis will be placed on devloping practical solutions to generic measurement problems so that resulting techniques and instrumentation will have widespread utility. Projected levels of safeguards assurance, together with other vital - and cost-sensitive - plant operational factors such as process and quality control, criticality safety and waste management are examined in an evaluation of the impact of future advanced materials control systems on overall plant operations, efficiency and productivity. The task of implementing effective and stringent safeguards includes the transfer of new safeguards technology to the nuclear industry. Clearly the training of inspectors (both IAEA and national), plant people, etc., in the effective use of new NDA equipment is of paramount importance; thus in the United States, the Energy Research and Development

DNA comet assay as a rapid detection method of irradiated bovine meat by electron beam

International Nuclear Information System (INIS)

Marin-Huachaca, Nelida Simona; Villavicencio, Anna Lucia C.H.

2001-01-01

Full text: Introduction: The presence in food of pathogenic microorganisms, such as Salmonella species, Escherichia coli 0157:H7, Listeria Monocytogenes or Yersinia enterolitica, is a problem of growing concern to public health authorities all over the world. Thus, irradiation of certain prepackaged meat products such as ground beef, minced meat, and hamburgers may help in controlling meatborne pathogens and parasites. Pathogenic microorganisms and parasites in meat products, which are commonly consumed raw, are of particular importance, Up to now, only electron-beam accelerators and gamma-ray cells have been used for commercial applications. At the international conference on 'The Acceptance, Control of, and Trade in Irradiated Food', it was recommended that governments should encourage research into detection methods (Anon, 1989), Already five international standards are available to food control agencies. A number of physical, chemical, and biological techniques of detection of irradiated foods have been discussed in the literature. A rapid and inexpensive screening test employing DNA Comet Assay to identify radiation treatment of food has been described by Cerda et al. (1997). This method is restricted to foods that have not been subjected to heat or other treatments, which also induce DNA fragmentation. Advantages are its simplicity, low cost and speed of measurement. This method was proposed to the European Committee for Standardization (CEN) as a screening protocol (presumptive) and not as a proof (definitive). The DNA comet assay have been yielded good results with chicken, pork, fish meat, exotic meat, hamburgers, fruits and cereals. In this work we studied a DNA fragmentation of bovine meat irradiated by electron beam. Experimental: Bovine meat was purchased in local shops in Sao Paulo. Irradiation was performed with electron beam of accelerator facility of Radiation Dynamics Inc., USA (E=1,5 MeV, l=25 mA). The irradiation doses were 3,5; 4,5, 5,5, and 7

Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

Science.gov (United States)

William R. Kenealy; Thomas W. Jeffries

2003-01-01

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

Risk of iatrogenic human immunodeficiency virus infection through transfusion of blood tested by inappropriately stored or expired rapid antibody assays in a Zambian hospital

NARCIS (Netherlands)

Consten, E. C.; van der Meer, J. T.; de Wolf, F.; Heij, H. A.; Henny, P. C.; van Lanschot, J. J.

1997-01-01

BACKGROUND: The purpose of this study was to estimate the risk of human immunodeficiency virus (HIV) infection via the transfusion of blood tested by inappropriately stored or expired rapid antibody assays in Zambia. STUDY DESIGN AND METHODS: Surgical patients (n = 370) were tested with antibody

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts

OpenAIRE

Redmile-Gordon, M.A.; Armenise, E.; White, R.P.; Hirsch, P.R.; Goulding, K.W.T.

2013-01-01

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colori...

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

Science.gov (United States)

Ali, Akhtar

2017-11-01

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time. Copyright © 2017 Elsevier B.V. All rights reserved.

The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood

Science.gov (United States)

Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Zhang, Sean X.; Avornu, Gideon D.; Rounds, Megan A.; Carolan, Heather E.; Toleno, Donna M.; Moore, David; Hall, Thomas A.; Massire, Christian; Richmond, Gregory S.; Gutierrez, Jose R.; Sampath, Rangarajan; Ecker, David J.; Blyn, Lawrence B.

2016-01-01

Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States. PMID:27384540

A semiquantitative PCR assay for assessing Perkinsus marinus infections in the eastern oyster, Crassostrea virginica.

Science.gov (United States)

Marsh, A G; Gauthier, J D; Vasta, G R

1995-08-01

A 3.2-kb fragment of Perkinsus marinus DNA was cloned and sequenced. A noncoding domain was identified and targeted for the development of a semiquantitative polymerase chain reaction (PCR) assay for the presence of P. marinus in eastern oyster tissues. The assay involves extracting total DNA from oyster hemolymph and using 1 microgram of that DNA as template in a stringent PCR amplification with oligonucleotide primers that are specific for the P. marinus 3.2-kb fragment. With this assay, we can detect 10 pg of total P. marinus DNA per 1 microgram of oyster hemocyte DNA with ethidium bromide (EtBr) staining of agarose gels, 100 fg total P. marinus DNA with Southern blot autoradiography, and 10 fg of total P. marinus DNA with dot-blot hybridizations. We have used the sensitivity of the PCR assay to develop a method for estimating the level of P. marinus DNA in oyster hemolymph and have successfully applied this technique to gill tissues. Our semiquantitative assay uses a dilution series to essentially titrate the point at which a P. marinus DNA target is no longer amplified in a sample. We refer to this technique as "dilution endpoint" PCR. Using hemocytes obtained by withdrawing a 1-ml sample of hemolymph, this assay provides a nondestructive methodology for rapidly screening large numbers of adult oysters for the presence and quantification of P. marinus infection levels. This technique is applicable to other tissues (gills) and could potentially be applied to DNA extracts of whole larvae or spat.

Linearization of the Bradford Protein Assay

OpenAIRE

Ernst, Orna; Zor, Tsaffrir

2010-01-01

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

Rapid, actionable diagnosis of urban epidemic leptospirosis using a pathogenic Leptospira lipL32-based real-time PCR assay.

Science.gov (United States)

Riediger, Irina N; Stoddard, Robyn A; Ribeiro, Guilherme S; Nakatani, Sueli M; Moreira, Suzana D R; Skraba, Irene; Biondo, Alexander W; Reis, Mitermayer G; Hoffmaster, Alex R; Vinetz, Joseph M; Ko, Albert I; Wunder, Elsio A

2017-09-01

With a conservatively estimated 1 million cases of leptospirosis worldwide and a 5-10% fatality rate, the rapid diagnosis of leptospirosis leading to effective clinical and public health decision making is of high importance, and yet remains a challenge. Based on parallel, population-based studies in two leptospirosis-endemic regions in Brazil, a real-time PCR assay which detects lipL32, a gene specifically present in pathogenic Leptospira, was assessed for the diagnostic effectiveness and accuracy. Patients identified by active hospital-based surveillance in Salvador and Curitiba during large urban leptospirosis epidemics were tested. Real-time PCR reactions were performed with DNA-extracted samples obtained from 127 confirmed and 23 unconfirmed cases suspected of leptospirosis, 122 patients with an acute febrile illness other than leptospirosis, and 60 healthy blood donors. The PCR assay had a limit of detection of 280 Leptospira genomic equivalents/mL. Sensitivity for confirmed cases was 61% for whole blood and 29% for serum samples. Sensitivity was higher (86%) for samples collected within the first 6 days after onset of illness compared to those collected after 7 days (34%). The real-time PCR assay was able to detect leptospiral DNA in blood from 56% of serological non-confirmed cases. The overall specificity of the assay was 99%. These findings indicate that real-time PCR may be a reliable tool for early diagnosis of leptospirosis, which is decisive for clinical management of severe and life-threatening cases and for public health decision making.

Monte carlo feasibility study of an active neutron assay technique for full-volume UF{sub 6} cylinder assay using a correlated interrogation source

Energy Technology Data Exchange (ETDEWEB)

Miller, Karen A., E-mail: kamiller@lanl.gov [Los Alamos National Laboratory, Los Alamos, P.O. Box 1663 MS E540, NM 87545 (United States); Menlove, Howard O.; Swinhoe, Martyn T.; Marlow, Johnna B. [Los Alamos National Laboratory, Los Alamos, P.O. Box 1663 MS E540, NM 87545 (United States)

2013-03-01

Uranium cylinder assay plays an important role in the nuclear material accounting at gas centrifuge enrichment plants. The Passive Neutron Enrichment Meter (PNEM) was designed to determine uranium mass and enrichment in 30B and 48Y cylinders using total neutron and coincidence counting in the passive mode. 30B and 48Y cylinders are used to hold bulk UF{sub 6} feed, product, and tails at enrichment plants. In this paper, we report the results of a Monte-Carlo-based feasibility study for an active uranium cylinder assay system based on the PNEM design. There are many advantages of the active technique such as a shortened count time and a more direct measure of {sup 235}U content. The active system is based on a modified PNEM design and uses a {sup 252}Cf source as the correlated, active interrogation source. We show through comparison with a random AmLi source of equal strength how the use of a correlated driver significantly boosts the active signal and reduces the statistical uncertainty. We also discuss ways in which an active uranium cylinder assay system can be optimized to minimize background from {sup 238}U fast-neutron induced fission and direct counts from the interrogation source.

Fundamental and clinical evaluation of ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen

International Nuclear Information System (INIS)

Koizumi, Mitsuru; Endo, Keigo; Nakajima, Kotoko

1987-01-01

A commercial ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen (SCC antigen) was fundamentally and clinically evaluated. Laboratory performance was satisfactory for intra-assay and inter-assay reproducibility, recovery, and dilution, with rapid and simple measurement techniques. Seropositivity for SCC antigen was significantly higher for squamous cell carcinoma of the liver and uterine cervix than the other histology types. In the case of cervical squamous cell carcinoma, it increased with progressing disease. Post-treatment serum levels of SCC antigen returned to negative. SCC antigen is considered to be a useful tumor marker for these diseases. There was a good correlation between the measurement values obtained from the present and conventional (SCC RIAKIT) assays. The present assay remarkably decreased false-positive cases of pulmonary benign diseases. The results showed a ''SCC RIABEAD'' to be a favorable kit for immunoradiometric assay of SSC antigen, as compared with conventional assay kit. (Namekawa, K.)

Appendix: a solution hybridization assay to detect radioactive globin messenger RNA nucleotide sequences

Energy Technology Data Exchange (ETDEWEB)

Ross, J

1976-09-15

In view of the sensitivity and specificity of the solution hybridization assay for unlabeled globin mRNA a similar technique has been devised to detect radioactive globin mRNA sequences with unlabeled globin cDNA. Several properties of the hybridization reaction are presented since RNA kinetic experiments reported recently depend on the validity of this assay. Data on hybridization analysis of (/sup 3/H)RNA from mouse fetal liver or erythroleukemia cell cytoplasm are presented. These data indicate that the excess cDNA solution assay for radioactive globin mRNA detection is specific for globin mRNA sequences. It can be performed rapidly and is highly reproducible from experiment. It is at least 500-fold less sensitive than the assay for unlabeled globin mRNA, due to the RNAase backgrounds of 0.05 to 0.15 %. However, this limitation has not affected kinetic experiments with non-dividing fetal liver erythroid cells, which synthesize relatively large quantities of globin mRNA.

Variables affecting viral plaque formation in microculture plaque assays using homologous antibody in a liquid overlay.

Science.gov (United States)

Randhawa, A S; Stanton, G J; Green, J A; Baron, S

1977-05-01

A liquid antibody microculture plaque assay and the variables that govern its effectiveness are described. The assay is based on the principle that low concentrations of homologous antibody can inhibit secondary plaque formation without inhibiting formation of primary plaques. Thus, clear plaques that followed a linear dose response were produced. The assay was found to be more rapid, less cumbersome, and less expensive than assays using agar overlays and larger tissue culture plates. It was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique in measuring infectious coxsackievirus type B-3. It was more sensitive in assaying adenovirus type 3 and Western equine encephalomyelitis, vesicular stomatitis, Semliki forest, Sendai, Sindbis, and Newcastle disease viruses than were liquid, carboxymethylcellulose, and methylcellulose microculture plaque assays. The variables influencing sensitivity and accuracy, as determined by using coxsackievirus type B-3, were: (i) the inoculum volume of virus; (ii) the incubation period of virus; and (iii) the incubation temperature.

Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of fasciolosis in sheep and its application under field conditions

OpenAIRE

Mart?nez-Valladares, Mar?a; Rojo-V?zquez, Francisco Antonio

2016-01-01

Background Loop-mediated isothermal amplification (LAMP) is a very specific, efficient, and rapid gene amplification procedure in which the reaction can run at a constant temperature. In the current study we have developed a LAMP assay to improve the diagnosis of Fasciola spp. in the faeces of sheep. Findings After the optimisation of the LAMP assay we have shown similar results between this technique and the standard PCR using the outer primers of the LAMP reaction. In both cases the limit o...

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

Science.gov (United States)

Kurosaki, Yohei; Magassouba, N’Faly; Oloniniyi, Olamide K.; Cherif, Mahamoud S.; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

2016-01-01

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure. PMID:26900929

Identification of irradiated refrigerated pork with the DNA comet assay

Energy Technology Data Exchange (ETDEWEB)

Araujo, M.M. E-mail: villavic@net.ipen.br; Marin-Huachaca, N.S.; Mancini-Filho, J. E-mail: jmancini@usp.br; Delincee, H.; Villavicencio, A.L.C.H. E-mail: henry.delincee@bfe.uni-karlsruhe.de

2004-10-01

Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells ('Comet Assay') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a {sup 60}Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sao Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5 kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6 deg. C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA 'Comet Assay'. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

Identification of irradiated refrigerated pork with the DNA comet assay

Science.gov (United States)

Araújo, M. M.; Marin-Huachaca, N. S.; Mancini-Filho, J.; Delincée, H.; Villavicencio, A. L. C. H.

2004-09-01

Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells (``Comet Assay'') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a 60Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sa~o Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6°C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA ``Comet Assay''. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

Energy Technology Data Exchange (ETDEWEB)

Frank F. Roberto

2008-08-01

Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

Issues in the analyze of low content gold mining samples by fire assay technique

Science.gov (United States)

Cetean, Valentina

2016-04-01

The classic technique analyze of samples with low gold content - below 0.1 g/t (=100 ppb = parts per billion), either ore or gold sediments, involves the preparation of sample by fire assay extraction, followed by the chemical attack with aqua regia (hydrochloric and nitric acid) and measuring the gold content by atomic absorption spectrometry or inductively coupled mass spectrometry. The issues raised by this analysis are well known for the world laboratories, commercial or research ones. The author's knowledge regarding this method of determining the gold content, accumulated in such laboratory from Romania (with more than 40 years of experience, even if not longer available from 2014) confirms the obtaining of reliable results required a lot of attention, amount of work and the involving of an experienced fire assayer specialist. The analytical conclusion for a research laboratory is that most reliable and statistically valid results are till reached for samples with more than 100 ppb gold content; the degree of confidence below this value is lower than 90%. Usually, for samples below 50 ppb, it does not exceed 50-70 %, unless without very strictly control of each stage, that involve additional percentage of hours allocated for successive extracting tests and knowing more precisely the other compounds that appear in the sample (Cu, Sb, As, sulfur / sulphides, Te, organic matter, etc.) or impurities. The most important operation is the preparation, namely: - grinding and splitting of sample (which can cause uneven distribution of gold flakes in the double samples for analyzed); - pyro-metallurgical recovery of gold = fire assay stage, involving the more precise temperature control in furnace during all stages (fusion and cupellation) and adjusting of the fire assay flux components to produce a successful fusion depending of the sample matrix and content; - reducing the sample weight to decrease the amount of impurities that can be concentrated in the lead button

Microbead agglutination based assays

KAUST Repository

Kodzius, Rimantas

2013-01-21

We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

Science.gov (United States)

Howson, E L A; Armson, B; Madi, M; Kasanga, C J; Kandusi, S; Sallu, R; Chepkwony, E; Siddle, A; Martin, P; Wood, J; Mioulet, V; King, D P; Lembo, T; Cleaveland, S; Fowler, V L

2017-06-01

Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV. © 2015 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.

Rapid, actionable diagnosis of urban epidemic leptospirosis using a pathogenic Leptospira lipL32-based real-time PCR assay.

Directory of Open Access Journals (Sweden)

Irina N Riediger

2017-09-01

Full Text Available With a conservatively estimated 1 million cases of leptospirosis worldwide and a 5-10% fatality rate, the rapid diagnosis of leptospirosis leading to effective clinical and public health decision making is of high importance, and yet remains a challenge.Based on parallel, population-based studies in two leptospirosis-endemic regions in Brazil, a real-time PCR assay which detects lipL32, a gene specifically present in pathogenic Leptospira, was assessed for the diagnostic effectiveness and accuracy. Patients identified by active hospital-based surveillance in Salvador and Curitiba during large urban leptospirosis epidemics were tested. Real-time PCR reactions were performed with DNA-extracted samples obtained from 127 confirmed and 23 unconfirmed cases suspected of leptospirosis, 122 patients with an acute febrile illness other than leptospirosis, and 60 healthy blood donors.The PCR assay had a limit of detection of 280 Leptospira genomic equivalents/mL. Sensitivity for confirmed cases was 61% for whole blood and 29% for serum samples. Sensitivity was higher (86% for samples collected within the first 6 days after onset of illness compared to those collected after 7 days (34%. The real-time PCR assay was able to detect leptospiral DNA in blood from 56% of serological non-confirmed cases. The overall specificity of the assay was 99%.These findings indicate that real-time PCR may be a reliable tool for early diagnosis of leptospirosis, which is decisive for clinical management of severe and life-threatening cases and for public health decision making.

Simple and rapid detection of the porcine reproductive and respiratory syndrome virus from pig whole blood using filter paper.

Science.gov (United States)

Inoue, Ryo; Tsukahara, Takamitsu; Sunaba, Chinatsu; Itoh, Mitsugi; Ushida, Kazunari

2007-04-01

The combination of Flinders Technology Associates filter papers (FTA cards) and real-time PCR was examined to establish a simple and rapid technique for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) from whole pig blood. A modified live PRRS vaccine was diluted with either sterilised saline or pig whole blood, and the suspensions were applied onto the FTA cards. The real-time RT-PCR detection of PRRSV was performed directly with the samples applied to the FTA card without the RNA extraction step. Six whole blood samples from at random selected piglets in the PRRSV infected farm were also assayed in this study. The expected PCR product was successfully amplified from either saline diluted or pig whole blood diluted vaccine. The same PCR ampliocon was detected from all blood samples assayed in this study. This study suggested that the combination of an FTA card and real-time PCR is a rapid and easy technique for the detection of PRRSV. This technique can remarkably shorten the time required for PRRSV detection from whole blood and makes the procedure much easier.

Microbead agglutination based assays

KAUST Repository

Kodzius, Rimantas; Castro, David; Foulds, Ian G.; Parameswaran, Ash M.; Sumanpreet, K. Chhina

2013-01-01

We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling

New Strategies for Powder Compaction in Powder-based Rapid Prototyping Techniques

OpenAIRE

Budding, A.; Vaneker, T.H.J.

2013-01-01

In powder-based rapid prototyping techniques, powder compaction is used to create thin layers of fine powder that are locally bonded. By stacking these layers of locally bonded material, an object is made. The compaction of thin layers of powder mater ials is of interest for a wide range of applications, but this study solely focuses on the application for powder -based three-dimensional printing (e.g. SLS, 3DP). This research is primarily interested in powder compaction for creating membrane...

Rapid screening for targeted genetic variants via high-resolution melting curve analysis.

Science.gov (United States)

Chambliss, Allison B; Resnick, Molly; Petrides, Athena K; Clarke, William A; Marzinke, Mark A

2017-03-01

Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP. Intra-assay (n=5) precision of Tms for all SNPs was ≤0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/μL. The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are

DEVELOPMENT OF RAPID TECHNIQUE FOR DETERMINATION OF THE TOTAL MINERALIZATION OF NATURAL WATERS

Directory of Open Access Journals (Sweden)

T. A. Kuchmenko

2015-01-01

Full Text Available A new approach has been proposed for rapid and easy evaluation of a indicator of quality and properties of natural water - soluble salt content (mineralization. The method of quartz crystal microbalance is employed at load of the mass-sensitive resonator electrode (BAW-type with investigated water. The degree of correlation between the various indicators related to the contents of salts and insoluble compounds and the level of mineralization obtained by the standard method (gravimetry has been studied. A procedure for salt weighing by single sensor at unilateral load with small sample of natural water has been developed. The optimal conditions for measurement is established using the design of experiment by model 23 . The possibilities of quartz crystal microbalance for determination of non-volatile compounds in the water are described. The calibration of piezosensor is produced by standard solution NaCl (c = 1.000 g / dm3 at optimal conditions of experiment. The adequacy and accuracy of proposed technique is assessed by the correlation between the results of quartz crystal microbalance and conductometry. The correlation between indicators of mineralization established by quartz crystal microbalance and gravimetry is found. It has been obtained an equation that can be used to calculate the standard indicator of the mineralization by the results of a quartz crystal microbalance using single sensor. The approaches to enhance the analytical capabilities of the developed technique for water with low and high mineralization are proposed. The metrological characteristics of quartz crystal microbalance of insoluble compounds in natural water are estimated. A new technique of determination of the mass concentration of the dry residue in water with a conductivity of 0.2 mS or above has been developed, which can be used for rapid analysis of the water at nonlaboratory conditions and in the laboratory for rapid obtaining the information about a sample.

A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

International Nuclear Information System (INIS)

Goodall, A.H.; Meek, F.L.; Waters, J.A.; Miescher, G.C.; Janossy, G.; Thomas, H.C.

1982-01-01

A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available. (Auth.)

Nondestructive assay measurements applied to reprocessing plants

International Nuclear Information System (INIS)

Ruhter, Wayne D.; Lee, R. Stephen; Ottmar, Herbert; Guardini, Sergio

1999-01-01

Nondestructive assay for reprocessing plants relies on passive gamma-ray spectrometry for plutonium isotopic and plutonium mass values of medium-to-low-density samples and holdup deposits; on active x-ray fluorescence and densitometry techniques for uranium and plutonium concentrations in solutions; on calorimetry for plutonium mass in product; and passive neutron techniques for plutonium mass in spent fuel, product, and waste. This paper will describe the radiation-based nondestructive assay techniques used to perform materials accounting measurements. The paper will also discuss nondestructive assay measurements used in inspections of reprocessing plants [ru

A new assay for cytotoxic lymphocytes, based on a radioautographic readout of 111In release, suitable for rapid, semi-automated assessment of limit-dilution cultures

International Nuclear Information System (INIS)

Shortman, K.; Wilson, A.

1981-01-01

A new assay for cytotoxic T lymphocytes is described, of general application, but particularly suitable for rapid, semi-automated assessment of multiple microculture tests. Target cells are labelled with high efficiency and to high specific activity with the oxine chelate of 111 indium. After a 3-4 h incubation of test cells with 5 X 10 3 labelled target cells in V wells of microtitre trays, samples of the supernatant are spotted on paper (5 μl) or transferred to soft-plastic U wells (25-50 μl) and the 111 In release assessed by radioautography. Overnight exposure of X-ray film with intensifying screens at -70 0 C gives an image which is an intense dark spot for maximum release, a barely visible darkening with the low spontaneous release, and a definite positive with 10% specific lysis. The degree of film darkening, which can be quantitated by microdensitometry, shows a linear relationship with cytotoxic T lymphocyte dose up to the 40% lysis level. The labelling intensity and sensitivity can be adjusted over a wide range, allowing a single batch of the short half-life isotope to serve for 2 weeks. The 96 assays from a single tray are developed simultaneously on a single small sheet of film. Many trays can be processed together, and handling is rapid if 96-channel automatic pipettors are used. The method allows rapid visual scanning for positive and negative limit dilution cultures in cytotoxic T cell precursor frequency and specificity studies. In addition, in conjunction with an automated densitometer designed to scan microtitre trays, the method provides an efficient alternative to isotope counting in routine cytotoxic assays. (Auth.)

A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

Directory of Open Access Journals (Sweden)

Helene Andersson-Svahn

2011-11-01

Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

Development of an analytical theory to describe the PNAR and CIPN nondestructive assay techniques

International Nuclear Information System (INIS)

Bolind, Alan Michael

2014-01-01

Highlights: • Neutron albedo is modeled by a discrete and iterative reflection process. • The theory enables the PNAR and CIPN NDA techniques to be compared quantitatively. • Improvements to the data analysis and to the CIPN instrument design are suggested. • A correction to translate real no-reflection PNAR data into ideal data is provided. - Abstract: This paper develops an analytical theory to describe how neutron albedo (reflection) increases the multiplication of neutrons by a used fuel assembly. With this theory, the two nondestructive assay (NDA) techniques of Passive Neutron Albedo Reactivity (PNAR) and Californium-252 Interrogation with Prompt Neutron Detection (CIPN) can be compared directly. Specifically, the theory derives expressions for the PNAR and CIPN metrics in terms of the physical properties of the used fuel assembly, such as the neutron multiplications and fate probabilities. The theory thus clarifies the interpretation of these two NDA techniques and suggests ways to improve both the design of the NDA instruments and the algorithms for analyzing the measurement results

Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk.

Science.gov (United States)

Bosward, Katrina L; House, John K; Deveridge, Amber; Mathews, Karen; Sheehy, Paul A

2016-03-01

Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Validation of a Rapid Bacteria Endospore Enumeration System for Planetary Protection Application

Science.gov (United States)

Chen, Fei; Kern, Roger; Kazarians, Gayane; Venkateswaran, Kasthuri

NASA monitors spacecraft surfaces to assure that the presence of bacterial endospores meets strict criteria at launch, to minimize the risk of inadvertent contamination of the surface of Mars. Currently, the only approved method for enumerating the spores is a culture based assay that requires three days to produce results. In order to meet the demanding schedules of spacecraft assembly, a more rapid spore detection assay is being considered as an alternate method to the NASA standard culture-based assay. The Millipore Rapid Microbiology Detection System (RMDS) has been used successfully for rapid bioburden enumeration in the pharmaceutical and food industries. The RMDS is rapid and simple, shows high sensitivity (to 1 colony forming unit [CFU]/sample), and correlates well with traditional culture-based methods. It combines membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and image analysis based on photon detection with a Charge Coupled Device (CCD) camera. In this study, we have optimized the assay conditions and evaluated the use of the RMDS as a rapid spore detection tool for NASA applications. In order to select for spores, the samples were subjected to a heat shock step before proceeding with the RMDS incubation protocol. Seven species of Bacillus (nine strains) that have been repeatedly isolated from clean room environments were assayed. All strains were detected by the RMDS in 5 hours and these assay times were repeatedly demonstrated along with low image background noise. Validation experiments to compare the Rapid Sore Assay (RSA) and NASA standard assay (NSA) were also performed. The evaluation criteria were modeled after the FDA Guideline of Process Validation, and Analytical Test Methods. This body of research demonstrates that the Rapid Spore Assay (RSA) is quick, and of equivalent sensitivity to the NASA standard assay, potentially reducing the assay time for bacterial endospores from over 72 hours to less than 8 hours

Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan.

Science.gov (United States)

Mizukami, Keijiro; Chang, Hye-Sook; Yabuki, Akira; Kawamichi, Takuji; Kawahara, Natsuko; Hayashi, Daisuke; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Yamato, Osamu

2011-11-01

Neuronal ceroid lipofuscinosis (NCL) constitutes a group of recessively inherited lysosomal storage diseases that primarily affect neuronal cells. Such diseases share certain clinical and pathologic features in human beings and animals. Neuronal ceroid lipofuscinosis in Border Collie dogs was first detected in Australia in the 1980s, and the pathogenic mutation was shown to be a nonsense mutation (c.619C>T) in exon 4 in canine CLN5 gene. In the present study, novel rapid genotyping assays including polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR primer-induced restriction analysis, mutagenically separated PCR, and real-time PCR with TaqMan minor groove binder probes, were developed. The utility of microchip electrophoresis was also evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies in Japan using these assays to determine the current allele frequency in Japan, providing information to control and prevent this disease in the next stage. All assays developed in the current study are available to discriminate these genotypes, and microchip electrophoresis showed a timesaving advantage over agarose gel electrophoresis. Of all assays, real-time PCR was the most suitable for large-scale examination because of its high throughput. The genotyping survey demonstrated that the carrier frequency was 8.1%. This finding suggested that the mutant allele frequency of NCL in Border Collies is high enough in Japan that measures to control and prevent the disease would be warranted. The genotyping assays developed in the present study could contribute to the prevention of NCL in Border Collies.

Rapid real-time PCR assay for culture and tissue identification of Geomyces destructans: the etiologic agent of bat geomycosis (white nose syndrome).

Science.gov (United States)

Chaturvedi, Sudha; Rudd, Robert J; Davis, April; Victor, Tanya R; Li, Xiaojiang; Appler, Kim A; Rajkumar, Sunanda S; Chaturvedi, Vishnu

2011-10-01

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.

Radioactive waste package assay facility. Volume 1. Application of assay technology

International Nuclear Information System (INIS)

Findlay, D.J.S.; Green, T.H.; Molesworth, T.V.; Staniforth, D.; Strachan, N.R.; Rogers, J.D.; Wise, M.O.; Forrest, K.R.

1992-01-01

This report, in three volumes, covers the work carried out by Taylor Woodrow Construction Ltd., and two major sub-contractors: Harwell Laboratory (AEA Technology) and Siemens Plessey Controls Ltd., on the development of a radioactive waste package assay facility, for cemented 500 litre intermediate level waste drums. In volume 1, the reasons for assay are considered together with the various techniques that can be used, and the information that can be obtained. The practical problems associated with the use of the various techniques in an integrated assay facility are identified, and the key parameters defined. Engineering and operational features are examined and provisional designs proposed for facilities at three throughput levels: 15,000, 750 and 30 drums per year respectively. The capital and operating costs for such facilities have been estimated. A number of recommendations are made for further work. 16 refs., 14 figs., 13 tabs

Development and validation of a thallium flux-based functional assay for the sodium channel NaV1.7 and its utility for lead discovery and compound profiling.

Science.gov (United States)

Du, Yu; Days, Emily; Romaine, Ian; Abney, Kris K; Kaufmann, Kristian; Sulikowski, Gary; Stauffer, Shaun; Lindsley, Craig W; Weaver, C David

2015-06-17

Ion channels are critical for life, and they are targets of numerous drugs. The sequencing of the human genome has revealed the existence of hundreds of different ion channel subunits capable of forming thousands of ion channels. In the face of this diversity, we only have a few selective small-molecule tools to aid in our understanding of the role specific ion channels in physiology which may in turn help illuminate their therapeutic potential. Although the advent of automated electrophysiology has increased the rate at which we can screen for and characterize ion channel modulators, the technique's high per-measurement cost and moderate throughput compared to other high-throughput screening approaches limit its utility for large-scale high-throughput screening. Therefore, lower cost, more rapid techniques are needed. While ion channel types capable of fluxing calcium are well-served by low cost, very high-throughput fluorescence-based assays, other channel types such as sodium channels remain underserved by present functional assay techniques. In order to address this shortcoming, we have developed a thallium flux-based assay for sodium channels using the NaV1.7 channel as a model target. We show that the assay is able to rapidly and cost-effectively identify NaV1.7 inhibitors thus providing a new method useful for the discovery and profiling of sodium channel modulators.

Evaluation of irradiation in foods using DNA comet assay

International Nuclear Information System (INIS)

Khawar, Affaf; Bhatti, Ijaz Ahmad; Khan, Q.M.; Ali, T.; Khan, A.I.; Asi, M.R.

2011-01-01

Comet assay is a rapid, inexpensive and sensitive biological technique to detect DNA damage in food stuffs by irradiation. In this study the Comet assay is applied on foods of plant and animal origins. Samples were irradiated by using 60 Co gamma-radiation source. The applied doses were 2, 6 and 10 kGy for food of plant origin and 0.5, 1 and 2 kGy for meat items. The un-irradiated and irradiated samples were clearly differentiated on the basis of DNA fragmentation. During the electrophoresis study, it was found that in un-irradiated cells DNA remained intact and appeared as Comets without tail whereas in irradiated cells Comets with tails were visible due to stretching of fragmented DNA. Moreover, it was also revealed that the DNA tail length was dose dependent. Dry food stuffs (seeds) showed good results as compared to moist foods (meat, fruits and vegetables) due to the absence of background damage. (author)

A gp41-based heteroduplex mobility assay provides rapid and accurate assessment of intrasubtype epidemiological linkage in HIV type 1 heterosexual transmission Pairs.

Science.gov (United States)

Manigart, Olivier; Boeras, Debrah I; Karita, Etienne; Hawkins, Paulina A; Vwalika, Cheswa; Makombe, Nathan; Mulenga, Joseph; Derdeyn, Cynthia A; Allen, Susan; Hunter, Eric

2012-12-01

A critical step in HIV-1 transmission studies is the rapid and accurate identification of epidemiologically linked transmission pairs. To date, this has been accomplished by comparison of polymerase chain reaction (PCR)-amplified nucleotide sequences from potential transmission pairs, which can be cost-prohibitive for use in resource-limited settings. Here we describe a rapid, cost-effective approach to determine transmission linkage based on the heteroduplex mobility assay (HMA), and validate this approach by comparison to nucleotide sequencing. A total of 102 HIV-1-infected Zambian and Rwandan couples, with known linkage, were analyzed by gp41-HMA. A 400-base pair fragment within the envelope gp41 region of the HIV proviral genome was PCR amplified and HMA was applied to both partners' amplicons separately (autologous) and as a mixture (heterologous). If the diversity between gp41 sequences was low (<5%), a homoduplex was observed upon gel electrophoresis and the transmission was characterized as having occurred between partners (linked). If a new heteroduplex formed, within the heterologous migration, the transmission was determined to be unlinked. Initial blind validation of gp-41 HMA demonstrated 90% concordance between HMA and sequencing with 100% concordance in the case of linked transmissions. Following validation, 25 newly infected partners in Kigali and 12 in Lusaka were evaluated prospectively using both HMA and nucleotide sequences. Concordant results were obtained in all but one case (97.3%). The gp41-HMA technique is a reliable and feasible tool to detect linked transmissions in the field. All identified unlinked results should be confirmed by sequence analyses.

The rapid determination of americium curium, and uranium in urine by ultrafiltration

International Nuclear Information System (INIS)

Stradling, G.N.; Popplewell, D.S.; Ham, G.J.; Griffin, R.

1975-01-01

The rapid ultrafiltration method developed for the assay of plutonium has been extended to the determination of americium, curium and uranium in urine. The limits of detection for americium and curium, and uranium are 0.09 and 0.12 dm -1 l -1 respectively, and the analysis time excluding counting less than 2 hours. The method can therefor be effectively used as a rapid screening procedure. When the reference level for plutonium is exceeded, the α activity may require to be characterised. The single ultrafiltration technique must be modified for turbid urine samples. The method is inappropriate, except for uranium, when the urine contains DTPA. (author)

Some target assay uncertainties for passive neutron coincidence counting

International Nuclear Information System (INIS)

Ensslin, N.; Langner, D.G.; Menlove, H.O.; Miller, M.C.; Russo, P.A.

1990-01-01

This paper provides some target assay uncertainties for passive neutron coincidence counting of plutonium metal, oxide, mixed oxide, and scrap and waste. The target values are based in part on past user experience and in part on the estimated results from new coincidence counting techniques that are under development. The paper summarizes assay error sources and the new coincidence techniques, and recommends the technique that is likely to yield the lowest assay uncertainty for a given material type. These target assay uncertainties are intended to be useful for NDA instrument selection and assay variance propagation studies for both new and existing facilities. 14 refs., 3 tabs

Multimodality characterization of nuclear waste drums using emerging techniques for nondestructive examination and assay

International Nuclear Information System (INIS)

Bernardi, R.T.

1993-01-01

We are developing an x-ray imaging system that incorporates several inspection technologies for complete, nondestructive evaluation of containers of nuclear waste. In Phase I and Phase II SBIR programs for the DOE, we proved the feasibility of using x-ray computed tomography (CT) and digital radiography (DR)-imaging techniques using x-rays transmitted through the object-for container inspection. Now, with further funding from DOE and working with scientists at Lawrence Livermore National Lab., we are designing a mobile inspection system that will use CT and DR as well as two x-ray emission imaging techniques-single photon emission computed tomography and nondestructive assay. This system will provide much more information about the contents of containers than currently used inspection methods, and will provide archiving of digital data. In this paper, we describe inspection system and present recent results from the CT and DR evaluations

Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation.

Science.gov (United States)

Dittmer, W U; de Kievit, P; Prins, M W J; Vissers, J L M; Mersch, M E C; Martens, M F W C

2008-09-30

A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions as well as the bound/free separation are entirely controlled by magnetic forces induced by electromagnets above and below the sensor chip. During the assay, particles conjugated with tracer antibodies are actuated through the sample for target capture, and rapidly brought to the sensor surface where they bind to immobilized capture antibodies. Weakly or unbound labels are removed with a magnetic force oriented away from the GMR sensor surface. For the measurement of parathyroid hormone (PTH), a detection limit in the 10 pM range is obtained with a total assay time of 15 min when 300 nm particles are used. The same sensitivity can be achieved in 5 min when 500 nm particles are used. If 500 nm particles are employed in a 15-minute assay, then 0.8 pM of PTH is detectable. The low sample volume, high analytical performance and high speed of the test coupled with the compact GMR biosensor make the system especially suitable for sensitive testing outside of laboratory environments.

Specific binding assay technique; standardization of reagent

International Nuclear Information System (INIS)

Huggins, K.G.; Roitt, I.M.

1979-01-01

The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

Persistent Graves' hyperthyroidism despite rapid negative conversion of thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results: a case report.

Science.gov (United States)

Ohara, Nobumasa; Kaneko, Masanori; Kitazawa, Masaru; Uemura, Yasuyuki; Minagawa, Shinichi; Miyakoshi, Masashi; Kaneko, Kenzo; Kamoi, Kyuzi

2017-02-06

Graves' disease is an autoimmune thyroid disorder characterized by hyperthyroidism, and patients exhibit thyroid-stimulating hormone receptor antibody. The major methods of measuring circulating thyroid-stimulating hormone receptor antibody include the thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Although the diagnostic accuracy of these assays has been improved, a minority of patients with Graves' disease test negative even on second-generation and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulins. We report a rare case of a thyroid-stimulating hormone-binding inhibitory immunoglobulin-positive patient with Graves' disease who showed rapid lowering of thyroid-stimulating hormone-binding inhibitory immunoglobulin levels following administration of the anti-thyroid drug thiamazole, but still experienced Graves' hyperthyroidism. A 45-year-old Japanese man presented with severe hyperthyroidism (serum free triiodothyronine >25.0 pg/mL; reference range 1.7 to 3.7 pg/mL) and tested weakly positive for thyroid-stimulating hormone-binding inhibitory immunoglobulins on second-generation tests (2.1 IU/L; reference range hyperthyroidism for more than 8 years, requiring 15 mg/day of thiamazole to correct. During that period, he tested negative on all first-generation, second-generation, and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, but thyroid scintigraphy revealed diffuse and increased uptake, and thyroid ultrasound and color flow Doppler imaging showed typical findings of Graves' hyperthyroidism. The possible explanations for serial changes in the thyroid-stimulating hormone-binding inhibitory immunoglobulin results in our patient include the presence of thyroid-stimulating hormone receptor antibody, which is bioactive but less reactive on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, or the effect of reduced levels of circulating thyroid

Conceptual design of the special nuclear material nondestructive assay and accountability system for the HTGR fuel refabrication pilot plant

International Nuclear Information System (INIS)

Jenkins, J.D.; McNeany, S.R.; Rushton, J.E.

1975-07-01

The conceptual design of the fissile material assay and accountability system for the HTGR refabrication pilot plant has been established. The primary feature affecting the design is the high, time varying, gamma activity of the process material due to the unavoidable presence of uranium-232. This imposes stringent requirements for remote operation and remote maintainability of system components. At the same time, the remote operation lends itself to implementation of an automated data collection and processing system for real-time accountability. The high time-varying gamma activity of the material also precludes application of a number of techniques presently employed for light-water reactor fuel assay. The techniques selected for application in the refabrication facility are (1) active thermal neutron interrogation with fast-fission or delayed-neutron counting for fuel-rod and small-sample assay, (2) calorimetry for high-level waste assay, and (3) passive gamma scanning for low-level waste assay, and rapid on-line relative rod-loading measurements. The principal nondestructive assay subsystems are identified as (1) on-line devices for 100 percent product fuel rod assay and quality control, (2) a multipurpose device in the sample inspection laboratory for small- sample assay and secondary standards calibration, and (3) equipment for assay of high- and low-uranium content scrap and waste materials. A data processing system, which coordinates data from these subsystems with information from other process control sensors, is included to provide real-time material balance information. (U.S.)

The Attainment of High Sensitivity and Precision in Radioimmunoassay Techniques as Exemplified in a Simple Assay of Serum Insulin

Energy Technology Data Exchange (ETDEWEB)

Albano, Janet; Ekins, R. P. [Institute of Nuclear Medicine, Middlesex Hospital Medical School, London (United Kingdom)

1970-02-15

Recent controversy has underlined the fundamental confusion surrounding the concepts of assay ''sensitivity'' and ''precision'' and, in particular, their optimization in radioimmunoassay and other saturation assay procedures. Many formal definitions of sensitivity (e.g. that laid down by the American Chemical Society) express this concept in terms of the slope of the ''dose'' response curve; nevertheless, in common usage, the term is normally regarded as a synonym for the detection limit of the measurement technique. However, a technique which is ''sensitive'' in the formal sense may not display a low limit of detection, and it is readily demonstrable that, in radioimmunoassay systems in particular, there are circumstances in which increase in the slope of the response curve may lead to an increase in the detection limit of the assay. The authors have based their insulin assay protocols on mathematical principles specifically designed to lead to the minimization of the detection limit. The method depends on the use of (uncoated) charcoal for the separation of free and bound labelled insulin in incubation mixtures in which insulin-free human serum is used as diluent. The detection limit of the method is approximately 1 pg/ml of incubation mixture, corresponding to roughly 0.25 {mu}U/ml of serum at the serum dilutions used. In a formal comparative study, the method has been shown to be more sensitive, precise and accurate than other methods relying on double antibody or chromato-electrophoietic separation. The relevance of such factors as high specific activity labelled hormone to the attainment of high sensitivity is discussed. (author)

A rapid microassay for detecting antibodies against poliovirus based on [14C]thymidine uptake of treated cell cultures

International Nuclear Information System (INIS)

Hilfenhaus, J.; Damm, H.; Ziegelmaier, R.; Gruschkau, H.

1977-01-01

DNA synthesis of mammalian cells propagated in microplates can easily be measured if cell cultures incubated with [ 14 C]thymidine are harvested on to glass fibre filters by a semiautomatic harvesting technique. Soon after infection with poliovirus, [ 14 C]thymidine uptake of U cells (established, human amniotic cell line) is inhibited. This inhibition can be prevented by previous virus neutralization with antibody. Based on this effect a rapid, precise assay method was set up to determine neutralizing antibody titres against poliovirus. There was a good correlation between titres obtained by this assay and those obtained by 50% endpoint titrations in cytopathogenic effect inhibition assays

Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

Science.gov (United States)

Arnold, A R; Burnham, C-A D; Ford, B A; Lawhon, S D; McAllister, S K; Lonsway, D; Albrecht, V; Jerris, R C; Rasheed, J K; Limbago, B; Burd, E M; Westblade, L F

2016-03-01

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Improved assay for thymine base damage in E. coli using high performance liquid chromatography

International Nuclear Information System (INIS)

Claycamp, H.G.

1985-01-01

A simple high performance liquid chromatography (HPLC) technique has been established for the simultaneous assay of thymine and thymidine radiation damage products. The HPLC procedure uses an isocratic mobile phase of 4% acetonitrile in 0.2 M ammonium dihydrogen phosphate (pH 5.0), a reversed-phase octadecylsilicate (5 micro-spherical packing) 0.45 x 25 cm column, and a variable wavelength UV detector. This procedure affords much better resolution than other published procedures that use 10 micron columns or separate assays for bases and nucleosides. For example, irradiation of 5 x 10 -3 M thymidine solutions have been performed to calibrate the system for base damage assays in E. coli. This yields up to 15 resolvable residues within 20 minutes. Sensitivity of the system (at 2210 nm) for 5,6- dihydrothymine (DHT) is about 10 -10 moles. Preliminary results show that this translates to about 0.4 DHT residues per 10 6 daltons of E. coli DNA. This is comparable to the sensitivities of monoclonal assays to thymine damage products that have recently been reported by others. Since it is feasible that the sensitivity of this system can be improved by 2-3 times, this HPLC technique should provide a simple and rapid means of detecting E. coli base damage release and base damage in nucleoside hydrolysates of DNA

Evaluation of laser diode thermal desorption (LDTD) coupled with tandem mass spectrometry (MS/MS) for support of in vitro drug discovery assays: increasing scope, robustness and throughput of the LDTD technique for use with chemically diverse compound libraries.

Science.gov (United States)

Beattie, Iain; Smith, Aaron; Weston, Daniel J; White, Peter; Szwandt, Simon; Sealey, Laura

2012-02-05

Within the drug discovery environment, the key process in optimising the chemistry of a structural series toward a potential drug candidate is the design, make and test cycle, in which the primary screens consist of a number of in vitro assays, including metabolic stability, cytochrome P450 inhibition, and time-dependent inhibition assays. These assays are often carried out using multiple drug compounds with chemically diverse structural features, often in a 96 well-plate format for maximum time-efficiency, and are supported using rapid liquid chromatographic (LC) sample introduction with a tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) endpoint, taking around 6.5 h per plate. To provide a faster time-to-decision at this critical point, there exists a requirement for higher sample throughput and a robust, well-characterized analytical alternative. This paper presents a detailed evaluation of laser diode thermal desorption (LDTD), a relatively new ambient sample ionization technique, for compound screening assays. By systematic modification of typical LDTD instrumentation and workflow, and providing deeper understanding around overcoming a number of key issues, this work establishes LDTD as a practical, rapid alternative to conventional LC-MS/MS in drug discovery, without need for extensive sample preparation or expensive, scope-limiting internal standards. Analysis of both the five and three cytochrome P450 competitive inhibition assay samples by LDTD gave improved sample throughput (0.75 h per plate) and provided comparable data quality as the IC₅₀ values obtained were within 3 fold of those calculated from the LC-MS/MS data. Additionally when applied generically to a chemically diverse library of over 250 proprietary compounds from the AstraZeneca design, make and test cycle, LDTD demonstrated a success rate of 98%. Copyright © 2011 Elsevier B.V. All rights reserved.

Relative mass resolution technique for optimum design of a gamma nondestructive assay system

International Nuclear Information System (INIS)

Koh, Duck Joon

1995-02-01

Nondestructive assay(NDA) is a widely used nuclear technology for quantitative elemental and isotopic assay. Nondestructive assay is performed by the detection of an identifying radiation emerging from the sample, which can be unambiguously related to the element or isotope of interest. In every assay we can identify two distinct factors that lead to measurement uncertainty. We refer to these as statistical and spatial uncertainties. If the spatial distribution of the analyte and the matrix material in the sample are known and fairly constant from sample to sample, then the major source of measurement uncertainty is the statistical uncertainty resulting from randomness in the counting process. The spatial uncertainty is independent of the measurement time and therefore sets a lower limit to the measurement uncertainty, which is inherent in the assay system in conjunction with the population of samples to be measured. The only way to minimize the spatial uncertainty is an optimized design of the assay system. Therefore we have to decide on the type and number of detectors to be used, their deployment around the sample, the type of radiation to be measured, the duration of each measurement, the size and shape of the sample drum. The design procedure leading to the optimal assay system should be based on a quantitative(RMR:Relative Mass Resolution) comparison of the performance of each proposed design. For NDA system design of low level radwaste, a specific purpose Monte Carlo code has been developed to simulate point-source responses for sources within an assayed radwaste drum and to analyze the effect of scattered gammas from higher energy gammas on the spectrum of a low energy gamma-ray. We could use the well-known Monte Carlo code, such as MCNP for the simulation of NDA in the case of low level radwaste. But, MCNP is a multi-purpose Monte Carlo transport code for several geometries which requires large memory and long CPU time. For some cases in nuclear

An improved in vitro micronucleus assay to biological dosimetry

International Nuclear Information System (INIS)

Ocampo, Ivette Z.; Okazaki, Kayo; Vieira, Daniel P.

2013-01-01

The biological dosimetry is widely used to estimate the absorbed dose in people occupationally or accidentally exposed to the radiation for a better medical treatment, minimizing the harmful effects. Many techniques and methods have been proposed to detect and quantify the radioinduced lesions in genetic material, among them, the micronucleus (MN) assay. In the present study, we proposed an improved in vitro micronucleus technique that is rapid, sensitive and with minor cell manipulations. Assays were carried out with human tumor cells (MCF-7) seeded (3x10 4 cells) in slides placed into Petri dishes. Adherent cells were maintained with RPMI medium, supplemented with fetal calf serum, 1 % antibiotics, cytochalasin B (2 μg/mL), and incubated at 37 deg C in the presence of 5% CO2 for 72h. Cells were pre-treated for 24h with aminoguanidine, a nitric oxide synthase inhibitor. Nitric oxide is an intracellular free-radical, involved in DNA double-strand break repair mechanisms. After incubation, adherent cells on slides were briefly fixed with paraformaldehyde and stained with acridine orange (100 μg/mL) for analysis through fluorescence microscopy. Dye fluorescence permitted accurate discrimination between nuclei and micronuclei (bright green) and cytoplasm (red), and made possible a faster counting of binucleated cells. Aminoguanidine (2 mM) induced significant increase (p< 0.05) in frequencies of binucleated cells with micronuclei and in the number of micronuclei per binucleated cell. Data showed that proposed modifications permit to understand an early aspect of NO inhibition and suggested an improved protocol to MN assays. (author)

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

Science.gov (United States)

Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

2011-01-01

Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus. PMID:22363089

Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

Directory of Open Access Journals (Sweden)

Ya-Bing Duan

Full Text Available Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP with hydroxynaphthol blue dye (HNB. The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3 ng µL(-1 of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2 ng µL(-1. Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2% were confirmed as positive by LAMP, 172 (90.1% positive by the tissue separation, while 147 (77.0% positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

High-sensitive and rapid detection of Mycobacterium tuberculosis infection by IFN-γ release assay among HIV-infected individuals in BCG-vaccinated area

Directory of Open Access Journals (Sweden)

Jiang Weimin

2009-05-01

Full Text Available Abstract Background An accurate test for Mycobacterium tuberculosis infection is urgently needed in immunosuppressed populations. The aim of this study was to investigate the diagnostic power of enzyme-linked immunospot (ELISPOT-based IFN-γ release assay in detecting active and latent tuberculosis in HIV-infected population in bacillus Calmette-Guerin (BCG-vaccinated area. A total of 100 HIV-infected individuals including 32 active tuberculosis patients were recruited. An ELISPOT-based IFN-γ release assay, T-SPOT.TB, was used to evaluate the M. tuberculosis ESAT-6 and CFP-10 specific IFN-γ response. Tuberculin skin test (TST was performed for all recruited subjects. Results The subjects were divided into group HIV+ATB (HIV-infected individuals with active tuberculosis, n = 32, group HIV+LTB (HIV-infected individuals with positive results of T-SPOT.TB assay, n = 46 and group HIV only (HIV-infected individuals with negative results of T-SPOT.TB assay and without evidence of tuberculosis infection, n = 22. In group HIV+ATB and HIV+LTB, T-SPOT.TB positive rate in subjects with TST P 85% in patients with TB treatment for less than 1 month and CD4+ T cells ≥200/μl, while for patients treated for more than 3 months and CD4+ T cells Conclusion ELISPOT-based IFN-γ release assay is more sensitive and rapid for the diagnosis of TB infection in Chinese HIV-infected individuals with history of BCG vaccination, and could be an effective tool for guiding preventive treatment with isoniazid in latently infected people and for TB control in China.

A new real-time PCR assay for rapid identification of the S. aureus/MRSA strains

Directory of Open Access Journals (Sweden)

Ivan Manga

2013-01-01

Full Text Available The Methicillin-resistant Staphylococcus aureus (MRSA with the livestock-associated MRSA (LA-MRSA are of great interest to scientists and general public. The aim of our study was to present a new more rapid and reliable diagnostic method working on the RT-PCR platform applicable for monitoring of MRSA/S. aureus. The parallel testing of the S. aureus specific nuc gene sequence and the mecA gene sequence was utilised for this purpose. A collection of ten S. aureus/MRSA reference strains, fifteen genetically related non S. aureus reference strains and fifty-six environmental samples was employed for estimation of the assay performance and parameters. The environmental samples acquired in the Czech livestock farms were represented with the livestock and human nasal mucosae or skin swabs, the slaughter meat swabs and were chosen preferentially from individuals with previously confi rmed or suspected positive MRSA/S. aureus cases. The classic selective cultivation approach with the biochemical test and agar disk diffusion test was accepted as reference diagnostic method. As there were no culture positive samples that were negative using RT-PCR, our method featured with 100% sensitivity in comparison to reference method. The limit of detection allowed to identify from tens to hundreds copies of S. aureus/MRSA genome. Further, the RT-PCR assay featured with 100% inclusivity and 95% exclusivity at Cq value below 30. These parameters suggested on powerful and reliable diagnostic method with real potential of practical utilisation. We consider our method as ideal for testing of individual suspected colonies, when the results can be acquired in less than 1.5 hour.

Development of Experimental Setup of Metal Rapid Prototyping Machine using Selective Laser Sintering Technique

Science.gov (United States)

Patil, S. N.; Mulay, A. V.; Ahuja, B. B.

2018-04-01

Unlike in the traditional manufacturing processes, additive manufacturing as rapid prototyping, allows designers to produce parts that were previously considered too complex to make economically. The shift is taking place from plastic prototype to fully functional metallic parts by direct deposition of metallic powders as produced parts can be directly used for desired purpose. This work is directed towards the development of experimental setup of metal rapid prototyping machine using selective laser sintering and studies the various parameters, which plays important role in the metal rapid prototyping using SLS technique. The machine structure in mainly divided into three main categories namely, (1) Z-movement of bed and table, (2) X-Y movement arrangement for LASER movements and (3) feeder mechanism. Z-movement of bed is controlled by using lead screw, bevel gear pair and stepper motor, which will maintain the accuracy of layer thickness. X-Y movements are controlled using timing belt and stepper motors for precise movements of LASER source. Feeder mechanism is then developed to control uniformity of layer thickness metal powder. Simultaneously, the study is carried out for selection of material. Various types of metal powders can be used for metal RP as Single metal powder, mixture of two metals powder, and combination of metal and polymer powder. Conclusion leads to use of mixture of two metals powder to minimize the problems such as, balling effect and porosity. Developed System can be validated by conducting various experiments on manufactured part to check mechanical and metallurgical properties. After studying the results of these experiments, various process parameters as LASER properties (as power, speed etc.), and material properties (as grain size and structure etc.) will be optimized. This work is mainly focused on the design and development of cost effective experimental setup of metal rapid prototyping using SLS technique which will gives the feel of

Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations.

Science.gov (United States)

You, David J; Yoon, Jeong-Yeol

2012-09-04

A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of

Determining plutonium mass in spent fuel with non-destructive assay techniques - NGSU research overview and update on 6 NDA techniques

Energy Technology Data Exchange (ETDEWEB)

Tobin, Stephen J [Los Alamos National Laboratory; Conlin, Jeremy L [Los Alamos National Laboratory; Evans, Louise G [Los Alamos National Laboratory; Hu, Jianwei [Los Alamos National Laboratory; Blanc, Pauline C [Los Alamos National Laboratory; Lafleur, Adrienne M [Los Alamos National Laboratory; Menlove, Howard O [Los Alamos National Laboratory; Schear, Melissa A [Los Alamos National Laboratory; Swinhoe, Martyn T [Los Alamos National Laboratory; Croft, Stephen [Los Alamos National Laboratory; Fensin, Michael L [Los Alamos National Laboratory; Freeman, Corey R [Los Alamos National Laboratory; Koehler, William E [Los Alamos National Laboratory; Mozin, V [Los Alamos National Laboratory; Sandoval, N P [Los Alamos National Laboratory; Lee, T H [KAERI; Cambell, L W [PNNL; Cheatham, J R [ORNL; Gesh, C J [PNNL; Hunt, A [IDAHO STATE UNIV; Ludewigt, B A [LBNL; Smith, L E [PNNL; Sterbentz, J [INL

2010-09-15

This poster is one of two complementary posters. The Next Generation Safeguards Initiative (NGSI) of the U.S. DOE has initiated a multi-lab/university collaboration to quantify the plutonium (Pu) mass in, and detect the diversion of pins from, spent nuclear fuel assemblies with non-destructive assay (NDA). This research effort has the goal of quantifying the capability of 14 NDA techniques as well as training a future generation of safeguards practitioners. By November of 2010, we will be 1.5 years into the first phase (2.5 years) of work. This first phase involves primarily Monte Carlo modelling while the second phase (also 2.5 years) will focus on experimental work. The goal of phase one is to quantify the detection capability of the various techniques for the benefit of safeguard technology developers, regulators, and policy makers as well as to determine what integrated techniques merit experimental work, We are considering a wide range of possible technologies since our research horizon is longer term than the focus of most regulator bodies. The capability of all of the NDA techniques will be determined for a library of 64 17 x 17 PWR assemblies [burnups (15, 30, 45, 60 GWd/tU), initial enrichments (2, 3, 4, 5%) and cooling times (1, 5, 20, 80 years)]. The burnup and cooling time were simulated with each fuel pin being comprised of four radial regions. In this paper an overview of the purpose will be given as well as a technical update on the following 6 neutron techniques: {sup 252}Cf Interrogation with Prompt Neutron Detection, Delayed Neutrons, Differential Die-Away, Differential Die-Away Self-Interrogation, Passive Neutron Albedo Reactivity, Self-Integration Neutron Resonance Densitometry. The technical update will quantify the anticipated performance of each technique for the 64 assemblies of the spent fuel library.

Determining plutonium mass in spent fuel with non-destructive assay techniques - NGSI research overview and update on 6 NDA techniques

International Nuclear Information System (INIS)

Tobin, Stephen J.; Conlin, Jeremy L.; Evans, Louise G.; Hu, Kianwei; Blanc, P.C.; Lafleur, Am; Menlove, H.O.; Schear, M.A.; Swinhoe, M.T.; Croft, S.; Fensin, M.L.; Freeman, C.R.; Koehler, W.E.; Mozin, V.; Sandoval, N.P.; Lee, T.H.; Cambell, L.W.; Cheatham, J.R.; Gesh, C.J.; Hunt, A.; Ludewigt, B.A.; Smith, L.E.; Sterbentz, J.

2010-01-01

This poster is one of two complementary posters. The Next Generation Safeguards Initiative (NGSI) of the U.S. DOE has initiated a multi-lab/university collaboration to quantify the plutonium (Pu) mass in, and detect the diversion of pins from, spent nuclear fuel assemblies with non-destructive assay (NDA). This research effort has the goal of quantifying the capability of 14 NDA techniques as well as training a future generation of safeguards practitioners. By November of 2010, we will be 1.5 years into the first phase (2.5 years) of work. This first phase involves primarily Monte Carlo modelling while the second phase (also 2.5 years) will focus on experimental work. The goal of phase one is to quantify the detection capability of the various techniques for the benefit of safeguard technology developers, regulators, and policy makers as well as to determine what integrated techniques merit experimental work, We are considering a wide range of possible technologies since our research horizon is longer term than the focus of most regulator bodies. The capability of all of the NDA techniques will be determined for a library of 64 17 x 17 PWR assemblies (burnups (15, 30, 45, 60 GWd/tU), initial enrichments (2, 3, 4, 5%) and cooling times (1, 5, 20, 80 years)). The burnup and cooling time were simulated with each fuel pin being comprised of four radial regions. In this paper an overview of the purpose will be given as well as a technical update on the following 6 neutron techniques: 252 Cf Interrogation with Prompt Neutron Detection, Delayed Neutrons, Differential Die-Away, Differential Die-Away Self-Interrogation, Passive Neutron Albedo Reactivity, Self-Integration Neutron Resonance Densitometry. The technical update will quantify the anticipated performance of each technique for the 64 assemblies of the spent fuel library.

A rapid and sensitive method for measuring N-acetylglucosaminidase activity in cultured cells.

Directory of Open Access Journals (Sweden)

Victor Mauri

Full Text Available A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG, in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB, a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in

Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

Science.gov (United States)

Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

2010-05-26

Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

Rapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCR

Directory of Open Access Journals (Sweden)

Cattoir Vincent

2010-08-01

Full Text Available Abstract Background Pseudomonas aeruginosa is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR assay, using ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive blood cultures (BCs. Methods Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification. Results Thirty-three strains of P. aeruginosa, 53 strains of Enterobactericaeae, nine strains of Stenotrophomonas maltophilia and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing P. aeruginosa. All P. aeruginosa clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%. Conclusions This reliable technique may offer a rapid (

Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease.

Science.gov (United States)

Puenpa, Jiratchaya; Suwannakarn, Kamol; Chansaenroj, Jira; Vongpunsawad, Sompong; Poovorawan, Yong

2017-10-01

Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid, quantitative and sensitive immunochromatographic assay based on stripping voltammetric detection of a metal ion label

Energy Technology Data Exchange (ETDEWEB)

Lu, Fang; Wang, Kaihua; Lin, Yuehe

2005-10-10

A novel, sensitive immunochromatographic electrochemical biosensor (IEB) which combines an immunochromatographic strip technique with an electrochemical detection technique is demonstrated. The IEB takes advantages of the speed and low-cost of the conventional immunochromatographic test kits and high-sensitivity of stripping voltammetry. Bismuth ions (Bi3+) have been coupled with the antibody through the bifunctional chelating agent diethylenetriamine pentaacetic acid (DTPA). After immunoreactions, Bi3+ was released and quantified by anodic stripping voltammetry at a built-in single-use screen-printed electrode. As an example for the applications of such novel device, the detection of human chorionic gonadotronphin (HCG) in a specimen was performed. This biosensor provides a more user-friendly, rapid, clinically accurate, and less expensive immunoassay for such analysis in specimens than currently available test kits.

Rapid Detection of Enterobacter Sakazakii in milk Powder using amino modified chitosan immunomagnetic beads.

Science.gov (United States)

Zhu, Yinglian; Wang, Dongfeng

2016-12-01

Chitosan immunomagnetic beads (CIBs) were first prepared through converting hydroxyl groups of natural polymer material-chitosan into amino groups using epichlorohydrin and ethylenediamine as modification agent and then coupling with polyclonal antibodies of Enterobacter sakazakii using glutaraldehyde as cross-linking agent. The beads before coupling with antibodies were characterized by magnetic property measurement, FTIR, SEM and XRD technologies. In the assay a natural polysaccharide-chitosan, which has good biological and chemical properties such as non-toxicity, biocompatibility and high chemical reactivity was first used for synthesis of immunomagnetic beads. The detection method first established in this paper that combined the beads with chromogenic medium together to rapid detect E. sakazakii in milk powder could greatly improve the detection specificity and working efficiency. The beads exhibited a maximum capturing capacity of 1×10 6 cfu/g with the detection sensitivity of 4cfu/g. The results demonstrate that the assay is a straightforward, specific and sensitive alternative for rapid detection of E.sakazakii in food matrix. The total analysis time was as little as about 25h, which greatly shorten the detection time. The method can provides new ideas not only to preparation technique of immunomagnetic beads but to imunne detection technique in food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a rapid immunochromatographic assay to detect contamination of raw oysters with enteropathogenic Vibrio parahaemolyticus.

Science.gov (United States)

Sakata, Junko; Yonekita, Taro; Kawatsu, Kentaro

2018-01-02

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of enteropathogenic Vibrio parahaemolyticus. TDH and TRH are bacterial exotoxins, and their presence in culture medium serves as a specific marker for detecting this significant pathogen. Here, we developed and evaluated an immunochromatographic assay (TDH/TRH-ICA) to simultaneously or individually detect TDH and TRH. The TDH/TRH-ICA detected TDH in all broth cultures of 47 V. parahaemolyticus strains carrying tdh. The genes encoding TRH are classified as variants trh1 and trh2, and TRH was detected in all broth cultures of 25 V. parahaemolyticus strains carrying trh1 and certain proportion (5/31) of broth cultures of V. parahaemolyticus strains carrying trh2. In contrast, TDH and TRH were not detected in broth cultures of 12 non-enteropathogenic V. parahaemolyticus strains without tdh and trh. It was difficult to detect TRH2 using the TDH/TRH-ICA. However, TRH2 may not serve as a suitable marker for detecting enteropathogenic V. parahaemolyticus, and evidence indicates that TRH2 may not contribute to enteropathogenesis. Further, a screening method using a combination of TDH/TRH-ICA and SPP medium supplemented with 1.5% NaCl (modified-SPP medium) detected oyster samples artificially spiked with 1.1-22 colony-forming units of enteropathogenic V. parahaemolyticus per 25g of oysters within approximately 8.5h, including the enrichment culture. The assay may serve as a method that facilitates the rapid and easy detection of raw oysters contaminated with enteropathogenic V. parahaemolyticus. Copyright © 2017. Published by Elsevier B.V.

Evaluation of the BYG Carba Test, a New Electrochemical Assay for Rapid Laboratory Detection of Carbapenemase-Producing Enterobacteriaceae

Science.gov (United States)

Yunus, Sami; Massart, Marion; Huang, Te-Din; Glupczynski, Youri

2015-01-01

Accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) constitutes a major laboratory diagnostic challenge. We evaluated an electrochemical technique (the BYG Carba test) which allows detection of CPE in less than 35 min. The BYG Carba test was first validated in triplicate against 57 collection isolates with previously characterized β-lactam resistance mechanisms (OXA-48, n = 12; KPC, n = 8; NDM, n = 8; VIM, n = 8; IMP, n = 3; GIM, n = 1; GES-6, n = 1; no carbapenemase, n = 16) and against a panel of 10 isolates obtained from the United Kingdom National External Quality Assessment Service (NEQAS). The test was then evaluated prospectively against 324 isolates referred to the national reference center for suspicion of CPE. The BYG Carba test results were compared with those obtained with the Carba NP test using multiplex PCR sequencing as the gold standard. Of the 57 collection and the 10 NEQAS isolates, all but one GES-6-producing isolate were correctly identified by the Carba BYG test. Among the 324 consecutive Enterobacteriaceae isolates tested prospectively, 146 were confirmed as noncarbapenemase producers by PCR while 178 harbored a carbapenemase gene (OXA-48, n = 117; KPC, n = 25; NDM, n = 23; and VIM, n = 13). Prospectively, in comparison with PCR results, the BYG Carba test displayed 95% sensitivity and 100% specificity versus 89% and 100%, respectively, for the Carba NP test. The BYG Carba test is a novel, rapid, and efficient assay based on an electro-active polymer biosensing technology discriminating between CPE and non-CPE. The precise electrochemical signal (electrochemical impedance variations) allows the establishment of real-time objective measurement and interpretation criteria which should facilitate the accreditation process of this technology. PMID:26637378

Rapid Automated Dissolution and Analysis Techniques for Radionuclides in Recycle Process Streams

International Nuclear Information System (INIS)

Sudowe, Ralf; Roman, Audrey; Dailey, Ashlee; Go, Elaine

2013-01-01

The analysis of process samples for radionuclide content is an important part of current procedures for material balance and accountancy in the different process streams of a recycling plant. The destructive sample analysis techniques currently available necessitate a significant amount of time. It is therefore desirable to develop new sample analysis procedures that allow for a quick turnaround time and increased sample throughput with a minimum of deviation between samples. In particular, new capabilities for rapid sample dissolution and radiochemical separation are required. Most of the radioanalytical techniques currently employed for sample analysis are based on manual laboratory procedures. Such procedures are time- and labor-intensive, and not well suited for situations in which a rapid sample analysis is required and/or large number of samples need to be analyzed. To address this issue we are currently investigating radiochemical separation methods based on extraction chromatography that have been specifically optimized for the analysis of process stream samples. The influence of potential interferences present in the process samples as well as mass loading, flow rate and resin performance is being studied. In addition, the potential to automate these procedures utilizing a robotic platform is evaluated. Initial studies have been carried out using the commercially available DGA resin. This resin shows an affinity for Am, Pu, U, and Th and is also exhibiting signs of a possible synergistic effects in the presence of iron.

Large-volume constant-concentration sampling technique coupling with surface-enhanced Raman spectroscopy for rapid on-site gas analysis

Science.gov (United States)

Zhang, Zhuomin; Zhan, Yisen; Huang, Yichun; Li, Gongke

2017-08-01

In this work, a portable large-volume constant-concentration (LVCC) sampling technique coupling with surface-enhanced Raman spectroscopy (SERS) was developed for the rapid on-site gas analysis based on suitable derivatization methods. LVCC sampling technique mainly consisted of a specially designed sampling cell including the rigid sample container and flexible sampling bag, and an absorption-derivatization module with a portable pump and a gas flowmeter. LVCC sampling technique allowed large, alterable and well-controlled sampling volume, which kept the concentration of gas target in headspace phase constant during the entire sampling process and made the sampling result more representative. Moreover, absorption and derivatization of gas target during LVCC sampling process were efficiently merged in one step using bromine-thiourea and OPA-NH4+ strategy for ethylene and SO2 respectively, which made LVCC sampling technique conveniently adapted to consequent SERS analysis. Finally, a new LVCC sampling-SERS method was developed and successfully applied for rapid analysis of trace ethylene and SO2 from fruits. It was satisfied that trace ethylene and SO2 from real fruit samples could be actually and accurately quantified by this method. The minor concentration fluctuations of ethylene and SO2 during the entire LVCC sampling process were proved to be samples were achieved in range of 95.0-101% and 97.0-104% respectively. It is expected that portable LVCC sampling technique would pave the way for rapid on-site analysis of accurate concentrations of trace gas targets from real samples by SERS.

Scoping study to expedite development of a field deployable and portable instrument for UF6 enrichment assay

OpenAIRE

Chan, CYG; Valentine, JD; Russo, RE

2017-01-01

The primary objective of the present study is to identity the most promising, viable technologies that are likely to culminate in an expedited development of the next-generation, field-deployable instrument for providing rapid, accurate, and precise enrichment assay of uranium hexafluoride (UF6). UF6 is typically involved, and is arguably the most important uranium compound, in uranium enrichment processes. As the first line of defense against proliferation, accurate analytical techniques t...

Novel immunoradiometric assay of thyroglobulin in serum with use of monoclonal antibodies selected for lack of cross-reactivity with autoantibodies

International Nuclear Information System (INIS)

Piechaczyk, M.; Baldet, L.; Pau, B.; Bastide, J.M.

1989-01-01

A multisite immunoradiometric assay for measurement of serum thyroglobulin (Tg), designated Magnogel-IRMA-Tg, has been developed, involving magnetic microbeads (Magnogel). This assay is based on the use of five anti-Tg monoclonal antibodies (MAbs) directed against three antigenic regions on the Tg molecule that are not recognized by anti-Tg autoantibodies (aAbs). Four of these MAbs, directed against two antigenic domains, were coupled to the magnetic beads and were used to trap the serum antigen. Another MAb, directed against the third region, was iodinated and served as the labeled second antibody. The Magnogel-IRMA-Tg technique is reproducible, rapid, and sensitive (lower detection limit, 3 micrograms/L). The assay reliably measures serum Tg in the presence of anti-Tg aAbs

Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

Directory of Open Access Journals (Sweden)

Zhang PF

2015-09-01

Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

Science.gov (United States)

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics

Integration of novel low-cost colorimetric, laser photometric, and visual fluorescent techniques for rapid identification of falsified medicines in resource-poor areas: application to artemether-lumefantrine.

Science.gov (United States)

Green, Michael D; Hostetler, Dana M; Nettey, Henry; Swamidoss, Isabel; Ranieri, Nicola; Newton, Paul N

2015-06-01

The availability of falsified antimalarial drugs can be reduced with effective drug regulatory agencies and proper enforcement. Fundamental to these agencies taking action, rapid identification must be made as soon as they appear in the market place. Since falsified antimalarials occur mostly in developing countries, performing drug analysis presents itself with unique challenges. A fundamental factor in choosing a useful technique is affordability and simplicity. Therefore, we suggest a three-tiered drug evaluation strategy for identifying a falsified drug in resource-poor areas. Tier I is a simple comparison of a tablet's weight and dimensions with official specifications. Tier II uses inexpensive photometric devices (laser and fluorescence) to evaluate a tablet. Suspicious samples from Tier I and II assessments are then subjected to a colorimetric assay for active ingredients identification and quantification. In this article, we evaluate a novel colorimetric assay for the simultaneous assessment of both lumefantrine and artemether in co-formulated Coartem™ tablets, and integrate the method with two novel, low-cost, fluorescence and laser photometric devices. Image analysis software is used for the assessments. Although artemether-lumefantrine is used as an example, the strategy may be adapted to other medicines. © The American Society of Tropical Medicine and Hygiene.

Towards the development of rapid screening techniques for shale gas core properties

Science.gov (United States)

Cave, Mark R.; Vane, Christopher; Kemp, Simon; Harrington, Jon; Cuss, Robert

2013-04-01

Shale gas has been produced for many years in the U.S.A. and forms around 8% of total their natural gas production. Recent testing for gas on the Fylde Coast in Lancashire UK suggests there are potentially large reserves which could be exploited. The increasing significance of shale gas has lead to the need for deeper understanding of shale behaviour. There are many factors which govern whether a particular shale will become a shale gas resource and these include: i) Organic matter abundance, type and thermal maturity; ii) Porosity-permeability relationships and pore size distribution; iii) Brittleness and its relationship to mineralogy and rock fabric. Measurements of these properties require sophisticated and time consuming laboratory techniques (Josh et al 2012), whereas rapid screening techniques could provide timely results which could improve the efficiency and cost effectiveness of exploration. In this study, techniques which are portable and provide rapid on-site measurements (X-ray Fluorescence (XRF) and Infra-red (IR) spectroscopy) have been calibrated against standard laboratory techniques (Rock-Eval 6 analyser-Vinci Technologies) and Powder whole-rock XRD analysis was carried out using a PANalytical X'Pert Pro series diffractometer equipped with a cobalt-target tube, X'Celerator detector and operated at 45kV and 40mA, to predict properties of potential shale gas material from core material from the Bowland shale Roosecote, south Cumbria. Preliminary work showed that, amongst various mineralogical and organic matter properties of the core, regression models could be used so that the total organic carbon content could be predicted from the IR spectra with a 95 percentile confidence prediction error of 0.6% organic carbon, the free hydrocarbons could be predicted with a 95 percentile confidence prediction error of 0.6 mgHC/g rock, the bound hydrocarbons could be predicted with a 95 percentile confidence prediction error of 2.4 mgHC/g rock, mica content

Development of an in vitro Assay, based on the BioFilm Ring Test®, for Rapid Profiling of Biofilm-Growing Bacteria

Directory of Open Access Journals (Sweden)

Enea Gino Di Domenico

2016-09-01

Full Text Available Microbial biofilm represents a major virulence factor associated with chronic and recurrent infections. Pathogenic bacteria embedded in biofilms are highly resistant to environmental and chemical agents, including antibiotics and therefore difficult to eradicate. Thus, reliable tests to assess biofilm formation by bacterial strains as well as the impact of chemicals or antibiotics on biofilm formation represent desirable tools for a most effective therapeutic management and microbiological risk control. Current methods to evaluate biofilm formation are usually time-consuming, costly, and hardly applicable in the clinical setting.The aim of the present study was to develop and assess a simple and reliable in vitro procedure for the characterization of biofilm-producing bacterial strains for future clinical applications based on the BioFilm Ring Test® (BRT technology. The procedure developed for clinical testing (cBRT can provide an accurate and timely (5 hours measurement of biofilm formation for the most common pathogenic bacteria seen in clinical practice. The results gathered by the cBRT assay were in agreement with the traditional crystal violet (CV staining test, according to the kappa coefficient test (kappa = 0.623. However, the cBRT assay showed higher levels of specificity (92.2% and accuracy (88.1% as compared to CV. The results indicate that this procedure offers an easy, rapid and robust assay to test microbial biofilm and a promising tool for clinical microbiology.

Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment

Directory of Open Access Journals (Sweden)

I.C. Oliveira

2012-09-01

Full Text Available A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8 pfu/mL without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL, after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.

Solid phase radioimmunoassay for quantitation of IgM rheumatoid factor (RF). Comparison with agglutination techniques and radioimmunoprecipitation polyethylene glycol assay (RIPEGA)

Energy Technology Data Exchange (ETDEWEB)

Herrmann, D.; Jaeger, L.; Hein, G.; Henzgen, M.; Fiebig, H.; Schlenvoigt, G.; Vogelsang, H. (Friedrich-Schiller-Universitaet, Jena (German Democratic Republic). Bereich Medizin; Karl-Marx-Universitaet, Leipzig (German Democratic Republic). Sektion Biowissenschaften)

1985-01-01

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor using a monoclonal anti-..mu..-chain antibody is described. Human IgG did not interfere with the detection of IgM RF by this method. The small nonspecific binding of nonRF IgM to the human IgG coated tubes utilized in the assay must be corrected for by assaying samples in parallel bovine serum albumin coated control tubes only in cases of deviation of IgM from normal range. 69 coded and randomly arranged sera from patients with rheumatoid arthritis (RA), nonrheumatic joint diseases and healthy adult control subjects were investigated by this method, agglutination techniques as well as RIPEGA. A good correlation between solid-phase radioimmunoassay and agglutination techniques was found. Patients with seropositive RA had significantly higher concentrations of IgM RF than seronegative RA patients or control subjects.

ZyFISH: a simple, rapid and reliable zygosity assay for transgenic mice.

Directory of Open Access Journals (Sweden)

Donal McHugh

Full Text Available Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH. The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.

A rapid two dot filter assay for the detection of E. coli O157 in water samples.

Science.gov (United States)

Kamma, Sujatha; Tang, Lily; Leung, Kelvin; Ashton, Edie; Newman, Norman; Suresh, Mavanur R

2008-07-31

E. coli O157:H7 is an enterohemorrhagic bacteria that cause deadly water-borne infections implicated in outbreaks of a wide spectrum of human gastrointestinal diseases. It is therefore important to have a rapid convenient, simple and sensitive range of detection of E. coli O157:H7. A new E. coli O157 MAb designated P124 was developed for ultrasensitive detection of E. coli O157 in water, apple juice and beef for routine use. A prototype filter dot assay was designed with anti-E. coli O157 MAb bound to 0.2 microm nitrocellulose filter disk as the capture antibody. A 100 ml water sample spiked with 1-50 CFU of E. coli O157 either in the presence or absence of other non-specific bacteria were filtered for capture of the pathogen on the antibody coated nitrocellulose disk. The detection of the pathogen was successfully accomplished by the same antibody both as a capture and detecting antibody as a homosandwich. In a non-enriched format, detection of E. coli was possible with a sensitivity of 2500 CFU/100 ml. Ultrasensitive detection of ~1 CFU/100 ml sample could be achieved by a prior pathogen enrichment step before the addition of the labeled antibody. The design of this diagnostic test is based on the common architecture of all bacteria, viruses and spores, namely the manifestation of repeat lipopolysaccharide epitopes on the surface. We have developed an easy-to-use two dot visual filter assay for translation into current water testing in public health laboratories to detect E. coli O157:H7. In a 5 h assay approximately 1 CFU and approximately 5 CFU of E. coli O157 could be detected in 100 ml of water or juice and lake samples respectively. This simple homosandwich enrichment strategy can also be used to detect low levels of other water-borne pathogens.

The narrow therapeutic window of glycated hemoglobin and assay variability.

Science.gov (United States)

Hosseini, S S; Bibler, I; Charles, M A

1999-12-01

Glycated hemoglobin is measured by a variety of assays, each of which has a unique normal level. Our purpose is to show that among the different assays available in the United States, using the same patient's blood sample, assay results may vary widely and may more or less easily achieve a glycated hemoglobin value within the normal range. The following assays were compared using the same patient's blood sample for each pair of assays: glycohemoglobin affinity assay (GHB Reader; Isolab, Akron, OH) versus gel electrophoresis assay (n = 76); Isolab versus ion capture assay (IMX; Abbott Laboratories, Irving, TX) (n = 57); monoclonal antibody assay (DCA2000; Bayer Diagnostics, Pittsburgh, PA) versus IMX (n = 100); and high-performance liquid chromatography (HPLC) assay (Bio-Rad Variant A1c; Bio-Rad Laboratories, Richmond, CA) versus IMX assay (n = 55). Our analyses indicate that a relative ranking can be established for the ease of achieving a normal glycated hemoglobin level. The ranking indicates that the most stringent or difficult assays for achieving a normal level are the Isolab and DCA2000 assays. The intermediate assays are the IMX and Bio-Rad Variant, and the easiest method for achieving a normal value is the gel electrophoresis assay. Our results indicate that various glycated hemoglobin assays vary widely and are associated with more or less difficulty for an individual patient to achieve a glycated hemoglobin level within the normal range. These results are especially significant with respect to (1) the clinically narrow therapeutic window of glycated hemoglobin values in type 1 diabetes to avoid rapidly advancing severe hypoglycemia rates and chronic microvascular complication rates, and (2) the glycated hemoglobin threshold for rapidly advancing macrovascular disease in both type 1 and type 2 patients.

A novel rapid direct haemagglutination-inhibition assay for measurements of humoral immune response against non-haemagglutinating Fowlpox virus strains in vaccinated chickens.

Science.gov (United States)

Wambura, Philemon N; Mzula, Alexanda

2017-10-01

Fowlpox (FP) is a serious disease in chickens caused by Fowlpox virus (FPV). One method currently used to control FPV is vaccination followed by confirmation that antibody titres are protective using the indirect haemagglutination assay (IHA). The direct haemagglutination inhibition (HI) assay is not done because most FPV strains do not agglutinate chicken red blood cells (RBCs). A novel FPV strain TPV-1 which agglutinates chicken RBCs was discovered recently and enabled a direct HI assay to be conducted using homologous sera. This study is therefore aimed at assessing the direct HI assay using a recently discovered novel haemagglutinating FPV strain TPV-1 in chickens vaccinated with a commercial vaccine containing a non-haemagglutinating FPV.Chicks vaccinated with FPV at 1 day-old had antibody geometric mean titres (GMT) of log 2 3.7 at 7 days after vaccination and log 2 8.0 at 28 days after vaccination when tested in the direct HI. Chickens vaccinated at 6 weeks-old had antibody geometric mean titres (GMT) of log 2 5.0 at 7 days after vaccination and log 2 8.4 at 28 days after vaccination when tested in the direct HI. The GMT recorded 28 days after vaccination was slightly higher in chickens vaccinated at 6-week-old than in chicks vaccinated at one-day-old. However, this difference was not significant (P > 0.05). All vaccinated chickens showed "takes". No antibody response to FPV and "takes" were detected in unvaccinated chickens (GMT 0.05). These findings indicate that a simple and rapid direct HI assay using the FPV TPV-1 strain as antigen may be used to measure antibody levels in chickens vaccinated with non-haemagglutinating strains of FPV, and that the titres are comparable to those obtained by indirect IHA.

Rover waste assay system

International Nuclear Information System (INIS)

Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

1997-01-01

The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

Surface plasmon resonance based biosensor: A new platform for rapid diagnosis of livestock diseases

Directory of Open Access Journals (Sweden)

Pravas Ranjan Sahoo

2016-12-01

Full Text Available Surface plasmon resonance (SPR based biosensors are the most advanced and developed optical label-free biosensor technique used for powerful detection with vast applications in environmental protection, biotechnology, medical diagnostics, drug screening, food safety, and security as well in livestock sector. The livestock sector which contributes the largest economy of India, harbors many bacterial, viral, and fungal diseases impacting a great loss to the production and productive potential which is a major concern in both small and large ruminants. Hence, an accurate, sensitive, and rapid diagnosis is required for prevention of these above-mentioned diseases. SPR based biosensor assay may fulfill the above characteristics which lead to a greater platform for rapid diagnosis of different livestock diseases. Hence, this review may give a detail idea about the principle, recent development of SPR based biosensor techniques and its application in livestock sector.

Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

Directory of Open Access Journals (Sweden)

You David J

2012-09-01

Full Text Available Abstract A computer numerical control (CNC apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using “wire-guided” method (a pipette tip was used in this study. This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate. Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction. The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability, in rapid succession (using droplets

Reduction of the use of antimicrobial drugs following the rapid detection of Streptococcus agalactiae in the vagina at delivery by real-time PCR assay.

Science.gov (United States)

Poncelet-Jasserand, E; Forges, F; Varlet, M-N; Chauleur, C; Seffert, P; Siani, C; Pozzetto, B; Ros, A

2013-08-01

To assess whether the determination of the presence of group B streptococci (GBS) in the vagina using a rapid polymerase chain reaction (PCR) assay at delivery was able to spare useless antimicrobial treatments, as compared with conventional culture at 34-38 weeks of gestation. Practical evaluation and prospective cost-effectiveness analysis. A university hospital in France. A cohort of 225 women in labour at the University-Hospital of Saint-Etienne. Each woman had a conventional culture performed at 34-38 weeks of gestation. At the beginning of labour, two vaginal swabs were sampled for rapid PCR testing and culture. The decision to prescribe a prophylactic antimicrobial treatment or not was taken according to the result of the PCR test. A comparative cost-effectiveness analysis of the two diagnostic strategies was carried out. Number of women receiving inadequate prophylactic antimicrobial drugs following each testing strategy, costs of PCR testing and culture, frequency of vaginal GBS, and diagnostic performance of the PCR test at delivery. The percentage of unnecessarily treated women was significantly reduced using the rapid test versus conventional culture (4.5 and 13.6%, respectively; P < 0.001). The rate of vaginal GBS at delivery was 12.5%. The incremental cost-effectiveness ratio (ICER) for each inadequate management avoided was €36 and €173 from the point of view of the healthcare system and hospital, respectively. The PCR assay reduced the number of inadequate antimicrobial treatments aimed to prevent the early onset of GBS disease. However, this strategy generates extra costs that must be put into balance with its clinical benefits. © 2013 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2013 RCOG.

Evaluation of the diagnostic performance and operational characteristics of four rapid immunochromatographic syphilis tests in Burkina Faso.

Science.gov (United States)

Bocoum, Fadima Yaya; Ouédraogo, Henri; Tarnagda, Grissoum; Kiba, Alice; Tiendrebeogo, Simon; Bationo, Fabrice; Liestman, Benjamin; Diagbouga, Serge; Zarowsky, Christina; Traoré, Ramata Ouédraogo; Kouanda, Séni

2015-06-01

Little information is available on the rapid diagnostic testing for syphilis in Burkina Faso. The objectives of the study were (i) to assess the sensitivity and specificity of four on site rapid tests in comparison with Treponema pallidum haemagglutination assay (TPHA) as a gold standard and (ii) to evaluate the operational characteristics of those tests among health workers in a maternity unit. Four rapid syphilis tests commercially available in Burkina Faso were evaluated using archived serum samples and Treponema pallidum hemagglutination assay (TPHA) as the gold standard. Blood samples were collected between November 2011 and June 2012 from blood donors at the Regional Blood Transfusion Center of Ouagadougou. The sensitivity and specificity of the tests were calculated. Evaluation of operational characteristics such as clarity of pamphlet, complexity of technique, duration, was conducted in a first-level healthcare center with health workers in maternity unit. Alere DetermineTM Syphilis was the most sensitive of the four rapid syphilis tests evaluated. It was followed by SD Bioline Syphilis 3.0, Cypress Diagnostics Syphilis Quick test and Accu-Tell ® Rapid Anti-TP, which was the least sensitive. The four tests demonstrated a good diagnostic specificity for syphilis (95-98%), and healthcare workers found them easy to use. The study allowed confirming the good performance of three of four rapid syphilis tests in Burkina Faso. More research will be conducted to assess the feasibility of introducing selected rapid tests for syphilis in antenatal care services.

Rapid Active Assay for the Detection of Antibodies to West Nile Virus in Chickens

National Research Council Canada - National Science Library

Groves, Stephanie S; Turell, Michael J; Bailey, Charles L; Morozov, Victor N

2008-01-01

... by detection of bound IgM molecules with functionalized magnetic beads as active labels. This assay takes only 15 minutes and has the same sensitivity as a commercially available human WNV IgM antibody-capture enzyme-linked immunosorbent assay...

Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of fasciolosis in sheep and its application under field conditions.

Science.gov (United States)

Martínez-Valladares, María; Rojo-Vázquez, Francisco Antonio

2016-02-05

Loop-mediated isothermal amplification (LAMP) is a very specific, efficient, and rapid gene amplification procedure in which the reaction can run at a constant temperature. In the current study we have developed a LAMP assay to improve the diagnosis of Fasciola spp. in the faeces of sheep. After the optimisation of the LAMP assay we have shown similar results between this technique and the standard PCR using the outer primers of the LAMP reaction. In both cases the limit of detection was 10 pg; also, the diagnosis of fasciolosis was confirmed during the first week post-infection in experimental infected sheep by both techniques. In eight naturally infected sheep, the infection with F. hepatica was confirmed in all animals before a treatment with triclabendazole and on day 30 post treatment in two sheep using the LAMP assay; however, when we carried out the standard PCR with the outer primers, the results before treatment were the same but on day 30 post-treatment the infection was only confirmed in one out of the two sheep. On the other hand, the standard PCR took around 3 h to obtain a result, comparing with 1 h and 10 min for the LAMP assay. The LAMP assay described here could be a good alternative to conventional diagnostic methods to detect F. hepatica in faeces since it solves the drawbacks of the standard PCR.

Localized irradiations, Evaluation through ''comet assay''

International Nuclear Information System (INIS)

Giorgio, M.D.; Taja, M.R.; Nasazzi, N.B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.

2000-01-01

During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources Co 60 , Cs 137 or Ir 192 at workplaces for industrial gammagraphy. Severe skin reaction may develop at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models ''Contaminated Poisson'' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. This give an indication of the proportion of haemopoietic stem cell compartment involved in the overexposure. There are also different biophysical techniques that can give responses in biological dosimetry. The ''Comet Assay'' (single cell gel electrophoresis) is a sensitive and rapid method for DNA strand break detection in individual cells. The advantages of the technique include: collection of data at the level of individual cell; the need for small numbers of cells per sample; its sensitivity for detecting DNA damage and that virtually any eukaryote cell population is amenable to analysis. The objective of this work is to apply ''Comet Assay'' method to evaluate the effect of radiation on skin and subcutaneous tissues, differentiating irradiated from unirradiated body areas. It could provide a useful tool to estimate the extension and the dose in the irradiated region, contributing with the current techniques. In this first study, we evaluate the alkaline comet assay as a method for detection of DNA radiation induced damage in keratinocytes from primary culture obtained from full thickness skin biopsies of patients requiring grafts. Skin and, particularly, keratinocytes were selected as an appropriate cellular system due to: Skin, the first barrier

Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

Energy Technology Data Exchange (ETDEWEB)

Cary,; Bruce, R [Santa Fe, NM; Stubben, Christopher J [Los Alamos, NM

2011-03-22

The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

International Nuclear Information System (INIS)

Garaj-Vrhovac, V.; Kopjar, N.

2003-01-01

Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

Multiplex real-time PCR assay for Legionella species.

Science.gov (United States)

Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

2015-12-01

Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development and evaluation of a rapid dipstick assay for serodiagnosis of acute human brucellosis

NARCIS (Netherlands)

Smits, H. L.; Basahi, M. A.; Díaz, R.; Marrodan, T.; Douglas, J. T.; Rocha, A.; Veerman, J.; Zheludkov, M. M.; Witte, O. W.; de Jong, J.; Gussenhoven, G. C.; Goris, M. G.; van der Hoorn, M. A.

1999-01-01

A dipstick assay for the detection of brucella-specific immunoglobulin M antibodies was evaluated with 707 sera from 247 laboratory-confirmed brucellosis patients and 342 control sera from brucellosis-free individuals. These sera were collected from six different countries. The assay was found to be

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

Science.gov (United States)

Quareshy, Mussa; Uzunova, Veselina; Prusinska, Justyna M; Napier, Richard M

2017-01-01

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

Radiometric assays for glycerol, glucose, and glycogen

International Nuclear Information System (INIS)

Bradley, D.C.; Kaslow, H.R.

1989-01-01

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays

Microsomal aryl hydrocarbon hydroxylase comparison of the direct, indirect and radiometric assays

International Nuclear Information System (INIS)

Denison, M.S.; Murray, M.; Wilkinson, C.F.

1983-01-01

The direct fluorometric assay of aryl hydrocarbon hydroxlyase has been compared to the more commonly used indirect fluorometric and radiometric assays. Although rat hepatic microsomal activities measured by the direct assay were consistently higher than those obtained by the other assays, the relative changes in activity following enzyme induction and/or inhibition were similar. The direct assay provides an accurate and rapid measure of aryl hydrocarbon hydroxylase activity and avoids several problems inherent in the indirect and radiometric assays. 2 tables

Magnetic particle separation technique: a reliable and simple tool for RIA/IRMA and quantitative PCR assay

International Nuclear Information System (INIS)

Shen Rongsen; Shen Decun

1998-01-01

Five types of magnetic particles without or with aldehyde, amino and carboxyl functional groups, respectively were used to immobilize first or second antibody by three models, i. e. physical adsorption, chemical coupling and immuno-affinity, forming four types of magnetic particle antibodies. The second antibody immobilized on polyacrolein magnetic particles through aldehyde functional groups and the first antibodies immobilized on carboxylic polystyrene magnetic particles through carboxyl functional groups were recommended to apply to RIAs and/or IRMAs. Streptavidin immobilized on commercial magnetic particles through amino functional groups was successfully applied to separating specific PCR product for quantification of human cytomegalovirus. In the paper typical data on reliability of these magnetic particle ligands were reported and simplicity of the magnetic particle separation technique was discussed. The results showed that the technique was a reliable and simple tool for RIA/IRMA and quantitative PCR assay. (author)

Rover waste assay system

Energy Technology Data Exchange (ETDEWEB)

Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

1997-11-01

The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

Time-resolved immunofluorometric assay of serum ferritin

Energy Technology Data Exchange (ETDEWEB)

Yan, Yao [China Inst. of Atomic Energy, Beijing (China)

2007-06-15

This assay is a solid phase, two-site fluoroimmunometric assay based on the direct sandwish technique. Standards or samples containing ferritin are first reacted with immobilized anti-ferritin antibodies. Then the europium-lablled antibodies are reacted with the bound antigen. The range of this assay is 2-1000 ng/mL. The analytical sentivity is better than 0.05 ng/mL. The intra-assay variation and inter-assay variation are both below 5%; This kit was compared with Wallac DELFIA kit. The correlation is r=0.96. (authors)

Evaluation of the Cepheid Xpert Flu Assay for rapid identification and differentiation of influenza A, influenza A 2009 H1N1, and influenza B viruses.

Science.gov (United States)

Novak-Weekley, S M; Marlowe, E M; Poulter, M; Dwyer, D; Speers, D; Rawlinson, W; Baleriola, C; Robinson, C C

2012-05-01

The Xpert Flu Assay cartridge is a next-generation nucleic acid amplification system that provides multiplexed PCR detection of the influenza A, influenza A 2009 H1N1, and influenza B viruses in approximately 70 min with minimal hands-on time. Six laboratories participated in a clinical trial comparing the results of the new Cepheid Xpert Flu Assay to those of culture or real-time PCR with archived and prospectively collected nasal aspirate-wash (NA-W) specimens and nasopharyngeal (NP) swabs from children and adults. Discrepant results were resolved by DNA sequence analysis. After discrepant-result analysis, the sensitivities of the Xpert Flu Assay for prospective NA-W specimens containing the influenza A, influenza A 2009 H1N1, and influenza B viruses compared to those of culture were 90.0%, 100%, and 100%, respectively, while the sensitivities of the assay for prospective NP swabs compared to those of culture were 100%, 100%, and 100%, respectively. The sensitivities of the Xpert Flu Assay for archived NA-W specimens compared to those of Gen-Probe ProFlu+ PCR for the influenza A, influenza A 2009 H1N1, and influenza B viruses were 99.4%, 98.4%, and 100%, respectively, while the sensitivities of the Xpert Flu Assay for archived NP swabs compared to those of ProFlu+ were 98.1%, 100%, and 93.8%, respectively. The sensitivities of the Xpert Flu Assay with archived NP specimens compared to those of culture for the three targets were 97.5%, 100%, and 93.8%, respectively. We conclude that the Cepheid Xpert Flu Assay is an accurate and rapid method that is suitable for on-demand testing for influenza viral infection.

Development and Evaluation of a Rapid and Sensitive EBOV-RPA Test for Rapid Diagnosis of Ebola Virus Disease.

Science.gov (United States)

Yang, Mingjuan; Ke, Yuehua; Wang, Xuesong; Ren, Hang; Liu, Wei; Lu, Huijun; Zhang, Wenyi; Liu, Shiwei; Chang, Guohui; Tian, Shuguang; Wang, Lihua; Huang, Liuyu; Liu, Chao; Yang, Ruifu; Chen, Zeliang

2016-06-01

Confirming Ebola virus disease (EVD), a deadly infectious disease, requires real-time RT-PCR, which takes up to a few hours to yield results. Therefore, a rapid diagnostic assay is imperative for EVD diagnosis. A rapid nucleic acid test based on recombinase polymerase amplification (EBOV-RPA) was developed to specifically detect the 2014 outbreak strains. The EBOV-RPA assay was evaluated by testing samples from suspected EVD patients in parallel with RT-PCR. An EBOV-RPA, which could be completed in 20 min, was successfully developed. Of 271 patients who tested positive for Ebola virus by RT-PCR, 264 (sensitivity: 97%, 95% CI: 95.5-99.3%) were positive by EBOV-RPA; 101 of 104 patients (specificity: 97%, 95% CI: 93.9-100%) who tested negative by RT-PCR were also negative by EBOV-RPA. The sensitivity values for samples with a Ct value of RPA had significantly high Ct values. Results of external quality assessment samples with EBOV-RPA were 100%, consistent with those of RT-PCR. The EBOV-RPA assay showed 97% sensitivity and 97% specificity for all EVD samples tested, making it a rapid and sensitive test for EVD diagnosis.

Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

Directory of Open Access Journals (Sweden)

KHUSNUL YAQIN

2006-09-01

Full Text Available Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The applications of this assay suggest that it is a useful assay to assess aquatic genotoxicants. However, there are some factors, which should be taken into account when using this assay as aquatic ecotoxicological assessment device such as inter-animal and cell variability.

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

Science.gov (United States)

Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

2014-03-07

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Elements of nondestructive assay (NDA) technology

International Nuclear Information System (INIS)

Anon.

1981-01-01

This session provides an introduction to nondestructive assay methods and instruments as they are applied to nuclear safeguards. The purpose of the sessions is to enable participants to: (1) discuss the general principles and major applications of NDA; (2) describe situations in which NDA is particularly useful for nuclear safeguards purposes; (3) distinguish between various passive and active gamma-ray and neutron NDA methods; (4) describe several NDA instruments that measure gamma rays, and identify assay situations particularly suited to gamma-ray techniques; (5) describe several NDA instruments that measure neutrons, and identify assay situations particularly suited to neutron techniques; (6) discuss the role of calorimetry in the NDA of plutonium-bearing materials; and (7) compare the advantages and disadvantages of various NDA methods for different types of nuclear materials

The zygomatic implant perforated (ZIP) flap: a new technique for combined surgical reconstruction and rapid fixed dental rehabilitation following low-level maxillectomy.

Science.gov (United States)

Butterworth, C J; Rogers, S N

2017-12-01

This aim of this report is to describe the development and evolution of a new surgical technique for the immediate surgical reconstruction and rapid post-operative prosthodontic rehabilitation with a fixed dental prosthesis following low-level maxillectomy for malignant disease.The technique involves the use of a zygomatic oncology implant perforated micro-vascular soft tissue flap (ZIP flap) for the primary management of maxillary malignancy with surgical closure of the resultant maxillary defect and the installation of osseointegrated support for a zygomatic implant-supported maxillary fixed dental prosthesis.The use of this technique facilitates extremely rapid oral and dental rehabilitation within a few weeks of resective surgery, providing rapid return to function and restoring appearance following low-level maxillary resection, even in cases where radiotherapy is required as an adjuvant treatment post-operatively. The ZIP flap technique has been adopted as a standard procedure in the unit for the management of low-level maxillary malignancy, and this report provides a detailed step-by-step approach to treatment and discusses modifications developed over the treatment of an initial cohort of patients.

The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

Directory of Open Access Journals (Sweden)

Bas-jan Van Der Leede

2015-08-01

Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

Rapid molecular diagnostics for multi-drug resistant tuberculosis in India.

Science.gov (United States)

Ramachandran, Rajeswari; Muniyandi, M

2018-03-01

Rapid molecular diagnostic methods help in the detection of TB and Rifampicin resistance. These methods detect TB early, are accurate and play a crucial role in reducing the burden of drug resistant tuberculosis. Areas covered: This review analyses rapid molecular diagnostic tools used in the diagnosis of MDR-TB in India, such as the Line Probe Assay and GeneXpert. We have discussed the burden of MDR-TB and the impact of recent diagnostic tools on case detection and treatment outcomes. This review also discusses the costs involved in establishing these new techniques in India. Expert commentary: Molecular methods have considerable advantages for the programmatic management of drug resistant TB. These include speed, standardization of testing, potentially high throughput and reduced laboratory biosafety requirements. There is a desperate need for India to adopt modern, rapid, molecular tools with point-of-care tests being currently evaluated. New molecular diagnostic tests appear to be cost effective and also help in detecting missing cases. There is enough evidence to support the scaling up of these new tools in India.

Radioreceptor assay: theory and applications to pharmacology

International Nuclear Information System (INIS)

Perret, G.; Simon, P.

1984-01-01

The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, β-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising [fr

Operating characteristics of a qualitative troponin assay for the diagnosis of acute coronary syndrome.

Science.gov (United States)

Farsi, Davood; Pishbin, Elham; Abbasi, Saeed; Hafezimoghadam, Peyman; Fathi, Marzieh; Zare, Mohammad Amin

2013-04-01

The troponin I serum level is widely used in acute coronary syndrome patients for their classification. The qualitative assay is faster and more available than the quantitative assay. The objective was to determine the operating characteristics of a qualitative troponin I assay compared with a quantitative method. This is a prospective observational study and patients suspected to have acute coronary syndrome were enrolled. A rapid troponin I test and a quantitative assay were carried out for each patient on arrival and 6 h after admission. A total of 262 patients were enrolled. The degree of agreement between the second rapid qualitative and quantitative troponin I was excellent (κ=0.946; 95% confidence interval, 0.903-0.989). The sensitivity, specificity, negative predictive value, and positive predictive value of the rapid qualitative troponin I test were 92.6, 100, 96.8, and 100%, respectively. In conclusion, this study reveals an excellent agreement between quantitative and qualitative bedside assays 6 h after admission in a sample of Iranian patients in the emergency department.

Clonogenic assay: adherent cells.

Science.gov (United States)

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

2011-03-13

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

Nondestructive assay of sale materials

International Nuclear Information System (INIS)

Rodenburg, W.W.; Fleissner, J.G.

1981-01-01

This paper covers three primary areas: (1) reasons for performing nondestructive assay on SALE materials; (2) techniques used; and (3) discussion of investigators' revised results. The study shows that nondestructive calorimetric assay of plutonium offers a viable alternative to traditional wet chemical techniques. For these samples, the precision ranged from 0.4 to 0.6% with biases less than 0.2%. Thus, for those materials where sampling errors are the predominant source of uncertainty, this technique can provide improved accuracy and precision while saving time and money as well as reducing the amount of liquid wastes to be handled. In addition, high resolution gamma-ray spectroscopy measurements of solids can provide isotopic analysis data in a cost effective and timely manner. The timeliness of the method can be especially useful to the plant operator for production control and quality control measurements

Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

Directory of Open Access Journals (Sweden)

Yan Zhang

2009-01-01

Full Text Available To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver, we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2>0.95, which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

Rapid determination of ractopamine residues in edible animal products by enzyme-linked immunosorbent assay: development and investigation of matrix effects.

Science.gov (United States)

Zhang, Yan; Wang, Fengxia; Fang, Li; Wang, Shuo; Fang, Guozhen

2009-01-01

To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC(50) of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 mug/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R(2) > 0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

Evalution of NDA techniques and instruments for assay of nuclear waste at a waste terminal storage facility

International Nuclear Information System (INIS)

Blakeman, E.D.; Allen, E.J.; Jenkins, J.D.

1978-05-01

The use of Nondestructive Assay (NDA) instrumentation at a nuclear waste terminal storage facility for purposes of Special Nuclear Material (SNM) accountability is evaluated. Background information is given concerning general NDA techniques and the relative advantages and disadvantages of active and passive NDA methods are discussed. The projected characteristics and amounts of nuclear wastes that will be delivered to a waste terminal storage facility are presented. Wastes are divided into four categories: High Level Waste, Cladding Waste, Intermediate Level Waste, and Low Level Waste. Applications of NDA methods to the assay of these waste types is discussed. Several existing active and passive NDA instruments are described and, where applicable, results of assays performed on wastes in large containers (e.g., 55-gal drums) are given. It is concluded that it will be difficult to routinely achieve accuracies better than approximately 10--30% with ''simple'' NDA devices or 5--20% with more sohpisticated NDA instruments for compacted wastes. It is recommended that NDA instruments not be used for safeguards accountability at a waste storage facility. It is concluded that item accountability methods be implemented. These conclusions and recommendations are detailed in a concurrent report entitled ''Recommendations on the Safeguards Requirements Related to the Accountability of Special Nuclear Material at Waste Terminal Storage Facilities'' by J.D. Jenkins, E.J. Allen and E.D. Blakeman

A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

Science.gov (United States)

Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall'Olio, Raffaele; Nanetti, Antonio

2018-04-01

Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also. Copyright © 2018 Elsevier GmbH. All rights reserved.

In vitro assays for predicting tumor cell response to radiation by apoptotic pathways

International Nuclear Information System (INIS)

Algan, Oe.; Hanks, G.E.; Biade, S.; Chapman, J.D.

1995-01-01

were detected by gel electrophoresis procedures but this technique is laborious and difficult to quantify. Centrifugation procedures of irradiation cells which had been pre-labeled with 3 H-thymidine showed ∼18% of total cellular DNA to be fragmented within 12 hr, after which time the extent of DNA fragmentation plateaued. The labeling of 3'-OH ends in cellular DNA by the immunofluorescence reagent, ApopTag[reg], showed ∼15% of cells to undergo apoptotic degradation. Staining of irradiated cells with LIVE/DEAD[reg] EUKOLIGHT TM and trypan blue showed 20-25% cell death. Although the vital stain assays are not specific for apoptosis, the proportion of rapid cell death (within 24-48 hr) which they measure is close to that obtained with the apoptotic-specific assays. These studies indicate that 24 hr after irradiation with 10 Gy, approximately 20% of DU-145 cells undergo death by apoptosis. Survival curves constructed with different radiation doses indicate that this rapid mechanism of cell death follows single-hit kinetics and constitutes between 10-30% of the total α coefficient measured by clonogenic assays with this cell line. Conclusion: Two phases of cell death are observed after ionizing radiation of the DU-145 prostate cancer cell line. Rapid cell death occurs within ∼24 hr and appears to correlate with apoptotic cell death. The vital stains, LIVE/DEAD[reg] EUKOLIGHT TM and trypan blue, yield quantitatively similar estimates of rapid cell killing to the apoptosis-specific assays. We are currently extending these studies to other human prostate tumor cell lines and to tumor cells released from human prostate biopsies. Such assays may provide additional prognostic information for predicting radiotherapy outcome of patients receiving radiotherapy

Establishment of 60Co dose calibration curve using fluorescent in situ hybridization assay technique: Result of preliminary study

International Nuclear Information System (INIS)

Rahimah Abdul Rahim; Noriah Jamal; Noraisyah Mohd Yusof; Juliana Mahamad Napiah; Nelly Bo Nai Lee

2010-01-01

This study aims at establishing an in-vitro 60 Co dose calibration curve using Fluorescent In-Situ Hybridization assay technique for the Malaysian National Bio dosimetry Laboratory. Blood samples collected from a female healthy donor were irradiated with several doses of 60 Co radiation. Following culturing of lymphocytes, microscopic slides are prepared, denatured and hybridized. The frequencies of translocation are estimated in the metaphases. A calibration curve was then generated using a regression technique. It shows a good fit to a linear-quadratic model. The results of this study might be useful in estimating absorbed dose for the individual exposed to ionizing radiation retrospectively. This information may be useful as a guide for medical treatment for the assessment of possible health consequences. (author)

Development of flexural vibration inspection techniques to rapidly assess the structural health of rural bridge systems

Science.gov (United States)

Brian K. Brashaw; Robert Vatalaro; Xiping Wang; Kevin Sarvela; James P. Wacker

2008-01-01

Approximately 4,000 vehicle bridges in the State of Minnesota contain structural timber members. Recent research at the University of Minnesota Duluth Natural Resources Research Institute (UMD NRRI) has been conducted on vibration testing of timber bridges as a means of developing rapid in-place testing techniques for assessing the structural health of bridges. The...

Development of loop-mediated isothermal amplification (LAMP) assays for the rapid detection of allergic peanut in processed food.

Science.gov (United States)

Sheu, Shyang-Chwen; Tsou, Po-Chuan; Lien, Yi-Yang; Lee, Meng-Shiou

2018-08-15

Peanut is a widely and common used in many cuisines around the world. However, peanut is also one of the most important food allergen for causing anaphylactic reaction. To prevent allergic reaction, the best way is to avoid the food allergen or food containing allergic ingredient such as peanut before food consuming. Thus, to efficient and precisely detect the allergic ingredient, peanut or related product, is essential and required for maintain consumer's health or their interest. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of allergic peanut using specifically designed primer sets. Two sets of the specific LAMP primers respectively targeted the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions and the ara h1 gene sequence of Arachia hypogeae (peanut) were used to address the application of LAMP for detecting peanut in processed food or diet. The results demonstrated that the identification of peanut using the newly designed primers for ITS 1 sequence is more sensitive rather than primers for sequence of Ara h1 gene when performing LAMP assay. Besides, the sensitivity of LAMP for detecting peanut is also higher than the traditional PCR method. These LAMP primers sets showed high specificity for the identification of the peanut and had no cross-reaction to other species of nut including walnut, hazelnut, almonds, cashew and macadamia nut. Moreover, when minimal 0.1% peanuts were mixed with other nuts ingredients at different ratios, no any cross-reactivity was evident during performing LAMP. Finally, genomic DNAs extracted from boiled and steamed peanut were used as templates; the detection of peanut by LAMP was not affected and reproducible. As to this established LAMP herein, not only can peanut ingredients be detected but commercial foods containing peanut can also be identified. This assay will be useful and potential for the rapid detection of peanut in practical food

Validation of the Applied Biosystems RapidFinder Shiga Toxin-Producing E. coli (STEC) Detection Workflow.

Science.gov (United States)

Cloke, Jonathan; Matheny, Sharon; Swimley, Michelle; Tebbs, Robert; Burrell, Angelia; Flannery, Jonathan; Bastin, Benjamin; Bird, Patrick; Benzinger, M Joseph; Crowley, Erin; Agin, James; Goins, David; Salfinger, Yvonne; Brodsky, Michael; Fernandez, Maria Cristina

2016-11-01

The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the "Big 6" non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the "Big 6" non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the "Big 6" STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant

Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution

International Nuclear Information System (INIS)

Hincks, J.R.; Coulombe, R.A. Jr.

1989-01-01

Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA strand breaks and DNA cross-links. Conventional alkaline elution procedures have extensive equipment requirements and are tedious to perform. Our laboratory recently presented a rapid, simplified, and sensitive modification of the alkaline elution technique to detect carcinogen-induced DNA strand breaks. In the present study, we have further modified this technique to enable the rapid characterization of chemically induced DNA-interstrand and DNA-protein associated cross-links in cultured epithelial cells. Cells were exposed to three known DNA cross-linking agents, nitrogen mustard (HN 2 ), mitomycin C (MMC), or ultraviolet irradiation (UV). One hour exposures of HN 2 at 0.25, 1.0, and 4.0 microM or of MMC at 20, 40, and 60 microM produced a dose-dependent induction of total DNA cross-links by these agents. Digestion with proteinase K revealed that HN 2 and MMC induced both DNA-protein cross-links and DNA-interstrand cross-links. Ultraviolet irradiation induced both DNA cross-links and DNA strand breaks, the latter of which were either protein and nonprotein associated. The results demonstrate that gravity-flow alkaline elution is a sensitive and accurate method to characterize the molecular events of DNA cross-linking. Using this procedure, elution of DNA from treated cells is completed in 1 hr, and only three fractions per sample are analyzed. This method may be useful as a rapid screening assay for genotoxicity and/or as an adjunct to other predictive assays for potential mutagenic or carcinogenic agents

Radioenzymatic assay for trimethoprim in very small serum samples.

OpenAIRE

Yogev, R; Melick, C; Tan-Pong, L

1985-01-01

A modification of the methotrexate radioassay kit (supplied by New England Enzyme Center) enabled determination of trimethoprim levels in 5-microliter serum samples. An excellent correlation between this assay and high-pressure liquid chromatography assay was found. These preliminary results suggest that with this method rapid determination of trimethoprim levels in very small samples (5 to 10 microliters) can be achieved.

Radioenzymatic assay for trimethoprim in very small serum samples

International Nuclear Information System (INIS)

Yogev, R.; Melick, C.; Tan-Pong, L.

1985-01-01

A modification of the methotrexate radioassay kit (supplied by New England Enzyme Center) enabled determination of trimethoprim levels in 5-microliter serum samples. An excellent correlation between this assay and high-pressure liquid chromatography assay was found. These preliminary results suggest that with this method rapid determination of trimethoprim levels in very small samples (5 to 10 microliters) can be achieved

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

Science.gov (United States)

Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

2017-09-01

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B.V. All rights reserved.

Elements of nondestructive assay (NDA) technology

International Nuclear Information System (INIS)

Hatcher, C.R.; Smith, H.

1984-01-01

A thorough introduction to nondestructive assay methods and instruments as they are applied to nuclear safeguards is presented. The general principles and major applications of NDA are discussed and situations in which NDA is particularly useful for nuclear safeguards purposes are described. Various passive and active γ-ray and neutron methods are examined and assay situations particularly suited to γ-ray techniques, or to neutron techniques are identified. The role of calorimetry in the NDA of plutonium-bearing materials is also discussed. The advantages and disadvantages of various NDA methods for different types of nuclear materials are mentioned

Direct 125I-radioligand assays for serum progesterone compared with assays involving extraction of serum

International Nuclear Information System (INIS)

Ratcliffe, W.A.; Corrie, J.E.T.; Dalziel, A.H.; Macpherson, J.S.

1982-01-01

Two direct radioimmunoassays for progesterone in 50 μL of unextracted serum or plasma with assays involving extraction of serum were compared. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11α-hemisuccinyl conjugate and the radioligand 125 I-labeled progesterone 11α-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r > 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum

Linearization of the bradford protein assay.

Science.gov (United States)

Ernst, Orna; Zor, Tsaffrir

2010-04-12

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

Novel Risk Stratification Assays for Acute Coronary Syndrome.

Science.gov (United States)

Ahmed, Haitham M; Hazen, Stanley L

2017-08-01

Since identification of aspartate aminotransferase as the first cardiac biomarker in the 1950s, there have been a number of new markers used for myocardial damage detection over the decades. There have also been several generations of troponin assays, each with progressively increasing sensitivity for troponin detection. Accordingly, the "standard of care" for myocardial damage detection continues to change. The purpose of this paper is to review the clinical utility, biological mechanisms, and predictive value of these various biomarkers in contemporary clinical studies. As of this writing, a fifth "next" generation troponin assay has now been cleared by the US Food and Drug Administration for clinical use in the USA for subjects presenting with suspected acute coronary syndromes. Use of these high-sensitivity assays has allowed for earlier detection of myocardial damage as well as greater negative predictive value for infarction after only one or two serial measurements. Recent algorithms utilizing these assays have allowed for more rapid rule-out of myocardial infarction in emergency department settings. In this review, we discuss novel assays available for the risk assessment of subjects presenting with chest pain, including both the "next generation" cardiac troponin assays as well as other novel biomarkers. We review the biological mechanisms for these markers, and explore the positive and negative predictive value of the assays in clinical studies, where reported. We also discuss the potential use of these new markers within the context of future clinical care in the modern era of higher sensitivity troponin testing. Finally, we discuss advances in new platforms (e.g., mass spectrometry) that historically have not been considered for rapid in vitro diagnostic capabilities, but that are taking a larger role in clinical diagnostics, and whose prognostic value and power promise to usher in new markers with potential for future clinical utility in acute coronary

233U Assay A Neutron NDA System

International Nuclear Information System (INIS)

Hensley, D.C.; Lucero, A.J.; Pierce, L.

1998-01-01

The assay of highly enriched 233 U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched 235 U do not convert easily over to the assay of 233 U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with γ ray isotopics information should give a good overall determination of 233 U material now stored in bldg. 3019 at the Oak Ridge National Laboratory

XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity.

Science.gov (United States)

Ooi, Nicola; Miller, Keith; Hobbs, Joanne; Rhys-Williams, William; Love, William; Chopra, Ian

2009-10-01

XF-73 is a novel porphyrin antibacterial agent previously reported to inhibit a range of gram-positive bacterial species, including Staphylococcus aureus. Its mode of action is unknown. Using S. aureus as a model organism we sought to examine the basis of its antibacterial activity. The effects of XF-73 on the growth and survival of S. aureus SH1000 were investigated by viable count and culture absorbance techniques. Inhibition of macromolecular synthesis and disruption of membrane integrity after exposure to XF-73 were examined by radiolabelling experiments, the BacLight fluorescent dye assay and measurement of K(+) and ATP leakage from the cell. The effect of XF-73 on a staphylococcal coupled transcription-translation system was also investigated. XF-73 was rapidly bactericidal against S. aureus SH1000 and demonstrated more rapid killing kinetics than all other comparator agents when tested at an equivalent multiple (4x) of the MIC. Exposure of S. aureus to XF-73 for 10 min completely inhibited DNA, RNA and protein synthesis. XF-73 had no effect on transcription and translation in vitro. Cells exposed to XF-73 gave a positive response in the BacLight assay, which detects membrane damage. The drug also caused substantial loss of K(+) and ATP from the cell, but did not promote bacterial lysis. XF-73 exhibited rapid membrane-perturbing activity, which is likely to be responsible for inhibition of macromolecular synthesis and the death of staphylococci exposed to the drug.

A novel colorimetric assay for rapid detection of cysteine and Hg²⁺ based on gold clusters.

Science.gov (United States)

Wang, Yi-Wei; Tang, Shurong; Yang, Huang-Hao; Song, Hongbo

2016-01-01

Inhibition and recovery of the catalytic activity of bovine serum albumin-capped gold nanoclusters (BSA-AuNCs) is observed for the first time by introduction of cysteine and Hg(2+). The prepared BSA-AuNCs possess highly intrinsic peroxidase-like activity. It can catalyze the oxidation of 3, 3, 5, 5-tetramethylbenzidine by H2O2 to produce a blue colored product. Based on this phenomenon, a new colorimetric assay for rapid, selective and sensitive detection of cysteine and Hg(2+) in aqueous solution has been demonstrated. The interaction process between target molecule and BSA-AuNCs is very fast, so that the whole test can be completed within ten minutes. Moreover, the fabricated colorimetric sensor is simple and cost-effective, without the need of nucleic acid based recognition element and complicated washing, separation and labeling process, thus holds great promise for routine analysis of cysteine and Hg(2+) in real samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Localized irradiations, evaluation through 'Comet Assay'

International Nuclear Information System (INIS)

Di Giorgio, Marina; Taja, Maria R.; Nasazzi, Nora B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.

2000-01-01

During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources of Cobalt 60, Cesium 137 or Iridium 192 at work placed for industrial gammagraphy and other radiation sources. Severe skin reaction may developed at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models 'Contaminated Poisson' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. There are also different biophysical techniques that can give response in localized irradiations. Biological dosimetry is a necessary complement to physical and clinical dosimetries. Thus, there is increasing interest in the assessment of biological markers that permit the detection of radiation induced damage in the localized irradiations. The 'Comet Assay' (single cell gel electrophoresis) is a sensitive, rapid and relatively inexpensive method for measuring DNA damage in individual cells. Single cells are embedded in agarose on microscope slides, lysed to remove the majority of the proteins, electrophoresed, then stained with ethidium bromide in order to visualize the DNA. When visualized using a fluorescent microscope, DNA of undamaged cells appears as a spherical mass occupying the cavity formed by the lysed cell. Following radiation damage, the smaller the fragment size and the grater the number of fragments of DNA, the grater the percentage of DNA that it is able to migrate in an electric field, forming a comet image. The assay can be performed under alkaline conditions to examine DNA single strand breaks (SSBs), or in non denaturing (neutral) conditions to measure double strand breaks (DSBs) in individual

A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

Science.gov (United States)

Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

2016-06-01

The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.

Determination of Nitrate Reductase Assay Depending on the Microbial Growth

International Nuclear Information System (INIS)

El-Kabbany, H.M.

2012-01-01

A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

Lateral flow assays

NARCIS (Netherlands)

Posthuma-Trumpie, G.A.; Amerongen, van A.

2012-01-01

A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

Technique for rapid detection of phthalates in water and beverages

KAUST Repository

Zia, Asif I.

2013-05-01

The teratogenic and carcinogenic effects of phthalate esters on living beings are proven in toxicology studies. These ubiquitous food and environmental pollutants pose a great danger to the human race due to their extraordinary use as a plasticizer in the consumer product industry. Contemporary detection techniques used for phthalates require a high level of skills, expensive equipment and longer analysis time than the presented technique. Presented research work introduces a real time non-invasive detection technique using a new type of silicon substrate based planar interdigital (ID) sensor fabricated on basis of thin film micro-electromechanical system (MEMS) semiconductor device fabrication technology. Electrochemical impedance spectroscopy (EIS) was used in conjunction with the fabricated sensor to detect phthalates in deionized water. Various concentrations of di(2-ethylhexyl) phthalate (DEHP) as low as 2 ppb to a higher level of 2 ppm in deionized water were detected distinctively using new planar ID sensor based EIS sensing system. Dip testing method was used to obtain the conductance and dielectric properties of the bulk samples. Parylene C polymer coating was used as a passivation layer on the surface of the fabricated sensor to reduce the influence of Faradaic currents. In addition, inherent dielectric properties of the coating enhanced the sensitivity of the capacitive type sensor. Electrochemical spectrum analysis algorithm was used to model experimentally observed impedance spectrum to deduce constant phase element (CPE) equivalent circuit to analyse the kinetic processes taking place inside the electrochemical cell. Curve fitting technique was used to extract the values of the circuit components and explain experimental results on theoretical grounds. The sensor performance was tested by adding DEHP to an energy drink at concentrations above and below the minimal risk level (MRL) limit set by the ATSDR (Agency for Toxic Substances & Disease Registry

The use of calorimetry for plutonium assay

International Nuclear Information System (INIS)

Mason, J.A.

1982-12-01

Calorimetry is a technique for measuring the thermal power of heat-producing substances. The technique may be applied to the measurement of plutonium-bearing materials which evolve heat as a result of alpha and beta decay. A calorimetric measurement of the thermal power of a plutonium sample, combined with a knowledge or measurement of the plutonium isotopic mass ratios of the sample provides a convenient and accurate, non-destructive measure of the total plutonium mass of the sample. The present report provides a description, and an assessment of the calorimetry technique applied to the assay of plutonium-bearing materials. Types and characteristics of plutonium calorimeters are considered, as well as calibration and operating procedures. The instrumentation used with plutonium calorimeters is described and the use of computer control for calorimeter automation is discussed. A critical review and assessment of plutonium calorimetry literature since 1970 is presented. Both fuel element and plutonium-bearing material calorimeters are considered. The different types of plutonium calorimeters are evaluated and their relative merits are discussed. A combined calorimeter and gamma-ray measurement assay system is considered. The design principles of plutonium assay calorimeters are considered. An automatic, computer-based calorimeter control system is proposed in conjunction with a general plutonium assay calorimeter design. (author)

A rapid, sensitive, and cost-efficient assay to estimate viability of potato cyst nematodes.

Science.gov (United States)

van den Elsen, Sven; Ave, Maaike; Schoenmakers, Niels; Landeweert, Renske; Bakker, Jaap; Helder, Johannes

2012-02-01

Potato cyst nematodes (PCNs) are quarantine organisms, and they belong to the economically most relevant pathogens of potato worldwide. Methodologies to assess the viability of their cysts, which can contain 200 to 500 eggs protected by the hardened cuticle of a dead female, are either time and labor intensive or lack robustness. We present a robust and cost-efficient viability assay based on loss of membrane integrity upon death. This assay uses trehalose, a disaccharide present at a high concentration in the perivitelline fluid of PCN eggs, as a viability marker. Although this assay can detect a single viable egg, the limit of detection for regular field samples was higher, ≈10 viable eggs, due to background signals produced by other soil components. On the basis of 30 nonviable PCN samples from The Netherlands, a threshold level was defined (ΔA(trehalose) = 0.0094) below which the presence of >10 viable eggs is highly unlikely (true for ≈99.7% of the observations). This assay can easily be combined with a subsequent DNA-based species determination. The presence of trehalose is a general phenomenon among cyst nematodes; therefore, this method can probably be used for (for example) soybean, sugar beet, and cereal cyst nematodes as well.

Real-Time, Label-Free Detection of Biomolecular Interactions in Sandwich Assays by the Oblique-Incidence Reflectivity Difference Technique

Directory of Open Access Journals (Sweden)

Yung-Shin Sun

2014-12-01

Full Text Available One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR. The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.

Rapid screening of monoclonal antibodies: new 'microstick' radioimmunoassay

International Nuclear Information System (INIS)

Scheinberg, D.A.; Strand, M.; Wilsnack, R.

1983-01-01

A new system for assaying monoclonal antibodies consisting of an 8 x 12 array of sticks which fits into a 96-well microtiter plate is described. Tests using virus specific monoclonal antibodies and virus proteins demonstrated sensitivity equivalent to the conventional microtiter plate assay. Antibody production, antigen specific antibody, and immunoglobulin isotypes could be measured under sterile conditions directly in the original fusion mixture wells and much greater rapidity than with the microtiter plate assay. (Auth.)

Rapid genotyping assays for the 4-base pair deletion of canine MDR1/ABCB1 gene and low frequency of the mutant allele in Border Collie dogs.

Science.gov (United States)

Mizukami, Keijiro; Chang, Hye-Sook; Yabuki, Akira; Kawamichi, Takuji; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Yamato, Osamu

2012-01-01

P-glycoprotein, encoded by the MDR1 or ABCB1 gene, is an integral component of the blood-brain barrier as an efflux pump for xenobiotics crucial in limiting drug uptake into the central nervous system. Dogs homozygous for a 4-base pair deletion of the canine MDR1 gene show altered expression or function of P-glycoprotein, resulting in neurotoxicosis after administration of the substrate drugs. In the present study, the usefulness of microchip electrophoresis for genotyping assays detecting this deletion mutation was evaluated. Mutagenically separated polymerase chain reaction (MS-PCR) and real-time PCR assays were newly developed and evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies dogs in Japan to determine the allele frequency in this breed. Microchip electrophoresis showed advantages in detection sensitivity and time saving over other modes of electrophoresis. The MS-PCR assay clearly discriminated all genotypes. Real-time PCR assay was most suitable for a large-scale survey due to its high throughput and rapidity. The genotyping survey demonstrated that the carrier and mutant allele frequencies were 0.49% and 0.25%, respectively, suggesting that the mutant allele frequency in Border Collies is markedly low compared to that in the susceptible dog breeds such as rough and smooth Collies.

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.

Science.gov (United States)

Song, Y; Kato, N; Liu, C; Matsumiya, Y; Kato, H; Watanabe, K

2000-06-15

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

Solid-phase immunoradiometric assay for C-reactive protein using magnetisable cellulose particles

International Nuclear Information System (INIS)

Beer, F.C. de; Pepys, M.B.

1982-01-01

An immunoradiometric assay (IRMA) for C-reactive protein (CRP) was developed using magnetisable cellulose particles as the solid-phase support for anti-CRP antibodies. 125 I-labelled immunopurified anti-CRP antibody was used to quantitate the amount of CRP taken up by the solid phase. Unbound label was easily and rapidly removed by decantation after sedimenting the particles on a magnet. The assay could detect 1 μg CRP/l and had a range of up to 10 mg/l with the portion of the standard curve between 10 μg/l and 2-3 mg/l being linear. Fifty samples per hour could be processed manually from serum to CRP result with an intra-assay CV of 5.2% and an inter-assay CV of 10.0%, based on 5 replicates of 5 samples with CRP levels between 2 mg/l and 180 mg/l run in 5 separate assays. Fifty clinical samples were assayed in parallel with a standard electroimmunoassay and yielded a linear correlation coefficient (r) of 0.975 and a slope of 0.98. With its single, brief incubation step including all reagents and its simple phase separation procedure the present method may be the assay of choice when precise measurement of CRP concentrations is required rapidly. (Auth.)

Rapid nuclear forensics analysis via laser based microphotonic techniques coupled with chemometrics

International Nuclear Information System (INIS)

Bhatta, B.; Kalambuka, H.A.; Dehayem-Kamadjeu, A.

2017-01-01

Nuclear forensics (NF) is an important tool for analysis and attribution of nuclear and radiological materials (NRM) in support of nuclear security. The critical challenge in NF currently is the lack of suitable microanalytical methodologies for direct, rapid and minimally-invasive detection and quantification of NF signatures. Microphotonic techniques can achieve this task particularly when the materials are of limited size and under concealed condition. The purpose of this paper is to demonstrate the combined potential of chemometrics enabled LIBS and laser Raman spectromicroscopy (LRS) for rapid NF analysis and attribution. Using LIBS, uranium lines at 385.464 nm, 385.957 nm and 386.592 nm were identified as NF signatures in uranium ore surrogates. A multivariate calibration strategy using artificial neural network was developed for quantification of trace uranium. Principal component analysis (PCA) of LIBS spectra achieved source attribution of the ores. LRS studies on UCl3, UO3(NO3)2.6H2O, UO2SO4.3H2O and UO3 in pellet state identified the bands associated with different uranium molecules as varying in the range of (840 to 867) ± 15 cm-1. Using this signature, we have demonstrated spectral imaging of uranium under concealed conditions (author)

A new rapid immunohistochemical staining technique using the EnVision antibody complex.