Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

PubMed

Flenker, Katie S; Burghardt, Elliot L; Dutta, Nirmal; Burns, William J; Grover, Julia M; Kenkel, Elizabeth J; Weaver, Tyler M; Mills, James; Kim, Hyeon; Huang, Lingyan; Owczarzy, Richard; Musselman, Catherine A; Behlke, Mark A; Ford, Bradley; McNamara, James O

2017-06-07

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2014-10-01

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

Rapid surface enhanced Raman scattering detection method for chloramphenicol residues

NASA Astrophysics Data System (ADS)

Ji, Wei; Yao, Weirong

2015-06-01

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

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

PubMed Central

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

2011-01-01

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

Rapid, On-Site, Ultrasensitive Melamine Quantitation Method for Protein Beverages Using Time-Resolved Fluorescence Detection Paper.

PubMed

Li, Guanghua; Wang, Du; Zhou, Aijun; Sun, Yimin; Zhang, Qi; Poapolathep, Amnart; Zhang, Li; Fan, Zhiyong; Zhang, Zhaowei; Li, Peiwu

2018-06-06

To ensure protein beverage safety and prevent illegal melamine use to artificially increase protein content, a rapid, on-site, ultrasensitive detection method for melamine must be developed because melamine is detrimental to human health. Herein, an ultrasensitive time-resolved fluorescence detection paper (TFDP) was developed to detect melamine in protein beverages within 15 min using a one-step sample preparation. The lower limits of detection were 0.89, 0.94, and 1.05 ng/mL, and the linear ranges were 2.67-150, 2.82-150, and 3.15-150 ng/mL (R 2 > 0.982) for peanut, walnut, and coconut beverages, respectively. The recovery rates were 85.86-110.60% with a coefficient of variation <7.80% in the spiking experiment. A high specificity was observed in the interferent experiment. When detecting real protein beverage samples, the TFDP and ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) results were consistent. This method is a promising alternative for rapid, on-site detection of melamine in beverages.

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

PubMed Central

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

2009-01-01

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

Rapid surface enhanced Raman scattering detection method for chloramphenicol residues.

PubMed

Ji, Wei; Yao, Weirong

2015-06-05

Chloramphenicol (CAP) is a widely used amide alcohol antibiotics, which has been banned from using in food producing animals in many countries. In this study, surface enhanced Raman scattering (SERS) coupled with gold colloidal nanoparticles was used for the rapid analysis of CAP. Density functional theory (DFT) calculations were conducted with Gaussian 03 at the B3LYP level using the 3-21G(d) and 6-31G(d) basis sets to analyze the assignment of vibrations. Affirmatively, the theoretical Raman spectrum of CAP was in complete agreement with the experimental spectrum. They both exhibited three strong peaks characteristic of CAP at 1104 cm(-1), 1344 cm(-1), 1596 cm(-1), which were used for rapid qualitative analysis of CAP residues in food samples. The use of SERS as a method for the measurements of CAP was explored by comparing use of different solvents, gold colloidal nanoparticles concentration and absorption time. The method of the detection limit was determined as 0.1 μg/mL using optimum conditions. The Raman peak at 1344 cm(-1) was used as the index for quantitative analysis of CAP in food samples, with a linear correlation of R(2)=0.9802. Quantitative analysis of CAP residues in foods revealed that the SERS technique with gold colloidal nanoparticles was sensitive and of a good stability and linear correlation, and suited for rapid analysis of CAP residue in a variety of food samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-04-01

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

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

PubMed

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

2012-02-01

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

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2003-01-01

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

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

PubMed

Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

2012-08-01

Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

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

PubMed Central

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

2016-01-01

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

A Rapid Method to Achieve Aero-Engine Blade Form Detection

PubMed Central

Sun, Bin; Li, Bing

2015-01-01

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

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

PubMed

Sun, Bin; Li, Bing

2015-06-01

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

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

DOEpatents

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

1999-06-15

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

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

DOEpatents

Funsten, Herbert O.; McComas, David J.

1997-01-01

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

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

DOEpatents

Funsten, Herbert O.; McComas, David J.

1999-01-01

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

Rapid Magnetic Nanobiosensor for the detection of Serratia marcescen

NASA Astrophysics Data System (ADS)

Aljabali, Alaa A. A.; Hussein, Emad; Aljumaili, Omar; Zoubi, Mazhar Al; Altrad, Bahaa; Albatayneh, Khaled; Al-razaq, Mutaz A. Abd

2018-02-01

The development of rapid, sensitive, accurate and reliable bacterial detection methods are of keen interest to ensure food safety and hospital security. Therefore, the development of a fast, specific, low-cost and trusted methods is in high demand. Magnetic nanoparticles with their unique material properties have been utilized as a tool for pathogen detection. Here, we present a novel iron oxide nanoparticles labeled with specific targeting antibodies to improve specificity and extend the use of nanoparticles as nanosensors. The results indicated that antibody labeled iron oxide platform that binds specifically to Serriata marcescenst in a straightforward method is very specific and sensitive. The system is capable of rapid and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. The generic platform could be used to identify pathogens for a variety of applications rapidly.

Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

PubMed

Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

2017-10-27

Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

A semi-automated method for rapid detection of ripple events on interictal voltage discharges in the scalp electroencephalogram

PubMed Central

Chu, Catherine. J.; Chan, Arthur; Song, Dan; Staley, Kevin J.; Stufflebeam, Steven M.; Kramer, Mark A.

2017-01-01

Summary Background High frequency oscillations are emerging as a clinically important indicator of epileptic networks. However, manual detection of these high frequency oscillations is difficult, time consuming, and subjective, especially in the scalp EEG, thus hindering further clinical exploration and application. Semi-automated detection methods augment manual detection by reducing inspection to a subset of time intervals. We propose a new method to detect high frequency oscillations that co-occur with interictal epileptiform discharges. New Method The new method proceeds in two steps. The first step identifies candidate time intervals during which high frequency activity is increased. The second step computes a set of seven features for each candidate interval. These features require that the candidate event contain a high frequency oscillation approximately sinusoidal in shape, with at least three cycles, that co-occurs with a large amplitude discharge. Candidate events that satisfy these features are stored for validation through visual analysis. Results We evaluate the detector performance in simulation and on ten examples of scalp EEG data, and show that the proposed method successfully detects spike-ripple events, with high positive predictive value, low false positive rate, and high intra-rater reliability. Comparison with Existing Method The proposed method is less sensitive than the existing method of visual inspection, but much faster and much more reliable. Conclusions Accurate and rapid detection of high frequency activity increases the clinical viability of this rhythmic biomarker of epilepsy. The proposed spike-ripple detector rapidly identifies candidate spike-ripple events, thus making clinical analysis of prolonged, multielectrode scalp EEG recordings tractable. PMID:27988323

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokoyama, Yoko; Shimizu, Akira; Okada, Etsuko

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

An efficient and rapid transgenic pollen screening and detection method using flow cytometry.

PubMed

Moon, Hong S; Eda, Shigetoshi; Saxton, Arnold M; Ow, David W; Stewart, C Neal

2011-01-01

Assaying for transgenic pollen, a major vector of transgene flow, provides valuable information and essential data for the study of gene flow and assessing the effectiveness of transgene containment. Most studies have employed microscopic screening methods or progeny analyses to estimate the frequency of transgenic pollen. However, these methods are time-consuming and laborious when large numbers of pollen grains must be analyzed to look for rare transgenic pollen grains. Thus, there is an urgent need for the development of a simple, rapid, and high throughput analysis method for transgenic pollen analysis. In this study, our objective was to determine the accuracy of using flow cytometry technology for transgenic pollen quantification in practical application where transgenic pollen is not frequent. A suspension of non-transgenic tobacco pollen was spiked with a known amount of verified transgenic tobacco pollen synthesizing low or high amounts of green fluorescent protein (GFP). The flow cytometric method detected approximately 75% and 100% of pollen grains synthesizing low and high amounts of GFP, respectively. The method is rapid, as it is able to count 5000 pollen grains per minute-long run. Our data indicate that this flow cytometric method is useful to study gene flow and assessment of transgene containment.

Rapid detection, characterization, and enumeration of foodborne pathogens.

PubMed

Hoorfar, J

2011-11-01

As food safety management further develops, microbiological testing will continue to play an important role in assessing whether Food Safety Objectives are achieved. However, traditional microbiological culture-based methods are limited, particularly in their ability to provide timely data. The present review discusses the reasons for the increasing interest in rapid methods, current developments in the field, the research needs, and the future trends. The advent of biotechnology has introduced new technologies that led to the emergence of rapid diagnostic methods and altered food testing practices. Rapid methods are comprised of many different detection technologies, including specialized enzyme substrates, antibodies and DNA, ranging from simple differential plating media to the use of sophisticated instruments. The use of non-invasive sampling techniques for live animals especially came into focus with the 1990s outbreak of bovine spongiform encephalopathy that was linked to the human outbreak of Creutzfeldt Jakob's Disease. Serology is still an important tool in preventing foodborne pathogens to enter the human food supply through meat and milk from animals. One of the primary uses of rapid methods is for fast screening of large number of samples, where most of them are expected to be test-negative, leading to faster product release for sale. This has been the main strength of rapid methods such as real-time Polymerase Chain Reaction (PCR). Enrichment PCR, where a primary culture broth is tested in PCR, is the most common approach in rapid testing. Recent reports show that it is possible both to enrich a sample and enumerate by pathogen-specific real-time PCR, if the enrichment time is short. This can be especially useful in situations where food producers ask for the level of pathogen in a contaminated product. Another key issue is automation, where the key drivers are miniaturization and multiple testing, which mean that not only one instrument is flexible

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

PubMed

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

2010-07-31

Mycobacterium avium subsp. paratuberculosis (MAP) may have a role in the development of Crohn's disease in humans via the consumption of contaminated milk and milk products. Detection of MAP from milk and dairy products has been reported from countries on the European continent, Argentina, the UK and Australia. In this study three different methods (quantitative real time PCR, combined phage IS900 PCR and conventional cultivation) were used to detect the presence of MAP in bulk tank milk (BTM) and cheese originating from sheep, goat and mixed milks from farms and products in Cyprus. During the first survey the presence of MAP was detected in 63 (28.6%) of cows' BTM samples by quantitative real time PCR. A second survey of BTM used a new combined phage IS900 PCR assay, and in this case MAP was detected in 50 (22.2%) samples showing a good level of agreement by both methods. None of the herds tested were known to be affected by Johne's disease and the presence of viable MAP was confirmed by conventional culture in only two cases of cows BTM. This suggests that either rapid method used is more sensitive than the conventional culture when testing raw milk samples for MAP. The two isolates recovered from BTM were identified by IS1311 PCR REA as cattle and sheep strains, respectively. In contrast when cheese samples were tested, MAP DNA was detected by quantitative real time PCR in seven (25.0%) samples (n=28). However no viable MAP was detected when either the combined phage IS900 PCR or conventional culture methods were used. Copyright 2010 Elsevier B.V. All rights reserved.

Sensitive, Rapid Detection of Bacterial Spores

NASA Technical Reports Server (NTRS)

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

2009-01-01

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

Rapid Detection of Pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

David Perlin

2005-08-14

Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleicmore » acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe

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

DOEpatents

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

2013-01-22

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

A semi-automated method for rapid detection of ripple events on interictal voltage discharges in the scalp electroencephalogram.

PubMed

Chu, Catherine J; Chan, Arthur; Song, Dan; Staley, Kevin J; Stufflebeam, Steven M; Kramer, Mark A

2017-02-01

High frequency oscillations are emerging as a clinically important indicator of epileptic networks. However, manual detection of these high frequency oscillations is difficult, time consuming, and subjective, especially in the scalp EEG, thus hindering further clinical exploration and application. Semi-automated detection methods augment manual detection by reducing inspection to a subset of time intervals. We propose a new method to detect high frequency oscillations that co-occur with interictal epileptiform discharges. The new method proceeds in two steps. The first step identifies candidate time intervals during which high frequency activity is increased. The second step computes a set of seven features for each candidate interval. These features require that the candidate event contain a high frequency oscillation approximately sinusoidal in shape, with at least three cycles, that co-occurs with a large amplitude discharge. Candidate events that satisfy these features are stored for validation through visual analysis. We evaluate the detector performance in simulation and on ten examples of scalp EEG data, and show that the proposed method successfully detects spike-ripple events, with high positive predictive value, low false positive rate, and high intra-rater reliability. The proposed method is less sensitive than the existing method of visual inspection, but much faster and much more reliable. Accurate and rapid detection of high frequency activity increases the clinical viability of this rhythmic biomarker of epilepsy. The proposed spike-ripple detector rapidly identifies candidate spike-ripple events, thus making clinical analysis of prolonged, multielectrode scalp EEG recordings tractable. Copyright © 2016 Elsevier B.V. All rights reserved.

Aptamer-mediated colorimetric method for rapid and sensitive detection of chloramphenicol in food.

PubMed

Yan, Chao; Zhang, Jing; Yao, Li; Xue, Feng; Lu, Jianfeng; Li, Baoguang; Chen, Wei

2018-09-15

We report an aptamer-mediated colorimetric method for sensitive detection of chloramphenicol (CAP). The aptamer of CAP is immobilized by the hybridization with pre-immobilized capture probe in the microtiter plate. The horseradish peroxidase (HRP) is covalently attached to the aptamer by the biotin-streptavidin system for signal production. CAP will preferably bind with aptamer due to the high binding affinity, which attributes to the release of aptamer and HRP and thus, affects the optical signal intensity. Quantitative determination of CAP is successfully achieved in the wide range from 0.001 to 1000 ng/mL with detection limit of 0.0031 ng/mL, which is more sensitive than traditional immunoassays. This method is further validated by measuring the recovery of CAP spiked in two different food matrices (honey and fish). The aptamer-mediated colorimetric method can be a useful protocol for rapid and sensitive screening of CAP, and may be used as an alternative means for traditional immunoassays. Copyright © 2018 Elsevier Ltd. All rights reserved.

A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae.

PubMed

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

1998-05-01

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

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

PubMed Central

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

1998-01-01

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

Selective cultivation and rapid detection of Staphylococcus aureus by computer vision.

PubMed

Wang, Yong; Yin, Yongguang; Zhang, Chaonan

2014-03-01

In this paper, we developed a selective growth medium and a more rapid detection method based on computer vision for selective isolation and identification of Staphylococcus aureus from foods. The selective medium consisted of tryptic soy broth basal medium, 3 inhibitors (NaCl, K2 TeO3 , and phenethyl alcohol), and 2 accelerators (sodium pyruvate and glycine). After 4 h of selective cultivation, bacterial detection was accomplished using computer vision. The total analysis time was 5 h. Compared to the Baird-Parker plate count method, which requires 4 to 5 d, this new detection method offers great time savings. Moreover, our novel method had a correlation coefficient of greater than 0.998 when compared with the Baird-Parker plate count method. The detection range for S. aureus was 10 to 10(7) CFU/mL. Our new, rapid detection method for microorganisms in foods has great potential for routine food safety control and microbiological detection applications. © 2014 Institute of Food Technologists®

A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation.

PubMed

Jin, Xiao; Gou, Jin-Ying

2016-01-01

Protein phosphorylation is one of the most important post-translational modifications catalyzed by protein kinases in living organisms. The advance of genome sequencing provided the information of protein kinase families in many organisms, including both model and non-model plants. The development of proteomics technologies also enabled scientists to efficiently reveal a large number of protein phosphorylations of an organism. However, kinases and phosphorylation targets are still to be connected to illustrate the complicated network in life. Here we adapted Pro-Q ® Diamond (Pro-Q ® Diamond Phosphoprotein Gel Stain), a widely used phosphoprotein gel-staining fluorescence dye, to establish a rapid, economical and non-radioactive fluorescence detection in tube (FDIT) method to analyze phosphorylated proteins. Taking advantages of high sensitivity and specificity of Pro-Q ® diamond, the FDIT method is also demonstrated to be rapid and reliable, with a suitable linear range for in vitro protein phosphorylation. A significant and satisfactory protein kinase reaction was detected as fast as 15 min from Wheat Kinase START 1.1 (WKS1.1) on a thylakoid ascorbate peroxidase (tAPX), an established phosphorylation target in our earlier study. The FDIT method saves up to 95% of the dye consumed in a gel staining method. The FDIT method is remarkably quick, highly reproducible, unambiguous and capable to be scaled up to dozens of samples. The FDIT method could serve as a simple and sensitive alternative procedure to determine protein kinase reactions with zero radiation exposure, as a supplementation to other widely used radioactive and in-gel assays.

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

PubMed Central

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

2010-01-01

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

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Letant, S E; Kane, S R; Murphy, G A

2008-05-30

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

Development of Untargeted Metabolomics Methods for the Rapid Detection of Pathogenic Naegleria fowleri.

PubMed

Yu, Zhihao; Miller, Haylea C; Puzon, Geoffrey J; Clowers, Brian H

2017-04-18

Despite comparatively low levels of infection, primary amoebic meningoencephalitis (PAM) induced by Naegleria fowleri is extremely lethal, with mortality rates above 95%. As a thermophile, this organism is often found in moderate-to-warm climates and has the potential to colonize drinking water distribution systems (DWDSs). Current detection approaches require days to obtain results, whereas swift corrective action can maximize the benefit of public health. Presently, there is little information regarding the underlying in situ metabolism for this amoeba but the potential exists to exploit differentially expressed metabolic signatures as a rapid detection technique. This research outlines the biochemical profiles of selected pathogenic and nonpathogenic Naegleria in vitro using an untargeted metabolomics approach to identify a panel of diagnostically meaningful compounds that may enable rapid detection of viable pathogenic N. fowleri and augment results from traditional monitoring approaches.

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

PubMed

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

2015-11-01

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

Rapid Methods for the Detection of General Fecal Indicators

EPA Science Inventory

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

[Rapid detection of caffeine in blood by freeze-out extraction].

PubMed

Bekhterev, V N; Gavrilova, S N; Kozina, E P; Maslakov, I V

2010-01-01

A new method for the detection of caffeine in blood has been proposed based on the combination of extraction and freezing-out to eliminate the influence of sample matrix. Metrological characteristics of the method are presented. Selectivity of detection is achieved by optimal conditions of analysis by high performance liquid chromatography. The method is technically simple and cost-efficient, it ensures rapid performance of the studies.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene.

PubMed

Wang, Jiashun; Li, Yi; Chen, Jia; Hua, Deping; Li, Yi; Deng, Hui; Li, Ying; Liang, Zhixuan; Huang, Jinhai

Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 10 1 and 10 4 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10h, which is a promising rapid method to detect Salmonella in emergency. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Rapid detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis by the direct thin-layer agar method.

PubMed

Robledo, J; Mejia, G I; Paniagua, L; Martin, A; Guzmán, A

2008-12-01

We evaluated thin-layer agar (TLA) for the detection of resistance of Mycobacterium tuberculosis to rifampicin (RMP) and isoniazid (INH) as a direct method in patients at risk of multidrug-resistant tuberculosis (MDR-TB). Quadrant TLA plates contain 7H10 Middlebrook growth control, para-nitrobenzoic acid, INH and RMP. Detection of RMP and INH resistance by TLA was compared to that in indirect conventional drug susceptibility testing (DST) and conventional culture media. Median time for growth was respectively 22, 10 and 7.6 days for Löwenstein-Jensen, TLA and the Mycobacterial Growth Indicator Tube. TLA sensitivity, specificity and predictive values for RMP and INH resistance were 100%. Time to resistance detection was respectively 11 and 11.5 days for RMP and INH. TLA showed a rapid turnaround time and performance comparable to conventional DST methods.

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

DTIC Science & Technology

1997-11-01

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

Rapid detection of Listeria monocytogenes in raw milk and soft cheese by a redox potential measurement based method combined with real-time PCR.

PubMed

Erdősi, Orsolya; Szakmár, Katalin; Reichart, Olivér; Szili, Zsuzsanna; László, Noémi; Székely Körmöczy, Péter; Laczay, Péter

2014-09-01

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detect Listeria monocytogenes in artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. For Listeria species the measuring time was maximum 34 h. The absence of L. monocytogenes could reliably be proven by the redox potential measurement method, but Listeria innocua and Bacillus subtilis could not be differentiated from L. monocytogenes on the basis of the redox curves. The presence of L. monocytogenes had to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g of L. monocytogenes in a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection of L. monocytogenes in food.

Biosensors for rapid and sensitive detection of Staphylococcus aureus in food.

PubMed

Rubab, Momna; Shahbaz, Hafiz Muhammad; Olaimat, Amin N; Oh, Deog-Hwan

2018-05-15

Foodborne illness outbreaks caused by the consumption of food contaminated with harmful bacteria has drastically increased in the past decades. Therefore, detection of harmful bacteria in the food has become an important factor for the recognition and prevention of problems associated with food safety and public health. Staphylococcus aureus is one of the most commonly isolated foodborne pathogen and it is considered as a major cause of foodborne illnesses worldwide. A number of different methods have been developed for the detection and identification of S. aureus in food samples. However, some of these methods are laborious and time-consuming and are not suitable for on-site applications. Therefore, it is highly important to develop rapid and more approachable detection methods. In the last decade, biosensors have gained popularity as an attractive alternative method and now considered as one of most rapid and on-site applicable methods. An overview of the biosensor based methods used for the detection of S. aureus is presented herein. This review focuses on the state-of-the-art biosensor methods towards the detection and quantification of S. aureus, and discusses the most commonly used biosensor methods based on the transducing mode, such as electrochemical, optical, and mass-based biosensors. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid immunohistochemical detection of tumor cells in gastric carcinoma.

PubMed

Mönig, Stefan P; Luebke, Thomas; Soheili, Afsoon; Landsberg, Stephanie; Dienes, H P; Hölscher, Arnulf H; Baldus, Stephan E

2006-11-01

The detection of single tumor cells or tumor cell clusters represents an important issue in intraoperative frozen section analysis. For example, surgical margins may be evaluated in order to minimize the number of additional operations. Furthermore, intraoperative diagnosis of lymph node micrometastasis (LNM) may help to define the area of appropriate lymph node dissection. In addition to haematoxylin and eosin (H&E)-stained sections, immunohistochemical detection of single tumor cells or cell clusters may be helpful in this context. The aim of this study was to evaluate the clinical significance, reliability and sensitivity of intraoperative rapid immunostaining of frozen sections. Therefore, we compared the results of rapid immunohistochemical staining of frozen sections and paraffin sections applying the EnVision and Histofine(R) detection systems. In a prospective immunohistochemical study, paraffin and frozen sections of 20 gastric cancer specimens were analyzed. Paraffin as well as frozen sections were stained immunohistochemically applying the EnVision and Histofine detection systems. As primary antibodies, AE1/AE3 (anti-cytokeratin), EMA (anti-MUC1) and B lymphocyte marker anti-CD20 were applied. The rapid immunostaining procedure was able to be completed within 10-13 min. Rapid immunohistochemical staining of frozen and paraffin sections of the same tumors resulted in comparable immunoreactivity. The rapid EnVision and Histofine procedures allowed immunostaining of frozen sections in less than 13 min. These methods can represent useful additional tools in routine surgical pathology and research, enabling a more accurate frozen section diagnosis compared to staining with H&E alone. Intraoperative rapid immunostaining can be a simple and useful technique to detect LNM.

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

PubMed

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

2012-01-01

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

A novel strategy for rapid detection of NT-proBNP

NASA Astrophysics Data System (ADS)

Cui, Qiyao; Sun, Honghao; Zhu, Hui

2017-09-01

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

Rapid detection of methanol in artisanal alcoholic beverages

NASA Astrophysics Data System (ADS)

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

2015-09-01

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

A detection method in living plant cells for rapidly monitoring the response of plants to exogenous lanthanum.

PubMed

Cheng, Mengzhu; Wang, Lihong; Yang, Qing; Huang, Xiaohua

2018-08-30

The pollution of rare earth elements (REEs) in ecosystem is becoming more and more serious, so it is urgent to establish methods for monitoring the pollution of REEs. Monitoring environmental pollution via the response of plants to pollutants has become the most stable and accurate method compared with traditional methods, but scientists still need to find the primary response of plants to pollutants to improve the sensitivity and speed of this method. Based on the facts that the initiation of endocytosis is the primary cellular response of the plant leaf cells to REEs and the detection of endocytosis is complex and expensive, we constructed a detection method in living plant cells for rapidly monitoring the response of plants to exogenous lanthanum [La(III), a representative of REEs] by designing a new immuno-electrochemical method for detecting the content change in extracellular vitronectin-like protein (VN) that are closely related to endocytosis. Results showed that when 30 μM La(III) initiated a small amount of endocytosis, the content of extracellular VN increased by 5.46 times, but the structure and function of plasma membrane were not interfered by La(III); when 80 μM La(III) strongly initiated a large amount of endocytosis, the content of extracellular VN increased by 119 times, meanwhile, the structure and function of plasma membrane were damaged. In summary, the detection method can reflect the response of plants to La(III) via detecting the content change in extracellular VN, which provides an effective and convenient way to monitor the response of plants to exogenous REEs. Copyright © 2018. Published by Elsevier Inc.

Rapid Change Detection Algorithm for Disaster Management

NASA Astrophysics Data System (ADS)

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

2012-07-01

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

Development of a loop-mediated isothermal amplification method for rapid campylobacter jejuni detection

USDA-ARS?s Scientific Manuscript database

Introduction: Campylobacter jejuni is the leading foodborne pathogen that causes human bacterial gastroenteritis worldwide. Poultry products are regarded as a major source for human infection. Early, rapid detection of this microorganism in poultry products is necessary for contamination control ...

Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis

PubMed Central

Yzquierdo, Sergio Luis; Lemus, Dihadenys; Echemendia, Miguel; Montoro, Ernesto; McNerney, Ruth; Martin, Anandi; Palomino, Juan Carlos

2006-01-01

Background Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. Methods Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. Results Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. Conclusion Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem. PMID:16630356

[Principle of LAMP method--a simple and rapid gene amplification method].

PubMed

Ushikubo, Hiroshi

2004-06-01

So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.

Rapid detection of tuberculosis using droplet-based microfluidics

NASA Astrophysics Data System (ADS)

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

2014-03-01

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

Rapid detection of bacteria with miniaturized pyrolysis-gas chromatographic analysis

NASA Astrophysics Data System (ADS)

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

2002-02-01

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

Rapid combined assay for Salmonella detection in food samples.

PubMed

Gadó, I; Major, P; Király, M; Pláveczky, M G

2000-01-01

A rapid method was developed to detect salmonellae in food samples. The method gave a possibility to obtain results after 28 h 30 min. The preenrichment in buffered peptone water lasted for 6 h, the enrichment in Rappaport-Vassiliadis medium was applied for 18 h followed by PCR with INVA1-INVA2 primer pair, adapting Chiu and Ou's method. This procedure was suitable to demonstrate salmonella contamination at min. 10 cfu/25 g sample. Out of 18 samples there was a good agreement between the results of the conventional and rapid methods in case of 17 samples. PCR with SPVC1-SPVC2 primer pair informing about the presence of virulence plasmid was performed in separate tubes, because decreased sensitivity was observed in case of multiplex PCR.

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

USDA-ARS?s Scientific Manuscript database

The hyperspectral microscope imaging (HMI) method can reduce detection time within 8 hours including incubation process. The early and rapid detection with this method in conjunction with the high throughput capabilities makes HMI method a prime candidate for implementation for the food industry. Th...

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

USDA-ARS?s Scientific Manuscript database

The hyperspectral microscope imaging (HMI) method can reduce detection time within 8 hours including incubation process. The early and rapid detection with this method in conjunction with the high throughput capabilities makes HMI method a prime candidate for implementation for the food industry. Th...

Integrated optical biosensor for rapid detection of bacteria

NASA Astrophysics Data System (ADS)

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

2016-02-01

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

Integrated optical biosensor for rapid detection of bacteria

NASA Astrophysics Data System (ADS)

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

2015-12-01

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

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles

PubMed Central

Chang, Yi-Chung; Yang, Chia-Ying; Sun, Ruei-Lin; Cheng, Yi-Feng; Kao, Wei-Chen; Yang, Pan-Chyr

2013-01-01

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technologies. Thus, there is an unmet demand of new platform technology to improve the bacterial detection and identification in clinical practice. In this study, we developed a rapid, ultra-sensitive, low cost, and non-polymerase chain reaction (PCR)-based method for bacterial identification. Using this method, which measures the resonance light-scattering signal of aptamer-conjugated gold nanoparticles, we successfully detected single S. aureus cell within 1.5 hours. This new platform technology may have potential to develop a rapid and sensitive bacterial testing at point-of-care. PMID:23689505

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.

PubMed

Chang, Yi-Chung; Yang, Chia-Ying; Sun, Ruei-Lin; Cheng, Yi-Feng; Kao, Wei-Chen; Yang, Pan-Chyr

2013-01-01

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technologies. Thus, there is an unmet demand of new platform technology to improve the bacterial detection and identification in clinical practice. In this study, we developed a rapid, ultra-sensitive, low cost, and non-polymerase chain reaction (PCR)-based method for bacterial identification. Using this method, which measures the resonance light-scattering signal of aptamer-conjugated gold nanoparticles, we successfully detected single S. aureus cell within 1.5 hours. This new platform technology may have potential to develop a rapid and sensitive bacterial testing at point-of-care.

Development of molecular methods for the rapid detection of antibiotic susceptibility of Mycoplasma bovis.

PubMed

Sulyok, Kinga M; Bekő, Katinka; Kreizinger, Zsuzsa; Wehmann, Enikő; Jerzsele, Ákos; Rónai, Zsuzsanna; Turcsányi, Ibolya; Makrai, László; Szeredi, Levente; Jánosi, Szilárd; Nagy, Sára Ágnes; Gyuranecz, Miklós

2018-01-01

Determining the antibiotic susceptibility profile of Mycoplasma bovis isolates in vitro provides the basis for the appropriate choice of antibiotics in the therapy. Traditionally, the antibiotic susceptibility examination of mycoplasmas is technically demanding, time-consuming and rarely performed in diagnostic laboratories. The aim of the present study was to develop rapid molecular assays to determine mutations responsible for elevated minimal inhibitory concentrations (MICs) to fluoroquinolones, tetracyclines, aminocyclitols, macrolides, lincosamides, phenicols and pleuromutilins in M. bovis. The nine mismatch amplification mutation assays (MAMA) and seven high resolution melt (HRM) tests designed in the present study enable the simultaneous detection of these genetic markers. The sensitivity of the assays varied between 10 2 -10 5 copy numbers/reaction. Cross-reactions with other mycoplasmas occurring in cattle were detected in assays targeting universal regions (e.g. 16S rRNA). Nevertheless, results of the novel method were in accordance with sequence and MICs data of the M. bovis pure cultures. Also, the tests of clinical samples containing high amount of M. bovis DNA were congruent even in the presence of other Mycoplasma spp. The presented method is highly cost-effective and can provide an antibiogram to 12 antibiotics in approximately 3-4 days when previous isolation of M. bovis is applied. In order to assure the proper identification of the genetic markers at issue, the regions examined by the MAMA and HRM tests are overlapping. In conclusion, the developed assays have potential to be used in routine diagnostics for the detection of antibiotic susceptibility in M. bovis. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2013-01-01

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

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

PubMed Central

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

2014-01-01

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

Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee.

PubMed

von Hertwig, Aline Morgan; Sant'Ana, Anderson S; Sartori, Daniele; da Silva, Josué José; Nascimento, Maristela S; Iamanaka, Beatriz Thie; Pelegrinelli Fungaro, Maria Helena; Taniwaki, Marta Hiromi

2018-05-01

Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B 2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species. Copyright © 2018 Elsevier B.V. All rights reserved.

A sensitive and innovative detection method for rapid C-reactive proteins analysis based on a micro-fluxgate sensor system

PubMed Central

Yang, Zhen; Zhi, Shaotao; Feng, Zhu; Lei, Chong; Zhou, Yong

2018-01-01

A sensitive and innovative assay system based on a micro-MEMS-fluxgate sensor and immunomagnetic beads-labels was developed for the rapid analysis of C-reactive proteins (CRP). The fluxgate sensor presented in this study was fabricated through standard micro-electro-mechanical system technology. A multi-loop magnetic core made of Fe-based amorphous ribbon was employed as the sensing element, and 3-D solenoid copper coils were used to control the sensing core. Antibody-conjugated immunomagnetic microbeads were strategically utilized as signal tags to label the CRP via the specific conjugation of CRP to polyclonal CRP antibodies. Separate Au film substrates were applied as immunoplatforms to immobilize CRP-beads labels through classical sandwich assays. Detection and quantification of the CRP at different concentrations were implemented by detecting the stray field of CRP labeled magnetic beads using the newly-developed micro-fluxgate sensor. The resulting system exhibited the required sensitivity, stability, reproducibility, and selectivity. A detection limit as low as 0.002 μg/mL CRP with a linearity range from 0.002 μg/mL to 10 μg/mL was achieved, and this suggested that the proposed biosystem possesses high sensitivity. In addition to the extremely low detection limit, the proposed method can be easily manipulated and possesses a quick response time. The response time of our sensor was less than 5 s, and the entire detection period for CRP analysis can be completed in less than 30 min using the current method. Given the detection performance and other advantages such as miniaturization, excellent stability and specificity, the proposed biosensor can be considered as a potential candidate for the rapid analysis of CRP, especially for point-of-care platforms. PMID:29601593

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

PubMed

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

2015-02-01

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

Validation of the Applied Biosystems RapidFinder Shiga Toxin-Producing E. coli (STEC) Detection Workflow.

PubMed

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 potyviruses from crude plant extracts.

PubMed

Silva, Gonçalo; Oyekanmi, Joshua; Nkere, Chukwuemeka K; Bömer, Moritz; Kumar, P Lava; Seal, Susan E

2018-04-01

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named 'Direct RT-RPA', detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Rapid Radiochemical Methods for Asphalt Paving Material ...

EPA Pesticide Factsheets

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

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

PubMed

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

2015-11-15

A rapid method for the determination of 14 types of isoflavones in food using ultra-high performance liquid chromatography (UHPLC) was validated in terms of precision, accuracy, sensitivity and linearity. The UHPLC separation was performed on a reverse-phase C18 column (particle size 2 μm, i.d. 2 mm, length 100 mm) using a photo diode array detector that was fixed to 260 nm. The limits of detection and quantification of the UHPLC analyses ranged from 0.03 to 0.33 mg kg(-1). The intra-day and inter-day precision of the individual isoflavones were less than 11.77% and calibration curves exhibited good linearity (r(2) = 0.99) within the tested ranges. These results suggest that the rapid method used in this study could be available to determine of 14 types of isoflavones in a variety of food such as soy bean, black bean, red bean and soybean paste. Copyright © 2015 Elsevier Ltd. All rights reserved.

RAPID METHOD FOR DETERMINATION OF RADIOSTRONTIUM IN EMERGENCY MILK SAMPLES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maxwell, S.; Culligan, B.

2008-07-17

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

Rapid quantitative detection of glucose content in glucose injection by reaction headspace gas chromatography.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2017-10-20

This work investigates an automated technique for rapid detecting the glucose content in glucose injection by reaction headspace gas chromatography (HS-GC). This method is based on the oxidation reaction of glucose in glucose injection with potassium dichromate. The carbon dioxide (CO 2 ) formed from the oxidation reaction can be quantitatively detected by GC. The results show that the relative standard deviation (RSD) of the present method was within 2.91%, and the measured glucose contents in glucose injection closely match those quantified by the reference method (relative differences <6.45%). The new HS-GC technique is rapid, practical and can be used to the batch detection of the glucose content in glucose injection related applications. Copyright © 2017 Elsevier B.V. All rights reserved.

A rapid detection method for policy-sensitive amines real-time supervision.

PubMed

Zhang, Haixu; Shu, Jinian; Yang, Bo; Zhang, Peng; Ma, Pengkun

2018-02-01

Many organic amines that comprise a benzene ring are policy-sensitive because of their toxicity and links to social harm. However, to date, detection of such compounds mainly relies on offline methods. This study proposes an online pptv (parts per trillion by volume) level of detection method for amines, using the recently-built vacuum ultraviolet photoionization mass spectrometer (VUV-PIMS) combined with a new doping technique. Thus, the dichloromethane doping-assisted photoionization mass spectra of aniline, benzylamine, phenethylamine, amphetamine, and their structural isomers were recorded. The dominant characteristic mass peaks for all amines are those afforded by protonated amines and the amino radical-loss. The signal intensities of the amines were enhanced by 60-130 times compared to those recorded without doping assistance. Under 10s detection time, the sensitivities of aniline and benzylamine in the gas phase were determined as 4.0 and 2.7 countspptv -1 , with limits of detection (LODs) of 36 and 22 pptv, respectively. Notably, the detection efficiency of this method can be tenfold better in future applications since the ion transmission efficiency of the mass spectrometer was intentionally reduced to ~ 10% in this study. Therefore, dichloromethane doping-assisted photoionization mass spectrometry has proven to be a highly promising on-line approach to amine detection in environmental and judicial supervision and shows great potential for application in the biological field. Copyright © 2017 Elsevier B.V. All rights reserved.

Nanomaterial-enabled Rapid Detection of Water Contaminants.

PubMed

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

2015-10-28

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

Rapid Detection Method for the Four Most Common CHEK2 Mutations Based on Melting Profile Analysis.

PubMed

Borun, Pawel; Salanowski, Kacper; Godlewski, Dariusz; Walkowiak, Jaroslaw; Plawski, Andrzej

2015-12-01

CHEK2 is a tumor suppressor gene, and the mutations affecting the functionality of the protein product increase cancer risk in various organs. The elevated risk, in a significant percentage of cases, is determined by the occurrence of one of the four most common mutations in the CHEK2 gene, including c.470T>C (p.I157T), c.444+1G>A (IVS2+1G>A), c.1100delC, and c.1037+1538_1224+328del5395 (del5395). We have developed and validated a rapid and effective method for their detection based on high-resolution melting analysis and comparative-high-resolution melting, a novel approach enabling simultaneous detection of copy number variations. The analysis is performed in two polymerase chain reactions followed by melting analysis, without any additional reagents or handling other than that used in standard high-resolution melting. Validation of the method was conducted in a group of 103 patients with diagnosed breast cancer, a group of 240 unrelated patients with familial history of cancer associated with the CHEK2 gene mutations, and a 100-person control group. The results of the analyses for all three groups were fully consistent with the results from other methods. The method we have developed improves the identification of the CHEK2 mutation carriers, reduces the cost of such analyses, as well as facilitates their implementation. Along with the increased efficiency, the method maintains accuracy and reliability comparable to other more labor-consuming techniques.

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

PubMed Central

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

2016-01-01

Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 104–2.8 × 106 cells/mL with a detection limit (LOD) of 0.9 × 103 cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens. PMID:26976753

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7.

PubMed

Setterington, Emma B; Alocilja, Evangelyn C

2011-01-15

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense. Copyright © 2010 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-04-01

Acetamiprid is a neonicotinoid pesticide that is commonly used in modern farming. Acetamiprid residue in food commodities can be a potential harm to human and has been implicated in the honey bee hive die off crisis. In this study, we developed rapid, simple, and sensitive methods to detect acetamiprid in apple juice and on apple surfaces using surface-enhanced Raman spectroscopy (SERS). No pretreatment of apple juice sample was performed. A simple surface swab method was used to recover acetamiprid from the apple surface. Samples were incubated with silver dendrites for several minutes and SERS spectra were taken directly from the silver surface. Detection of a set of 5 apple juice samples can be done within 10 min. The swab-SERS method took 15 min for a set of 5 samples. Resulting spectral data were analyzed using principal component analysis. The highest acetamiprid peak at 634 cm(-1) was used to detect and quantify the amount of acetamiprid spiked in 1:1 water-methanol solvent, apple juice, and on apple surface. The SERS method was able to successfully detect acetamiprid at 0.5 μg/mL (0.5 ppm) in solvent, 3 μg/mL (3 ppm) in apple juice, and 0.125 μg/cm(2) on apple surfaces. The SERS methods provide simple, rapid, and sensitive ways to detect acetamiprid in beverages and on the surfaces of thick skinned fruits and vegetables. © 2014 Institute of Food Technologists®

Rapid detection of pandemic influenza in the presence of seasonal influenza

PubMed Central

2010-01-01

Background Key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. In addition, they must minimise false alarms during a normal influenza season. We develop a method that uses historical syndromic influenza data from the existing surveillance system 'SERVIS' (Scottish Enhanced Respiratory Virus Infection Surveillance) for influenza-like illness (ILI) in Scotland. Methods We develop an algorithm based on the weekly case ratio (WCR) of reported ILI cases to generate an alarm for pandemic influenza. From the seasonal influenza data from 13 Scottish health boards, we estimate the joint probability distribution of the country-level WCR and the number of health boards showing synchronous increases in reported influenza cases over the previous week. Pandemic cases are sampled with various case reporting rates from simulated pandemic influenza infections and overlaid with seasonal SERVIS data from 2001 to 2007. Using this combined time series we test our method for speed of detection, sensitivity and specificity. Also, the 2008-09 SERVIS ILI cases are used for testing detection performances of the three methods with a real pandemic data. Results We compare our method, based on our simulation study, to the moving-average Cumulative Sums (Mov-Avg Cusum) and ILI rate threshold methods and find it to be more sensitive and rapid. For 1% case reporting and detection specificity of 95%, our method is 100% sensitive and has median detection time (MDT) of 4 weeks while the Mov-Avg Cusum and ILI rate threshold methods are, respectively, 97% and 100% sensitive with MDT of 5 weeks. At 99% specificity, our method remains 100% sensitive with MDT of 5 weeks. Although the threshold method maintains its sensitivity of 100% with MDT of 5 weeks, sensitivity of Mov-Avg Cusum declines to 92% with increased MDT of 6 weeks. For a two-fold decrease in the case reporting rate (0.5%) and 99% specificity, the WCR and threshold methods

Rapid detection of Escherichia coli O157:H7 using tunneling magnetoresistance biosensor

NASA Astrophysics Data System (ADS)

Wu, Yuanzhao; Liu, Yiwei; Zhan, Qingfeng; Liu, J. Ping; Li, Run-Wei

2017-05-01

A rapid method for the sensitive detection of bacteria using magnetic immunoassay, which are measured with a tunneling magnetoresistance (TMR) sensor, is described. For the measurement of Escherichia coli O157:H7 (E. coli O157:H7) bacteria, the target was labeled by magnetic beads through magnetic immunoassay. The magnetic beads produce a weak magnetic fringe field when external field is applied, thus induce the magnetoresistance change of TMR sensor. A detection limit of 100 CFU/mL E. coli O157:H7 bacteria in 5 hours was obtained. With its high sensitive and rapid detection scheme based on the TMR biosensor, the detection system is an excellent candidate suitable and promising for food safety and biomedical detection.

[Comparison of detection sensitivity in rapid-diagnosis influenza virus kits].

PubMed

Tokuno, Osamu; Fujiwara, Miki; Nakajoh, Yoshimi; Yamanouchi, Sumika; Adachi, Masayo; Ikeda, Akiko; Kitayama, Shigeo; Takahashi, Toshio; Kase, Tetsuo; Kinoshita, Shouhiro; Kumagai, Shunichi

2009-09-01

Rapid-diagnosis kits able to detect influenza A and B virus by immunochromatography developed by different manufacturers, while useful in early diagnosis, may vary widely in detection sensitivity. We compared sensitivity results for eight virus-detection kits in current use--Quick Chaser FluA, B (Mizuho Medy), Espline Influenza A & B-N (Fujirebio), Capilia Flu A + B (Nippon Beckton Dickinson & Alfesa Pharma), Poctem Influenza A/B (Otsuka Pharma & Sysmex), BD Flu Examan (Nippon Beckton Dickinson), Quick Ex-Flu "Seiken" (Denka Seiken), Quick Vue Rapid SP Influ (DP Pharma Biomedical), and Rapid Testa FLU stick (Daiichi Pure Chemicals)--against influenza virus stocks, contained five vaccination strains (one A/H1N1, two A/H3N2, and two B) and six clinical strains (two A/H1N1, two A/H3N2, and two B). Minimum detection concentrations giving immunologically positive signals in serial dilution and RNA copies in positive dilution in real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were assayed for all kits and virus stock combinations. RNA log10 copy numbers/mL in dilutions within detection limits yielded 5.68-7.02, 6.37-7,17, and 6.5-8.13 for A/H1N1, A/H3N2, and B. Statistically significant differences in sensitivity were observed between some kit combinations. Detection sensitivity tended to be relatively higher for influenza A than B virus. This is assumed due to different principles in kit methods, such as monoclonal antibodies, specimen-extraction conditions, and other unknown factors.

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

PubMed Central

Setterington, Emma B.; Alocilja, Evangelyn C.

2012-01-01

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

Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification.

PubMed

Ge, Junwei; Shi, Yunjia; Cui, Xingyang; Gu, Shanshan; Zhao, Lili; Chen, Hongyan

2018-06-01

To date, the pathogenic role of mink circovirus (MiCV) remains unclear, and its prevalence and economic importance are unknown. Therefore, a rapid and sensitive molecular diagnosis is necessary for disease management and epidemiological surveillance. However, only PCR methods can identify MiCV infection at present. In this study, we developed a nested PCR and established a novel recombinase polymerase amplification (RPA) assay for MiCV detection. Sensitivity analysis showed that the detection limit of nested PCR and RPA assay was 10 1 copies/reaction, and these methods were more sensitive than conventional PCR, which has a detection limit of 10 5 copies/reaction. The RPA assay had no cross-reactivity with other related viral pathogens, and amplification was completed in less than 20 min with a simple device. Further assessment of clinical samples showed that the two assays were accurate in identifying positive and negative conventional PCR samples. The detection rate of MiCV by the RPA assay in clinical samples was 38.09%, which was 97% consistent with that by the nested PCR. The developed nested PCR is a highly sensitive tool for practical use, and the RPA assay is a simple, sensitive, and potential alternative method for rapid and accurate MiCV diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid test for the detection of hazardous microbiological material

NASA Astrophysics Data System (ADS)

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

2009-09-01

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

Rapid detection of Salmonella in milk by electrochemical magneto-immunosensing.

PubMed

Liébana, Susana; Lermo, Anabel; Campoy, Susana; Cortés, María Pilar; Alegret, Salvador; Pividori, María Isabel

2009-10-15

A very simple and rapid method for the detection of Salmonella in milk is reported. In this approach, the bacteria are captured and preconcentrated from milk samples with magnetic beads through an immunological reaction. A second polyclonal antibody labeled with peroxidase is used as serological confirmation with electrochemical detection based on a magneto-electrode. The 'IMS/m-GEC electrochemical immunosensing' approach shows a limit of detection of 5 x 10(3) and 7.5 x 10(3)CFU mL(-1) in LB and in milk diluted 1/10 in LB broth, respectively, in 50 min without any pretreatment. If the skimmed-milk is preenriched for 6h, the method is able to detect as low as 1.4 CFU mL(-1), while if it is preenriched for 8h, as low as 0.108 x CFU mL(-1) (2.7 x CFU in 25 g of milk, in 5 samples of 5 mL) are detected accordingly with the legislation. Moreover, the method is able to clearly distinguish between food pathogenic bacteria such as Salmonella and Escherichia coli. The features of this approach are discussed and compared with classical culture methods.

Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification

PubMed Central

Okafuji, Takao; Yoshida, Naoko; Fujino, Motoko; Motegi, Yoshie; Ihara, Toshiaki; Ota, Yoshinori; Notomi, Tsugunori; Nakayama, Tetsuo

2005-01-01

Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis. PMID:15814976

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

PubMed Central

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

2015-01-01

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

Rapid Assessment of Genotoxicity by Flow Cytometric Detection of Cell Cycle Alterations.

PubMed

Bihari, Nevenka

2017-01-01

Flow cytometry is a convenient method for the determination of genotoxic effects of environmental pollution and can reveal genotoxic compounds in unknown environmental mixtures. It is especially suitable for the analyses of large numbers of samples during monitoring programs. The speed of detection is one of the advantages of this technique which permits the acquisition of 10 4 -10 5 cells per sample in 5 min. This method can rapidly detect cell cycle alterations resulting from DNA damage. The outcome of such an analysis is a diagram of DNA content across the cell cycle which indicates cell proliferation, G 2 arrests, G 1 delays, apoptosis, and ploidy.Here, we present the flow cytometric procedure for rapid assessment of genotoxicity via detection of cell cycle alterations. The described protocol simplifies the analysis of genotoxic effects in marine environments and is suitable for monitoring purposes. It uses marine mussel cells in the analysis and can be adapted to investigations on a broad range of marine invertebrates.

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients

PubMed Central

Hoben, D. A.; Ashton, D. H.; Peterson, A. C.

1973-01-01

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory. PMID:4568884

Microfluidic devices for sample preparation and rapid detection of foodborne pathogens.

PubMed

Kant, Krishna; Shahbazi, Mohammad-Ali; Dave, Vivek Priy; Ngo, Tien Anh; Chidambara, Vinayaka Aaydha; Than, Linh Quyen; Bang, Dang Duong; Wolff, Anders

2018-03-10

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line. Copyright © 2018. Published by Elsevier Inc.

Rapid detection of hemagglutination using restrictive microfluidic channels equipped with waveguide-mode sensors

NASA Astrophysics Data System (ADS)

Ashiba, Hiroki; Fujimaki, Makoto; Awazu, Koichi; Fu, Mengying; Ohki, Yoshimichi; Tanaka, Torahiko; Makishima, Makoto

2016-02-01

Hemagglutination is utilized for various immunological assays, including blood typing and virus detection. Herein, we describe a method of rapid hemagglutination detection based on a microfluidic channel installed on an optical waveguide-mode sensor. Human blood samples mixed with hemagglutinating antibodies associated with different blood groups were injected into the microfluidic channel, and reflectance spectra of the samples were measured after stopping the flow. The agglutinated and nonagglutinated samples were distinguishable by the alterations in their reflectance spectra with time; the microfluidic channels worked as spatial restraints for agglutinated red blood cells. The demonstrated system allowed rapid hemagglutination detection within 1 min. The suitable height of the channels was also discussed.

A novel method for rapid in vitro radiobioassay

NASA Astrophysics Data System (ADS)

Crawford, Evan Bogert

minutes of sample preparation and counting time. Radiation detector spectra -- e.g. those from high-purity germanium (HPGe) gamma detectors and liquid scintillation detectors -- which contain decay signals from multiple isotopes often have overlapping signals: the counts from one isotope's decay can appear in energy channels associated with another isotope's decay, complicating the calculation of each isotope's activity. The uncertainties associated with analyzing these spectra have been traced in order to determine the effects of one isotope's count rate on the sensitivity and uncertainty associated with each other isotope. The method that was developed takes advantage of activated carbon filtration to eliminate quenching effects and to make the liquid scintillation spectra from different urine samples comparable. The method uses pulse-shape analysis to reduce the interference from beta emitters in the liquid scintillation spectrum and improve the minimum detectable activity (MDA) and minimum quantifiable activity (MQA) for alpha emitters. The method uses an HPGe detector to quantify the activity of gamma emitters, and subtract their isotopes' contributions to the liquid scintillation spectra via a calibration factor, such that the pure beta and pure alpha emitters can be identified and quantified from the resulting liquid scintillation spectra. Finally, the method optionally uses extractive scintillators to rapidly separate the alpha emitters from the beta emitters when the activity from the beta emitters is too great to detect or quantify the activity from the alpha emitters without such a separation. The method is able to detect and quantify all five isotopes, with uncertainties and biases usually in the 10-40% range, depending upon the isotopic mixtures and the activity ratios between each of the isotopes.

Evaluation of a Method for Rapid Detection of Listeria monocytogenes in Dry-Cured Ham Based on Impedanciometry Combined with Chromogenic Agar.

PubMed

Labrador, Mirian; Rota, María C; Pérez, Consuelo; Herrera, Antonio; Bayarri, Susana

2018-05-01

The food industry is in need of rapid, reliable methodologies for the detection of Listeria monocytogenes in ready-to-eat products, as an alternative to the International Organization of Standardization (ISO) 11290-1 reference method. The aim of this study was to evaluate impedanciometry combined with chromogenic agar culture for the detection of L. monocytogenes in dry-cured ham. The experimental setup consisted in assaying four strains of L. monocytogenes and two strains of Listeria innocua in pure culture. The method was evaluated according to the ISO 16140:2003 standard through a comparative study with the ISO reference method with 119 samples of dry-cured ham. Significant determination coefficients ( R 2 of up to 0.99) for all strains assayed in pure culture were obtained. The comparative study results had 100% accuracy, 100% specificity, and 100% sensitivity. Impedanciometry followed by chromogenic agar culture was capable of detecting 1 CFU/25 g of food. L. monocytogenes was not detected in the 65 commercial samples tested. The method evaluated herein represents a promising alternative for the food industry in its efforts to control L. monocytogenes. Overall analysis time is shorter and the method permits a straightforward analysis of a large number of samples with reliable results.

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

PubMed

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

2017-01-01

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

Rapid and potentially portable detection and quantification technologies for foodborne pathogens

USDA-ARS?s Scientific Manuscript database

Introduction Traditional microbial culture methods are able to detect and identify a single specific bacterium, but may require days or weeks and typically do not produce quantitative data. The quest for faster, quantitative results has spurred development of “rapid methods” which usually employ bio...

A Rapid and Specific Assay for the Detection of MERS-CoV

PubMed Central

Huang, Pei; Wang, Hualei; Cao, Zengguo; Jin, Hongli; Chi, Hang; Zhao, Jincun; Yu, Beibei; Yan, Feihu; Hu, Xingxing; Wu, Fangfang; Jiao, Cuicui; Hou, Pengfei; Xu, Shengnan; Zhao, Yongkun; Feng, Na; Wang, Jianzhong; Sun, Weiyang; Wang, Tiecheng; Gao, Yuwei; Yang, Songtao; Xia, Xianzhu

2018-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that can cause human respiratory disease. The development of a detection method for this virus that can lead to rapid and accurate diagnosis would be significant. In this study, we established a nucleic acid visualization technique that combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualization strip (RT-LAMP-VF) to detect the N gene of MERS-CoV. The RT-LAMP-VF assay was performed in a constant temperature water bath for 30 min, and the result was visible by the naked eye within 5 min. The RT-LAMP-VF assay was capable of detecting 2 × 101 copies/μl of synthesized RNA transcript and 1 × 101 copies/μl of MERS-CoV RNA. The method exhibits no cross-reactivities with multiple CoVs including SARS-related (SARSr)-CoV, HKU4, HKU1, OC43 and 229E, and thus exhibits high specificity. Compared to the real-time RT-PCR (rRT-PCR) method recommended by the World Health Organization (WHO), the RT-LAMP-VF assay is easy to handle, does not require expensive equipment and can rapidly complete detection within 35 min. PMID:29896174

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

PubMed Central

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

2014-01-01

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

Rapid optimization method of the strong stray light elimination for extremely weak light signal detection.

PubMed

Wang, Geng; Xing, Fei; Wei, Minsong; You, Zheng

2017-10-16

The strong stray light has huge interference on the detection of weak and small optical signals, and is difficult to suppress. In this paper, a miniaturized baffle with angled vanes was proposed and a rapid optimization model of strong light elimination was built, which has better suppression of the stray lights than the conventional vanes and can optimize the positions of the vanes efficiently and accurately. Furthermore, the light energy distribution model was built based on the light projection at a specific angle, and the light propagation models of the vanes and sidewalls were built based on the Lambert scattering, both of which act as the bias of a calculation method of stray light. Moreover, the Monte-Carlo method was employed to realize the Point Source Transmittance (PST) simulation, and the simulation result indicated that it was consistent with the calculation result based on our models, and the PST could be improved by 2-3 times at the small incident angles for the baffle designed by the new method. Meanwhile, the simulation result was verified by laboratory tests, and the new model with derived analytical expressions which can reduce the simulation time significantly.

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

PubMed

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

2011-10-15

Naegleria fowleri is a small free-living amoebo-flagellate found in natural and manmade thermal aquatic habitats worldwide. The organism is pathogenic to man causing fatal primary amoebic meningoencephalitis (PAM). Infection typically results from bathing in contaminated water and is usually fatal. It is, therefore, important to identify sites containing N. fowleri in the interests of preventive public health microbiology. Culture of environmental material is the conventional method for the isolation of N. fowleri but requires several days incubation and subsequent biochemical or molecular tests to confirm identification. Here, a nested one-step PCR test, in conjunction with a direct DNA extraction from water or sediment material, was developed for the rapid and reliable detection of N. fowleri from the environment. Here, the assay detected N, fowleri in 18/109 river water samples associated with a nuclear power plant in South West France and 0/10 from a similar site in the UK. Although culture of samples yielded numerous thermophilic free-living amoebae, none were N. fowleri or other thermophilic Naegleria spp. The availability of a rapid, reliable and sensitive one-step nested PCR method for the direct detection of N. fowleri from the environment may aid ecological studies and enable intervention to prevent PAM cases. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-08-01

Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Use of rapid-scan EPR to improve detection sensitivity for spin-trapped radicals.

PubMed

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

2013-07-16

The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1-6.0 μM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2016-02-01

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

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

NASA Astrophysics Data System (ADS)

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

2015-09-01

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

Development of Rapid Detection and Genetic Characterization of Salmonella in Poultry Breeder Feeds

PubMed Central

Jarquin, Robin; Hanning, Irene; Ahn, Soohyoun; Ricke, Steven C.

2009-01-01

Salmonella is a leading cause of foodborne illness in the United States, with poultry and poultry products being a primary source of infection to humans. Poultry may carry some Salmonella serovars without any signs or symptoms of disease and without causing any adverse effects to the health of the bird. Salmonella may be introduced to a flock by multiple environmental sources, but poultry feed is suspected to be a leading source. Detecting Salmonella in feed can be challenging because low levels of the bacteria may not be recovered using traditional culturing techniques. Numerous detection methodologies have been examined over the years for quantifying Salmonella in feeds and many have proven to be effective for Salmonella isolation and detection in a variety of feeds. However, given the potential need for increased detection sensitivity, molecular detection technologies may the best candidate for developing rapid sensitive methods for identifying small numbers of Salmonella in the background of large volumes of feed. Several studies have been done using polymerase chain reaction (PCR) assays and commercial kits to detect Salmonella spp. in a wide variety of feed sources. In addition, DNA array technology has recently been utilized to track the dissemination of a specific Salmonella serotype in feed mills. This review will discuss the processing of feeds and potential points in the process that may introduce Salmonella contamination to the feed. Detection methods currently used and the need for advances in these methods also will be discussed. Finally, implementation of rapid detection for optimizing control methods to prevent and remove any Salmonella contamination of feeds will be considered. PMID:22346699

Lab on a chip sensor for rapid detection and antibiotic resistance determination of Staphylococcus aureus.

PubMed

Abeyrathne, Chathurika D; Huynh, Duc H; Mcintire, Thomas W; Nguyen, Thanh C; Nasr, Babak; Zantomio, Daniela; Chana, Gursharan; Abbott, Iain; Choong, Peter; Catton, Mike; Skafidas, Efstratios

2016-03-21

The Gram-positive bacterium, Staphylococcus aureus (S. aureus), is a major pathogen responsible for a variety of infectious diseases ranging from cellulitis to more serious conditions such as septic arthritis and septicaemia. Timely treatment with appropriate antibiotic therapy is essential to ensure clinical defervescence and to prevent further complications such as infective endocarditis or organ impairment due to septic shock. To date, initial antibiotic choice is empirical, using a "best guess" of likely organism and sensitivity- an approach adopted due to the lack of rapid identification methods for bacteria. Current culture based methods take up to 5 days to identify the causative bacterial pathogen and its antibiotic sensitivity. This paper provides proof of concept for a biosensor, based on interdigitated electrodes, to detect the presence of S. aureus and ascertain its sensitivity to flucloxacillin rapidly (within 2 hours) in a cost effective manner. The proposed method is label-free and uses non-faradic measurements. This is the first study to successfully employ interdigitated electrodes for the rapid detection of antibiotic resistance. The method described has important potential outcomes of faster definitive antibiotic treatment and more rapid clinical response to treatment.

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

PubMed

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

2013-06-01

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

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

PubMed

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

PubMed Central

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

2014-01-01

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

An FPGA-Based Rapid Wheezing Detection System

PubMed Central

Lin, Bor-Shing; Yen, Tian-Shiue

2014-01-01

Wheezing is often treated as a crucial indicator in the diagnosis of obstructive pulmonary diseases. A rapid wheezing detection system may help physicians to monitor patients over the long-term. In this study, a portable wheezing detection system based on a field-programmable gate array (FPGA) is proposed. This system accelerates wheezing detection, and can be used as either a single-process system, or as an integrated part of another biomedical signal detection system. The system segments sound signals into 2-second units. A short-time Fourier transform was used to determine the relationship between the time and frequency components of wheezing sound data. A spectrogram was processed using 2D bilateral filtering, edge detection, multithreshold image segmentation, morphological image processing, and image labeling, to extract wheezing features according to computerized respiratory sound analysis (CORSA) standards. These features were then used to train the support vector machine (SVM) and build the classification models. The trained model was used to analyze sound data to detect wheezing. The system runs on a Xilinx Virtex-6 FPGA ML605 platform. The experimental results revealed that the system offered excellent wheezing recognition performance (0.912). The detection process can be used with a clock frequency of 51.97 MHz, and is able to perform rapid wheezing classification. PMID:24481034

Quantum Dot-Fullerene Based Molecular Beacon Nanosensors for Rapid, Highly Sensitive Nucleic Acid Detection.

PubMed

Liu, Ye; Kannegulla, Akash; Wu, Bo; Cheng, Li-Jing

2018-05-15

Spherical fullerene (C 60 ) can quench the fluorescence of a quantum dot (QD) through energy transfer and charge transfer processes, with the quenching efficiency regulated by the number of proximate C 60 on each QD. With the quenching property and its small size compared with other nanoparticle-based quenchers, it is advantageous to group a QD reporter and multiple C 60 -labeled oligonucleotide probes to construct a molecular beacon (MB) probe for sensitive, robust nucleic acid detection. We demonstrated a rapid, high-sensitivity DNA detection method using the nanosensors composed of QD-C 60 based MBs carried by magnetic nanoparticles (MNPs). The assay was accelerated by first dispersing the nanosensors in analytes for highly efficient DNA capture resulting from short-distance 3-dimensional diffusion of targets to the sensor surface and then concentrating the nanosensors to a substrate by magnetic force to amplify the fluorescence signal for target quantification. The enhanced mass transport enabled a rapid detection (< 10 min) with a small sample volume (1-10 µl). The high signal-to-noise ratio produced by the QD-C 60 pairs and magnetic concentration yielded a detection limit of 100 fM (~106 target DNA copies for a 10 µl analyte). The rapid, sensitive, label-free detection method will benefit the applications in point-of-care molecular diagnostic technologies.

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

PubMed

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.

Detection of Streptococcus pyogenes using rapid visual molecular assay.

PubMed

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

2015-09-01

Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64°C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

NASA Astrophysics Data System (ADS)

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

2016-04-01

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

Rapid detection of fungal keratitis with DNA-stabilizing FTA filter paper.

PubMed

Menassa, Nardine; Bosshard, Philipp P; Kaufmann, Claude; Grimm, Christian; Auffarth, Gerd U; Thiel, Michael A

2010-04-01

Purpose. Polymerase chain reaction (PCR) is increasingly important for the rapid detection of fungal keratitis. However, techniques of specimen collection and DNA extraction before PCR may interfere with test sensitivity. The purpose of this study was to investigate the use of DNA-stabilizing FTA filter paper (Indicating FTA filter paper; Whatman International, Ltd., Maidstone, UK) for specimen collection without DNA extraction in a single-step, nonnested PCR for fungal keratitis. Methods. Specimens were collected from ocular surfaces with FTA filter discs, which automatically lyse collected cells and stabilize nucleic acids. Filter discs were directly used in single-step PCR reactions to detect fungal DNA. Test sensitivity was evaluated with serial dilutions of Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus cultures. Test specificity was analyzed by comparing 196 and 155 healthy individuals from Switzerland and Egypt, respectively, with 15 patients with a diagnosis of microbial keratitis. Results. PCR with filter discs detected 3 C. albicans, 25 F. oxysporum, and 125 A. fumigatus organisms. In healthy volunteers, fungal PCR was positive in 1.0% and 8.4% of eyes from Switzerland and Egypt, respectively. Fungal PCR remained negative in 10 cases of culture-proven bacterial keratitis, became positive in 4 cases of fungal keratitis, but missed 1 case of culture-proven A. fumigatus keratitis. Conclusions. FTA filter paper for specimen collection together with direct PCR is a promising method of detecting fungal keratitis. The analytical sensitivity is high without the need for a semi-nested or nested second PCR, the clinical specificity is 91.7% to 99.0%, and the method is rapid and inexpensive.

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

PubMed

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

2014-05-01

Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification.

PubMed

Guo, Xu-Guang; Zhou, Yong-Zhuo; Li, Qin; Wang, Wei; Wen, Jin-Zhou; Zheng, Lei; Wang, Qian

2018-04-18

To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.

Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

PubMed

Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

2015-05-01

Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid detection of α-thalassaemia variants using droplet digital PCR.

PubMed

Lee, T-Y; Lai, M-I; Ramachandran, V; Tan, J A M A; Teh, L-K; Othman, R; Hussein, N H; George, E

2016-08-01

Alpha thalassaemia is a highly prevalent disease globally and is a well-known public health problem in Malaysia. The deletional forms of the mutation are the most common forms found in alpha thalassaemia. The three most common deletional alpha thalassaemia found in this region include --(SEA) deletion, -α(3.7) rightward and -α(4.2) leftward deletions. The prevalence rate of triplication alpha cases such as ααα(anti3.7) and ααα(anti4.2) is not known in Malaysia although it plays a role in exacerbating the clinical phenotypes in beta thalassaemia carriers. Recently, there have been more reported cases of rare alpha thalassaemia mutations due to the advancement of molecular techniques involved in thalassaemia detections. Therefore, it is essential to develop a new method which allows the detection of different alpha thalassaemia mutations including the rare ones simultaneously and accurately. The purpose of this study was to design an assay for the detection of triplications, common and rare deletional alpha thalassaemia using droplet digital PCR (ddPCR). This is a quantitative detection method to measure the changes of copy number which can detect deletions, duplications and triplications of the alpha globin gene simultaneously. In conclusion, ddPCR is an alternative method for rapid detection of alpha thalassaemia variants in Malaysia. © 2016 John Wiley & Sons Ltd.

A Direct and Rapid Method to Determine Cyanide in Urine by Capillary Electrophoresis

PubMed Central

Zhang, Qiyang; Maddukuri, Naveen; Gong, Maojun

2015-01-01

Cyanides are poisonous chemicals that widely exist in nature and industrial processes as well as accidental fires. Rapid and accurate determination of cyanide exposure would facilitate forensic investigation, medical diagnosis, and chronic cyanide monitoring. Here, a rapid and direct method was developed for the determination of cyanide ions in urinary samples. This technique was based on an integrated capillary electrophoresis system coupled with laser-induced fluorescence (LIF) detection. Cyanide ions were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and a primary amine (glycine) for LIF detection. Three separate reagents, NDA, glycine, and cyanide sample, were mixed online, which secured uniform conditions between samples for cyanide derivatization and reduced the risk of precipitation formation of mixtures. Conditions were optimized; the derivatization was completed in 2-4 minutes, and the separation was observed in 25 s. The limit of detection (LOD) was 4.0 nM at 3-fold signal-to-noise ratio for standard cyanide in buffer. The cyanide levels in urine samples from smokers and non-smokers were determined by using the method of standard addition, which demonstrated significant difference of cyanide levels in urinary samples from the two groups of people. The developed method was rapid and accurate, and is anticipated to be applicable to cyanide detection in waste water with appropriate modification. PMID:26342870

A direct and rapid method to determine cyanide in urine by capillary electrophoresis.

PubMed

Zhang, Qiyang; Maddukuri, Naveen; Gong, Maojun

2015-10-02

Cyanides are poisonous chemicals that widely exist in nature and industrial processes as well as accidental fires. Rapid and accurate determination of cyanide exposure would facilitate forensic investigation, medical diagnosis, and chronic cyanide monitoring. Here, a rapid and direct method was developed for the determination of cyanide ions in urinary samples. This technique was based on an integrated capillary electrophoresis system coupled with laser-induced fluorescence (LIF) detection. Cyanide ions were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and a primary amine (glycine) for LIF detection. Three separate reagents, NDA, glycine, and cyanide sample, were mixed online, which secured uniform conditions between samples for cyanide derivatization and reduced the risk of precipitation formation of mixtures. Conditions were optimized; the derivatization was completed in 2-4min, and the separation was observed in 25s. The limit of detection (LOD) was 4.0nM at 3-fold signal-to-noise ratio for standard cyanide in buffer. The cyanide levels in urine samples from smokers and non-smokers were determined by using the method of standard addition, which demonstrated significant difference of cyanide levels in urinary samples from the two groups of people. The developed method was rapid and accurate, and is anticipated to be applicable to cyanide detection in waste water with appropriate modification. Published by Elsevier B.V.

Rapid assessment of assignments using plagiarism detection software.

PubMed

Bischoff, Whitney R; Abrego, Patricia C

2011-01-01

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

Nucleic Acid Detection Methods

DOEpatents

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

1998-05-19

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

Nucleic acid detection methods

DOEpatents

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

1998-05-19

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

Performance of three rapid screening methods in the detection of Schistosoma haematobium infection in school-age children in Southeastern Nigeria

PubMed Central

Okeke, Ogochukwu Caroline; Ubachukwu, Patience Obiageli

2014-01-01

Background A cross-sectional study of primary school children was conducted to evaluate and compare the performance of some rapid screening methods in the detection of Schistosoma haematobium infection in Nigeria Cement Factory (NigerCem) and Nike Lake areas of Southeastern Nigeria. Methods Urine samples of school children were examined for macro-haematuria and tested for micro-haematuria and proteinuria using reagent strips followed by egg microscopy. Self-reported haematuria was assessed using simple questionnaire. The performances of these rapid diagnoses singly and in combination were calculated using egg microscopy as gold standard. Results The prevalence of the infection was 26.6% in NigerCem and 5.1% in Nike Lake area, classifying these areas as moderate- and low-prevalence areas (MPA and LPA); while in the subsample used for self-reported haematuria, the prevalence was 27.2 and 4.2% in MPA and LPA, respectively. The positive predictive value (PPV) of micro-haematuria was comparable in MPA (55.26%) and LPA (57.89%). Overall PPV of macro-haematuria was 87.50% in MPA and 66.70% in LPA while in the detection of heavy infection; PPV was higher in LPA (75%) than in MPA (66.67%). In LPA and MPA, combination of micro-haematuria and proteinuria, and concomitant presence of macro-haematuria, micro-haematuria, and proteinuria had PPV of 83.33 and 63.16%, and 100 versus 66.67%, respectively. Generally, the rapid screening tests had lower negative predictive values (NPVs) in MPA than in LPA. The use of simple questionnaire increased the PPV of heavy infection in MPA (77.78%). This was further increased to 80% when self-reported haematuria was combined with micro-haematuria. Conclusion The result suggests that in MPA with chronic infections, combination of self-reported haematuria and micro-haematuria may reduce the chance of missing those who should be treated. PMID:24593687

Detection of Bordetella pertussis by rapid-cycle PCR and colorimetric microwell hybridization.

PubMed Central

Buck, G E

1996-01-01

The use of rapid-cycle PCR combined with colorimetric microwell hybridization for detecting Bordetella pertussis was investigated. Rapid cycling was performed with an air thermocycler (model 1605; Idaho Technology, Idaho Falls, Idaho). Although the instrument was originally designed to be used with capillary tubes, an adapter that allows this instrument to be used with PCR tubes has recently been introduced. Because of the low heat capacity of air, the thermocycler has rapid transition rates between temperatures. The combination of a rapid temperature transition rate, small sample volume (10 microliters), and overshooting or undershooting of the temperature set points allowed the cycles to be reduced to 5 s for denaturation and 10 s for extension and annealing. Thus, the amplification could be completed in a total of approximately 35 min. Amplified DNA was detected with biotin-labeled primers and by hybridization to a capture probe immobilized in microwell plates. When simulated clinical specimens consisting of pooled nasopharyngeal washes with known numbers of B. pertussis organisms were examined by this procedure, as little as one organism per 5 microliters of sample could be detected. Six nasopharyngeal aspirates or washes from culture-positive patients were positive by PCR, as were two of seven specimens obtained from patients that were negative by culture and direct fluorescent-antibody assay. The two patients who were PCR positive but culture and direct fluorescent-antibody assay negative had clinical disease compatible with pertussis. This method appears to be a sensitive, convenient means of detecting B. pertussis in clinical specimens. The total time required for specimen processing, amplification, and detection is about 2.5 h. PMID:8735080

Rapid Detection of Enterobacter Sakazakii in milk Powder using amino modified chitosan immunomagnetic beads.

PubMed

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

PubMed

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

2016-07-01

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

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.

PubMed

Feng, Xiao-Li; Ren, Hong-Lin; Li, Yan-Song; Hu, Pan; Zhou, Yu; Liu, Zeng-Shan; Yan, Dong-Ming; Hui, Qi; Liu, Dong; Lin, Chao; Liu, Nan-Nan; Liu, Yan-Yan; Lu, Shi-Ying

2014-08-15

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA. Copyright © 2014 Elsevier Inc. All rights reserved.

Simple, Rapid, Sensitive, and Versatile SWNT-Paper Sensor for Environmental Toxin Detection Competitive with ELISA

PubMed Central

Wang, Libing; Chen, Wei; Xu, Dinghua; Shim, Bong Sup; Zhu, Yingyue; Sun, Fengxia; Liu, Liqiang; Peng, Chifang; Jin, Zhengyu; Xu, Chuanlai; Kotov, Nicholas A.

2009-01-01

Safety of water was for a long time and still is one of the most pressing needs for many countries and different communities. Despite the fact that there are potentially many methods to evaluate water safety, finding a simple, rapid, versatile, and inexpensive method for detection of toxins in everyday items is still a great challenge. In this study, we extend the concept of composites obtained impregnation of porous fibrous materials, such as fabrics and papers, by single walled carbon-nanotubes (SWNTs) toward very simple but high-performance biosensors. They utilize the strong dependence of electrical conductivity through nanotubes percolation network on the width of nanotubes-nanotube tunneling gap and can potentially satisfy all the requirements outlined above for the routine toxin monitoring. An antibody to the microcystin-LR (MC-LR), one of the common culprits in mass poisonings, was dispersed together with SWNTs. This dispersion was used to dip-coat the paper rendering it conductive. The change in conductivity of the paper was used to sense the MC-LR in the water rapidly and accurately. The method has the linear detection range up to 10 nmol/L and non-linear detection up to 40 nmol/L. The limit of detection was found to be 0.6 nmol/L (0.6 ng/mL), which satisfies the strictest World Health Organization standard for MC-LR content in drinking water (1 ng/mL), and is comparable to the detection limit of traditional ELISA method of MC-LR detection, while drastically reducing the time of analysis by more than an order of magnitude, which is one of the major hurdles in practical applications. Similar technology of sensor preparation can also be used for a variety of other rapid environmental sensors. PMID:19928776

Rapid detection of chemical hazards (toxins, dioxins, and PCBs) in seafood.

PubMed

Arvanitoyannis, Ioannis S; Kotsanopoulos, Konstantinos V; Papadopoulou, Anna

2014-01-01

Among the various hazards occurring in fish and seafood chemical hazards and in particular toxins (ciguatera, scombroid fish poisoning, paralytic shellfish poisoning, neurotoxic (brevetoxic) shellfish poisoning, puffer fish poisoning, diarrhetic shellfish poisoning) have an important place in food poisoning cases. On the other hand, some of the chemical hazards are often due to the pollution of the environment (heavy metals, dioxins, polychlorinated biphenyls, and halogenated aromatic hydrocarbons) and their detection is neither rapid nor facile. As a result there was a great need for developing new rapid and effective methods toward the chemical hazards determination mainly because of their high toxicity. The aim of this review is to provide the information about the new up-to-date detection techniques (Immunological, Chemical and Biochemical, and Molecular assays) in conjunction with detection limits. The latter is made possible by means of inclusion of seven comprehensive and, in most case cases, very extended tables. A reference is also made on the risk characterization of toxins as regards their importance to food contamination or poisoning.

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

NASA Technical Reports Server (NTRS)

McFeters, G. A.; Pyle, B. H.; Lisle, J. T.; Broadaway, S. C.

1999-01-01

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between

A colloidal gold nanoparticle-based immunochromatographic test strip for rapid and convenient detection of Staphylococcus aureus.

PubMed

Niu, Kaili; Zheng, Xiaoping; Huang, Chusen; Xul, Kuan; Zhi, Yuan; Shen, Hebai; Jia, Nengqin

2014-07-01

An immunochromatographic test strip using gold nanoparticles-staphylococcus aureus monoclonal antibody conjugates was developed for the rapid and convenient detection of staphylococcus aureus based on a double-antibody sandwich format. The detection limit and the detection rate of this test strip is 10(3) CFU /mL and 98.7%, respectively. It could be used for the rapid detection of staphylococcus aureus in food and the results can be visually identified by the naked eye within 10 min. Compared with conventional bacterial detection methods, this developed immunochromatographic assay based test strip has several advantages including simple, fast, low-cost, favorable sensitivity and specificity, exhibiting a great potential for application in food safety control systems and clinical diagnosis.

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

PubMed

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

2017-05-01

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

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

PubMed Central

Cohen, Roy; Lata, James P.; Lee, Yurim; Hernández, Jean C. Cruz; Nishimura, Nozomi; Schaffer, Chris B.; Mukai, Chinatsu; Nelson, Jacquelyn L.; Brangman, Sharon A.; Agrawal, Yash; Travis, Alexander J.

2015-01-01

Background Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices. Methods and findings We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA. Conclusions Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using

Rapid detection of Streptococcus pneumoniae by real-time fluorescence loop-mediated isothermal amplification

PubMed Central

Guo, Xu-Guang; Zhou, Shan

2014-01-01

Background and aim of study A significant human pathogenic bacterium, Streptococcus pneumoniae was recognized as a major cause of pneumonia, and is the subject of many humoral immunity studies. Diagnosis is generally made based on clinical suspicion along with a positive culture from a sample from virtually any place in the body. But the testing time is too long. This study is to establish a rapid diagnostic method to identification of Streptococcus pneumoniae. Methods Our laboratory has recently developed a new platform called real-amp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of Streptococcus pneumonia. Two pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the Streptococcus pneumoniae. The amplification was carried out at 63 degree Celsius using SYBR Green for 60 minutes with the tube scanner set to collect fluorescence signals. Clinical samples of Streptococcus pneumoniae and other bacteria were used to determine the sensitivity and specificity of the primers by comparing with traditional culture method. Results The new set of primers consistently detected in laboratory-maintained isolates of Streptococcus pneumoniae from our hospital. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting Streptococcus pneumoniae. Conclusions This study demonstrates that the Streptococcus pneumoniae LAMP primers developed here have the ability to accurately detect Streptococcus pneumoniae infections by real-time fluorescence LAMP. PMID:25276360

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

PubMed

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

2015-02-01

A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry. Copyright © 2014 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1997-01-01

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

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Round Rock, TX

2011-07-05

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

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Livermore, CA

2006-08-01

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

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

PubMed

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.

Rapid specific and visible detection of porcine circovirus type 3 using loop-mediated isothermal amplification (LAMP).

PubMed

Zheng, S; Wu, X; Shi, J; Peng, Z; Gao, M; Xin, C; Liu, Y; Wang, S; Xu, S; Han, H; Yu, J; Sun, W; Cong, X; Li, J; Wang, J

2018-06-01

In this study, a rapid and specific assay for the detection of porcine circovirus type 3 (PCV3) was established using loop-mediated isothermal amplification (LAMP). Four primers were specifically designed to amplify PCV3. The LAMP assay was effectively optimized to amplify PCV3 by water bath at 60°C for 60 min. The detection limit was approximately 1 × 10 1 copy in this LAMP assay. Compared to porcine circovirus type 2 (PCV2), both gE and gD genes of pseudorabies virus (PRV) and porcine parvovirus (PPV), the LAMP assay showed a high specific detection of PCV3. A visible detection method was developed using SYBR Green I to recognize the results rapidly. Based on the detection of 20 clinical tissue samples, the LAMP assay was more practical and convenient than classical PCR due to its simplicity, high sensitivity, rapidity, specificity, visibility and cost efficiency. © 2018 Blackwell Verlag GmbH.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2013-05-01

We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rapid, Efficient Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis in Sputum by Microscopic Observation of Broth Cultures

PubMed Central

Caviedes, Luz; Lee, Tien-Shun; Gilman, Robert H.; Sheen, Patricia; Spellman, Emily; Lee, Ellen H.; Berg, Douglas E.; Montenegro-James, Sonia

2000-01-01

Inexpensive, rapid, and reliable methods of detecting infection by and drug susceptibility of Mycobacterium tuberculosis (MTB) are crucial to the control of tuberculosis. The novel microscopic observation broth-drug susceptibility assay (MODS) detects early growth of MTB in liquid medium, allowing more timely diagnosis and drug susceptibility testing. Sputum samples from hospitalized patients in Peru were analyzed by using stains, culture, and PCR. Sensitivity of MODS (92%) compared favorably with the most sensitive of the other culture methods (93%). Sputum samples positive for tuberculosis were tested for susceptibility to isoniazid and rifampin with the microwell alamar blue assay (MABA) and MODS. In 89% of cases, there was concordance between MODS and MABA. Of the diagnostic and susceptibility testing methods used, MODS yielded results most rapidly (median, 9.0 and 9.5 days, respectively). MODS is a rapid, inexpensive, sensitive, and specific method for MTB detection and susceptibility testing; it is particularly appropriate for use in developing countries burdened by significant infection rates and increasing numbers of multiple-drug-resistant cases. PMID:10699023

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

PubMed

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

2015-12-01

Hookworm infection contributes around 700 million infections worldwide especially in developing nations due to increased use of wastewater for crop production. The effective recovery of hookworm ova from wastewater matrices is difficult due to their low concentrations and heterogeneous distribution. In this study, we compared the recovery rates of (i) four rapid hookworm ova concentration methods from municipal wastewater, and (ii) two concentration methods from sludge samples. Ancylostoma caninum ova were used as surrogate for human hookworm (Ancylostoma duodenale and Necator americanus). Known concentration of A. caninum hookworm ova were seeded into wastewater (treated and raw) and sludge samples collected from two wastewater treatment plants (WWTPs) in Brisbane and Perth, Australia. The A. caninum ova were concentrated from treated and raw wastewater samples using centrifugation (Method A), hollow fiber ultrafiltration (HFUF) (Method B), filtration (Method C) and flotation (Method D) methods. For sludge samples, flotation (Method E) and direct DNA extraction (Method F) methods were used. Among the four methods tested, filtration (Method C) method was able to recover higher concentrations of A. caninum ova consistently from treated wastewater (39-50%) and raw wastewater (7.1-12%) samples collected from both WWTPs. The remaining methods (Methods A, B and D) yielded variable recovery rate ranging from 0.2 to 40% for treated and raw wastewater samples. The recovery rates for sludge samples were poor (0.02-4.7), although, Method F (direct DNA extraction) provided 1-2 orders of magnitude higher recovery rate than Method E (flotation). Based on our results it can be concluded that the recovery rates of hookworm ova from wastewater matrices, especially sludge samples, can be poor and highly variable. Therefore, choice of concentration method is vital for the sensitive detection of hookworm ova in wastewater matrices. Crown Copyright © 2015. Published by Elsevier

Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures.

PubMed

Schwalbe, E C; Hicks, D; Rafiee, G; Bashton, M; Gohlke, H; Enshaei, A; Potluri, S; Matthiesen, J; Mather, M; Taleongpong, P; Chaston, R; Silmon, A; Curtis, A; Lindsey, J C; Crosier, S; Smith, A J; Goschzik, T; Doz, F; Rutkowski, S; Lannering, B; Pietsch, T; Bailey, S; Williamson, D; Clifford, S C

2017-10-18

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

Electrochemical Biosensors for Rapid Detection of Foodborne Salmonella: A Critical Overview

PubMed Central

Cinti, Stefano; Volpe, Giulia; Piermarini, Silvia; Delibato, Elisabetta; Palleschi, Giuseppe

2017-01-01

Salmonella has represented the most common and primary cause of food poisoning in many countries for at least over 100 years. Its detection is still primarily based on traditional microbiological culture methods which are labor-intensive, extremely time consuming, and not suitable for testing a large number of samples. Accordingly, great efforts to develop rapid, sensitive and specific methods, easy to use, and suitable for multi-sample analysis, have been made and continue. Biosensor-based technology has all the potentialities to meet these requirements. In this paper, we review the features of the electrochemical immunosensors, genosensors, aptasensors and phagosensors developed in the last five years for Salmonella detection, focusing on the critical aspects of their application in food analysis. PMID:28820458

Rapid, cost-effective, sensitive and quantitative detection of Acinetobacter baumannii from pneumonia patients

PubMed Central

Nomanpour, B; Ghodousi, A; Babaei, A; Abtahi, HR; Tabrizi, M; Feizabadi, MM

2011-01-01

Background and Objectives Pneumonia with Acinetobacter baumannii has a major therapeutic problem in health care settings. Decision to initiate correct antibiotic therapy requires rapid identification and quantification of organism. The aim of this study was to develop a rapid and sensitive method for direct detection of A. baumannii from respiratory specimens. Materials and Methods A Taqman real time PCR based on the sequence of bla oxa-51 was designed and used for direct detection of A. baumannii from 361 respiratory specimens of patients with pneumonia. All specimens were checked by conventional bacteriology in parallel. Results The new real time PCR could detect less than 200 cfu per ml of bacteria in specimens. There was agreement between the results of real time PCR and culture (Kappa value 1.0, p value<0.001). The sensitivity, specificity and predictive values of real time PCR were 100%. The prevalence of A. baumannii in pneumonia patients was 10.53 % (n=38). Poly-microbial infections were detected in 65.71% of specimens. Conclusion Acinetobacter baumannii is the third causative agent in nosocomial pneumonia after Pseudomonas aeroginosa (16%) and Staphylococcus aureus (13%) at Tehran hospitals. We recommend that 104 CFU be the threshold for definition of infection with A. baumannii using real time PCR. PMID:22530083

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

NASA Astrophysics Data System (ADS)

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

2012-11-01

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

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

PubMed Central

2016-01-01

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

Testing the Rapid Detection Capabilities of the Quake-Catcher Network

NASA Astrophysics Data System (ADS)

Chung, A. I.; Cochran, E.; Yildirim, B.; Christensen, C. M.; Kaiser, A. E.; Lawrence, J. F.

2013-12-01

The Quake-Catcher Network (QCN) is a versatile network of MEMS accelerometers that are used in combination with distributed volunteer computing to detect earthquakes around the world. Using a dense network of QCN stations installed in Christchurch, New Zealand after the 2010 M7.1 Darfield earthquake, hundreds of events in the Christchurch area were detected and rapidly characterized. When the M6.3 Christchurch event occurred on 21 February 2011, QCN sensors recorded the event and calculated its magnitude, location, and created a map of estimated shaking intensity within 7 seconds of the earthquake origin time. Successive iterations improved the calculations and, within 24 seconds of the earthquake, magnitude and location values were calculated that were comparable to those provided by GeoNet. We have rigorously tested numerous methods to create a working magnitude scaling relationship. In this presentation, we show a drastic improvement in the magnitude estimates using the maximum acceleration at the time of the first trigger and updated ground accelerations from one to three seconds after the initial trigger. 75% of the events rapidly detected and characterized by QCN are within 0.5 magnitude units of the official GeoNet reported magnitude values, with 95% of the events within 1 magnitude unit. We also test the QCN detection algorithms using higher quality data from the SCSN network in Southern California. We examine a dataset of M5 and larger earthquakes that occurred since 1995. We present the performance of the QCN algorithms for this dataset, including time to detection as well as location and magnitude accuracy.

Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye

PubMed Central

2014-01-01

Background The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. Results The detection limit of LAMP using in vitro RNA transcripts was 3.6×10 copies·μL-1, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples. Conclusions The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test. PMID:24524254

Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

PubMed

Yang, Bo-Yun; Liu, Xiao-Lu; Wei, Yu-Mei; Wang, Jing-Qi; He, Xiao-Qing; Jin, Yi; Wang, Zi-Jian

2014-02-14

The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. The detection limit of LAMP using in vitro RNA transcripts was 3.6 × 10 copies·μL⁻¹, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples. The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test.

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

PubMed

Abernethy, Grant; Higgs, Kerianne

2013-05-03

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-12-01

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

Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification.

PubMed

Wang, Xuan; Yin, Fenggui; Bi, Yuhai; Cheng, Gong; Li, Jing; Hou, Lidan; Li, Yunlong; Yang, Baozhi; Liu, Wenjun; Yang, Limin

2016-12-01

Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKV clinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid and sensitive detection of ketamine in blood using novel fluorescence genosensor.

PubMed

Ding, Yanjun; Li, Xingmei; Guo, Yadong; Yan, Jie; Ling, Jiang; Li, Weichen; Lan, Lingmei; Chang, Yunfeng; Cai, Jifeng; Zha, Lagabaiyla

2017-12-01

In recent years, drug abuse has been considered as a most challenging social problem that aroused public attention. Ketamine has increased in unregulated use as a 'recreational drug' in teenagers. However, there is no suitable and maneuverable detection method for ketamine in situ at the moment. Fluorescence sensor technique, with predominant recognition and simple operation, is a good potential application in drug detection. Here, we first reported a highly sensitive and selective fluorescence genosensor for rapid detection of ketamine based on DNA-templated silver nanoclusters (DNA-AgNCs) probes, in which the DNA sequence could specially recognize ketamine with high affinity. Parameters affecting detection efficiency were investigated and optimized. Under optimum conditions, the as-prepared genosensor can allow for the determination of ketamine in the concentration range of 0.0001-20 μg/mL with two linear equations: one is y = 2.84x-7.139 (R 2 = 0.987) for 0.0001-0.1 μg/mL, and the other is y = 1.87x-0.091 (R 2 = 0.962) for 0.1-20 μg/mL, and the estimated detection limit of ketamine is 0.06 ng/mL. Moreover, the feasibility of this proposed method was also demonstrated by analyzing forensic blood samples. Compared with official gas chromatography/mass spectrometry (GC/MS), this fluorescence genosensor is simple, rapid, and accurate for quantitative determination of ketamine in blood for pharmaceutical and forensic analysis. Overall, it is the first report on a fluorescence genosensor for detecting ketamine directly in blood. This research may provide a new insight for the analyst to band fluorescence genosensor technology together with drug monitoring in the battle against drug abuse and forensic examination. Graphical abstract High selectively detection of ketamine using a novel fluorescence genosensor based on DNA-AgNCs probe.

Romer Labs RapidChek®Listeria monocytogenes Test System for the Detection of L. monocytogenes on Selected Foods and Environmental Surfaces.

PubMed

Juck, Gregory; Gonzalez, Verapaz; Allen, Ann-Christine Olsson; Sutzko, Meredith; Seward, Kody; Muldoon, Mark T

2018-04-27

The Romer Labs RapidChek ® Listeria monocytogenes test system (Performance Tested Method ℠ 011805) was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (USDA-FSIS/MLG), U.S. Food and Drug Association Bacteriological Analytical Manual (FDA/BAM), and AOAC Official Methods of Analysis ℠ (AOAC/OMA) cultural reference methods for the detection of L. monocytogenes on selected foods including hot dogs, frozen cooked breaded chicken, frozen cooked shrimp, cured ham, and ice cream, and environmental surfaces including stainless steel and plastic in an unpaired study design. The RapidChek method uses a proprietary enrichment media system, a 44-48 h enrichment at 30 ± 1°C, and detects L. monocytogenes on an immunochromatographic lateral flow device within 10 min. Different L. monocytogenes strains were used to spike each of the matrixes. Samples were confirmed based on the reference method confirmations and an alternate confirmation method. A total of 140 low-level spiked samples were tested by the RapidChek method after enrichment for 44-48 h in parallel with the cultural reference method. There were 88 RapidChek presumptive positives. One of the presumptive positives was not confirmed culturally. Additionally, one of the culturally confirmed samples did not exhibit a presumptive positive. No difference between the alternate confirmation method and reference confirmation method was observed. The respective cultural reference methods (USDA-FSIS/MLG, FDA/BAM, and AOAC/OMA) produced a total of 63 confirmed positive results. Nonspiked samples from all foods were reported as negative for L. monocytogenes by all methods. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the RapidChek method and the respective cultural reference method.

Rapid detection of EBOLA VP40 in microchip immunofiltration assay

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

Ion mobility spectrometry fingerprints: A rapid detection technology for adulteration of sesame oil.

PubMed

Zhang, Liangxiao; Shuai, Qian; Li, Peiwu; Zhang, Qi; Ma, Fei; Zhang, Wen; Ding, Xiaoxia

2016-02-01

A simple and rapid detection technology was proposed based on ion mobility spectrometry (IMS) fingerprints to determine potential adulteration of sesame oil. Oil samples were diluted by n-hexane and analyzed by IMS for 20s. Then, chemometric methods were employed to establish discriminant models for sesame oils and four other edible oils, pure and adulterated sesame oils, and pure and counterfeit sesame oils, respectively. Finally, Random Forests (RF) classification model could correctly classify all five types of edible oils. The detection results indicated that the discriminant models built by recursive support vector machine (R-SVM) method could identify adulterated sesame oil samples (⩾ 10%) with an accuracy value of 94.2%. Therefore, IMS was shown to be an effective method to detect the adulterated sesame oils. Meanwhile, IMS fingerprints work well to detect the counterfeit sesame oils produced by adding sesame oil essence into cheaper edible oils. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2011-07-01

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

Rapid detection of bacteria in foods and biological fluids

NASA Technical Reports Server (NTRS)

Fealey, R. D.; Renner, W.

1973-01-01

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

Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loop-mediated isothermal amplification.

PubMed

Zeng, Jing; Wei, Haiyan; Zhang, Lei; Liu, Xuefeng; Zhang, Haiyu; Cheng, Jinxia; Ma, Dan; Zhang, Ximeng; Fu, Pubo; Liu, Li

2014-03-17

The objective of this study was to develop a method that combined nanoparticle-based immunomagnetic separation (IMS) with real-time loop-mediated isothermal amplification (LAMP) for the rapid detection of Vibrio parahaemolyticus. Magnetic nanoparticles were functionalized with monoclonal antibodies that were produced against flagella from V. parahaemolyticus to capture and separate the target cells from raw oysters. After optimization, the immunomagnetic nanoparticles (IMNPs) presented a capture efficiency of 87.3% for 10(5) colony-forming unit (CFU)/mL of V. parahaemolyticus using 2.5μg of IMNPs within 30min. Although a very low level of non-specific binding was seen among 8 non-V. parahaemolyticus Vibrio spp. and 5 non-Vibrio strains, the IMS-LAMP method identified 133 V. parahaemolyticus strains correctly without the amplification from 54 other strains. The detection limit was about 1.4×10(2)CFU/mL in pure culture and was unaffected by the presence of 10(8)CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9×10(3)CFU/g without enrichment. After enrichment for 6-8h, the limit of detectability could be improved to 1.9 to 0.19CFU/g. Hence, the IMS-LAMP assay provided a rapid, simple, and cost-effective method for total V. parahaemolyticus detection. This method will have important implications in the rapid detection of contaminated food in the early stage before distribution. Copyright © 2014 Elsevier B.V. All rights reserved.

PCR-Based Method for Detecting Viral Penetration of Medical Exam Gloves

PubMed Central

Broyles, John M.; O'Connell, Kevin P.; Korniewicz, Denise M.

2002-01-01

The test approved by the U.S. Food and Drug Administration for assessment of the barrier quality of medical exam gloves includes visual inspection and a water leak test. Neither method tests directly the ability of gloves to prevent penetration by microorganisms. Methods that use microorganisms (viruses and bacteria) to test gloves have been developed but require classical culturing of the organism to detect it. We have developed a PCR assay for bacteriophage φX174 that allows the rapid detection of penetration of gloves by this virus. The method is suitable for use with both latex and synthetic gloves. The presence of glove powder on either latex or synthetic gloves had no effect on the ability of the PCR assay to detect bacteriophage DNA. The assay is rapid, sensitive, and inexpensive; requires only small sample volumes; and can be automated. PMID:12149320

Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing.

PubMed

Deng, Yi-Mo; Caldwell, Natalie; Barr, Ian G

2011-01-01

Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.

Rapid Detection and Subtyping of Human Influenza A Viruses and Reassortants by Pyrosequencing

PubMed Central

Deng, Yi-Mo; Caldwell, Natalie; Barr, Ian G.

2011-01-01

Background Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. Methodology/Principal Findings A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. Conclusions/Significance In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches. PMID:21886790

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

PubMed

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

2015-04-01

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

Rapid detection and classification of Salmonella enterica shedding in feedlot cattle utilizing Roka Bioscience Atlas Salmonella detection assay for the analysis of rectoanal mucosal swabs

USDA-ARS?s Scientific Manuscript database

With an increasing focus on preharvest food safety, rapid methods are required for the detection and quantification of foodborne pathogens such as Salmonella enterica in beef cattle. We validated the Atlas Salmonella Detection Assay (SEN), a nucleic acid amplification technology that targets Salmone...

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

PubMed

Janczarek, Monika; Palusińska-Szysz, Marta

2016-05-01

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

A Coffee Ring Aptasensor for Rapid Protein Detection

PubMed Central

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

2013-01-01

We introduce a new biosensing platform for rapid protein detection that combines one of the simplest methods for biomolecular concentration, coffee ring formation, with a sensitive aptamer-based optical detection scheme. In this approach, aptamer beacons are utilized for signal transduction where a fluorescence signal is emitted in the presence of the target molecule. Signal amplification is achieved by concentrating aptamer-target complexes within liquid droplets, resulting in the formation of coffee ring “spots”. Surfaces with various chemical coatings were utilized to investigate the correlation between surface hydrophobicity, concentration efficiency and signal amplification. Based on our results, we found that the increase in coffee ring diameter with larger droplet volumes is independent of surface hydrophobicity. Furthermore, we show that highly hydrophobic surfaces produce enhanced particle concentration, via coffee ring formation, resulting in signal intensities 6-fold greater than those on hydrophilic surfaces. To validate this biosensing platform for the detection of clinical samples, we detected α-thrombin in human serum and 4x diluted whole blood. Based on our results, coffee ring spots produced detection signals 40x larger than samples in liquid droplets. Additionally, this biosensor exhibits a lower limit of detection of 2 ng/mL (54 pM) in serum, and 4 ng/mL (105 pM) in blood. Based on its simplicity and high performance, this platform demonstrates immense potential as an inexpensive diagnostic tool for the detection of disease biomarkers, particularly for use in developing countries that lack the resources and facilities required for conventional biodetection practices. PMID:23540796

Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

PubMed

Sergueev, Kirill V; He, Yunxiu; Borschel, Richard H; Nikolich, Mikeljon P; Filippov, Andrey A

2010-06-28

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

Development of loop-mediated isothermal amplification method for rapid detection of Streptococcus iniae, the causative agent of streptococcicosis in fish.

PubMed

Cai, Shuang-Hu; Wang, Bei; Lu, Yi-Shan; Jian, Ji-Chang; Wu, Zao-He

2012-04-01

Streptococcus iniae is a major pathogen that causes sever economic losses in tilapia aquaculture. A set of four specific primers was designed by targeting lctO gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64 °C in a simple water bath. The sensitivity of the LAMP assay for the detection of S. iniae is about 12.4 cells per reaction in both of pure cultures and added fish tissues cultures. LAMP products could be judged with agar gel or naked eye after addition of SYBR Green I. There were no cross-reactions with other bacterial strains indicating high specificity of the LAMP. The LAMP method was also applied to detect S. iniae-infected tilapia tissues effectively. The LAMP assay reported here indicates the potential usefulness of the technique as a valuable simple, rapid alternative procedure for the detection of S. iniae during streptococcicosis monitoring of cultured fish. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Rapid prototyping: a very promising method].

PubMed

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

2013-03-01

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

Rapid detection of undesired cosmetic ingredients by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Ouyang, Jie; An, Dongli; Chen, Tengteng; Lin, Zhiwei

2017-10-01

In recent years, cosmetic industry profits soared due to the widespread use of cosmetics, which resulted in illicit manufacturers and products of poor quality. Therefore, the rapid and accurate detection of the composition of cosmetics has become crucial. At present, numerous methods, such as gas chromatography and liquid chromatography-mass spectrometry, were available for the analysis of cosmetic ingredients. However, these methods present several limitations, such as failure to perform comprehensive and rapid analysis of the samples. Compared with other techniques, matrix-assisted laser desorption ionization time-of-flight mass spectrometry offered the advantages of wide detection range, fast speed and high accuracy. In this article, we briefly summarized how to select a suitable matrix and adjust the appropriate laser energy. We also discussed the rapid identification of undesired ingredients, focusing on antibiotics and hormones in cosmetics.

Rapid Waterborne Pathogen Detection with Mobile Electronics.

PubMed

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

2017-06-09

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

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

PubMed

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

2009-09-01

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

Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system.

PubMed

Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Deng, Jiao-Yu; Cui, Zong-Qiang; Yang, Rui-Fu; Wang, Xu-Ying; Wei, Hong-Ping; Zhang, Xian-En

2013-04-15

There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×10³-10⁶ CFU ml⁻¹ and reproducible detection limits of 200 spores mg⁻¹ milk powder and 130 spores mg⁻¹ soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

Adapting and Evaluating a Rapid, Low-Cost Method to Enumerate Flies in the Household Setting

PubMed Central

Wolfe, Marlene K.; Dentz, Holly N.; Achando, Beryl; Mureithi, MaryAnne; Wolfe, Tim; Null, Clair; Pickering, Amy J.

2017-01-01

Diarrhea is a leading cause of death among children under 5 years of age worldwide. Flies are important vectors of diarrheal pathogens in settings lacking networked sanitation services. There is no standardized method for measuring fly density in households; many methods are cumbersome and unvalidated. We adapted a rapid, low-cost fly enumeration technique previously developed for industrial settings, the Scudder fly grill, for field use in household settings. We evaluated its performance in comparison to a sticky tape fly trapping method at latrine and food preparation areas among households in rural Kenya. The grill method was more sensitive; it detected the presence of any flies at 80% (433/543) of sampling locations versus 64% (348/543) of locations by the sticky tape. We found poor concordance between the two methods, suggesting that standardizing protocols is important for comparison of fly densities between studies. Fly species identification was feasible with both methods; however, the sticky tape trap allowed for more nuanced identification. Both methods detected a greater presence of bottle flies near latrines compared with food preparation areas (P < 0.01). The grill method detected more flies at the food preparation area compared with near the latrine (P = 0.014) while the sticky tape method detected no difference. We recommend the Scudder grill as a sensitive fly enumeration tool that is rapid and low cost to implement. PMID:27956654

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool.

PubMed

Mesquita, Flávio da Silva; Oliveira, Danielle Bruna Leal de; Crema, Daniela; Pinez, Célia Miranda Nunes; Colmanetti, Thaís Cristina; Thomazelli, Luciano Matsumia; Gilio, Alfredo Elias; Vieira, Sandra Elisabeth; Martinez, Marina Baquerizo; Botosso, Viviane Fongaro; Durigon, Edison Luiz

The aim of this study was to evaluate the QuickVue ® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue ® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue ® RSV Test and viral load or specific strain. The QuickVue ® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. This study demonstrated that the QuickVue ® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

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

PubMed

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.

Multicenter Evaluation of the Cepheid Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Test as a Rapid Screening Method for Detection of MRSA in Nares▿

PubMed Central

Wolk, D. M.; Picton, E.; Johnson, D.; Davis, T.; Pancholi, P.; Ginocchio, C. C.; Finegold, S.; Welch, D. F.; de Boer, M.; Fuller, D.; Solomon, M. C.; Rogers, B.; Mehta, M. S.; Peterson, L. R.

2009-01-01

The first U.S. multicenter clinical trial to assess the performance of the Cepheid Xpert MRSA assay (Xpert MRSA) was conducted. The assay is a qualitative test designed for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from nares swabs. This novel test combines integrated nucleic acid extraction and automated real-time PCR for the detection of a MRSA-specific signature sequence. A total of 1,077 nares specimens were collected from seven geographically distinct health care sites across the United States with prevalence rates ranging from 5.2% to 44%. Nares specimens were tested by (i) the Xpert MRSA assay, (ii) direct culture on CHROMagar MRSA medium (direct CM culture), and (iii) broth-enriched culture (Trypticase soy broth with 6.5% sodium chloride) followed by plating onto CHROMagar MRSA medium (broth-enriched CM culture). When direct CM culture was designated the reference method, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA assay were 94.3%, 93.2%, 73.0%, and 98.8%, respectively. When broth-enriched CM culture was used as the reference method, the clinical sensitivity, specificity, PPV, and NPV of the Xpert MRSA assay were 86.3%, 94.9%, 80.5%, and 96.6%, respectively. The BD GeneOhm MRSA (BDGO) assay was performed as a comparative molecular method. No statistical performance differences were observed between the Xpert MRSA and BDGO assays when they were compared to culture methods. From this large-scale, multicenter clinical comparison, we conclude that the Xpert MRSA assay is a simple, rapid, and accurate method for performing active surveillance for MRSA in a variety of health care populations. PMID:19129414

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

PubMed

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

2017-08-09

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

Detection of the Dinozoans Pfiesteria piscicida and P. shumwayae: a review of detection methods and geographic distribution.

PubMed

Rublee, Parke A; Remington, David L; Schaefer, Eric F; Marshall, Michael M

2005-01-01

Molecular methods, including conventional PCR, real-time PCR, denaturing gradient gel electrophoresis, fluorescent fragment detection PCR, and fluorescent in situ hybridization, have all been developed for use in identifying and studying the distribution of the toxic dinoflagellates Pfiesteria piscicida and P. shumwayae. Application of the methods has demonstrated a worldwide distribution of both species and provided insight into their environmental tolerance range and temporal changes in distribution. Genetic variability among geographic locations generally appears low in rDNA genes, and detection of the organisms in ballast water is consistent with rapid dispersal or high gene flow among populations, but additional sequence data are needed to verify this hypothesis. The rapid development and application of these tools serves as a model for study of other microbial taxa and provides a basis for future development of tools that can simultaneously detect multiple targets.

Rapid method to determine 89Sr/ 90Sr in large concrete samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maxwell, Sherrod L.; Culligan, Brian; Hutchison, Jay B.

Here, a new rapid method has been developed that provides high quality low-level measurements of 89,90Sr in concrete samples with an MDA (Minimum Detectable Activity) of <1 mBq g -1. The new method is fast, meets new decommissioning regulatory limits and is robust even if refractory particles are present. The method utilizes a rapid fusion to ensure total dissolution of samples and rapid preconcentration and separation of 89,90Sr from 5-10 g concrete samples. When, the 89Sr/ 90Sr ratio is high, Sr can be isolated from up to 5g concrete samples, total 89/90Sr measured, and then 90Sr determined via 90Y separatedmore » after a period of ingrowth. Another approach allows the immediate determination of 90Sr in 10 g concrete aliquots without waiting for 90Y ingrowth, in instances where the shorter lived 89Sr is unlikely to be encountered.« less

Rapid method to determine 89Sr/ 90Sr in large concrete samples

DOE PAGES

Maxwell, Sherrod L.; Culligan, Brian; Hutchison, Jay B.; ...

2016-03-24

Here, a new rapid method has been developed that provides high quality low-level measurements of 89,90Sr in concrete samples with an MDA (Minimum Detectable Activity) of <1 mBq g -1. The new method is fast, meets new decommissioning regulatory limits and is robust even if refractory particles are present. The method utilizes a rapid fusion to ensure total dissolution of samples and rapid preconcentration and separation of 89,90Sr from 5-10 g concrete samples. When, the 89Sr/ 90Sr ratio is high, Sr can be isolated from up to 5g concrete samples, total 89/90Sr measured, and then 90Sr determined via 90Y separatedmore » after a period of ingrowth. Another approach allows the immediate determination of 90Sr in 10 g concrete aliquots without waiting for 90Y ingrowth, in instances where the shorter lived 89Sr is unlikely to be encountered.« less

Rapid detection of Porcine circovirus 2 by recombinase polymerase amplification.

PubMed

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

2016-09-01

Porcine circovirus-associated disease, caused primarily by Porcine circovirus 2 (PCV-2), has become endemic in many pig-producing countries and has resulted in significant economic losses to the swine industry worldwide. Tests for PCV-2 infection include PCR, nested PCR, competitive PCR, and real-time PCR (rtPCR). Recombinase polymerase amplification (RPA) has emerged as an isothermal gene amplification technology for the molecular detection of infectious disease agents. RPA is performed at a constant temperature and therefore can be carried out in a water bath. In addition, RPA is completed in ~30 min, much faster than PCR, which usually takes >60 min. We developed a RPA-based method for the detection of PCV-2. The detection limit of RPA was 10(2) copies of PCV-2 genomic DNA. RPA showed the same sensitivity as rtPCR but was 10 times more sensitive than conventional PCR. Successful amplification of PCV-2 DNA, but not other viral templates, demonstrated high specificity of the RPA assay. This method was also validated using clinical samples. The results showed that the RPA assay had a diagnostic agreement rate of 93.7% with conventional PCR and 100% with rtPCR. These findings suggest that the RPA assay is a simple, rapid, and cost-effective method for PCV-2 detection, which could be potentially applied in clinical diagnosis and field surveillance of PCV-2 infection. © 2016 The Author(s).

RS-Forest: A Rapid Density Estimator for Streaming Anomaly Detection.

PubMed

Wu, Ke; Zhang, Kun; Fan, Wei; Edwards, Andrea; Yu, Philip S

Anomaly detection in streaming data is of high interest in numerous application domains. In this paper, we propose a novel one-class semi-supervised algorithm to detect anomalies in streaming data. Underlying the algorithm is a fast and accurate density estimator implemented by multiple fully randomized space trees (RS-Trees), named RS-Forest. The piecewise constant density estimate of each RS-tree is defined on the tree node into which an instance falls. Each incoming instance in a data stream is scored by the density estimates averaged over all trees in the forest. Two strategies, statistical attribute range estimation of high probability guarantee and dual node profiles for rapid model update, are seamlessly integrated into RS-Forest to systematically address the ever-evolving nature of data streams. We derive the theoretical upper bound for the proposed algorithm and analyze its asymptotic properties via bias-variance decomposition. Empirical comparisons to the state-of-the-art methods on multiple benchmark datasets demonstrate that the proposed method features high detection rate, fast response, and insensitivity to most of the parameter settings. Algorithm implementations and datasets are available upon request.

RS-Forest: A Rapid Density Estimator for Streaming Anomaly Detection

PubMed Central

Wu, Ke; Zhang, Kun; Fan, Wei; Edwards, Andrea; Yu, Philip S.

2015-01-01

Anomaly detection in streaming data is of high interest in numerous application domains. In this paper, we propose a novel one-class semi-supervised algorithm to detect anomalies in streaming data. Underlying the algorithm is a fast and accurate density estimator implemented by multiple fully randomized space trees (RS-Trees), named RS-Forest. The piecewise constant density estimate of each RS-tree is defined on the tree node into which an instance falls. Each incoming instance in a data stream is scored by the density estimates averaged over all trees in the forest. Two strategies, statistical attribute range estimation of high probability guarantee and dual node profiles for rapid model update, are seamlessly integrated into RS-Forest to systematically address the ever-evolving nature of data streams. We derive the theoretical upper bound for the proposed algorithm and analyze its asymptotic properties via bias-variance decomposition. Empirical comparisons to the state-of-the-art methods on multiple benchmark datasets demonstrate that the proposed method features high detection rate, fast response, and insensitivity to most of the parameter settings. Algorithm implementations and datasets are available upon request. PMID:25685112

Rapid and sensitive detection of Lily symptomless virus by reverse transcription loop-mediated isothermal amplification.

PubMed

He, Xiangfeng; Xue, Fei; Xu, Shufa; Wang, Wenhe

2016-12-01

Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl 2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR. Moreover, RT-LAMP detected LSV in not only symptomatic, but also in symptomless tissues of infected plants. These findings indicate that our RT-LAMP method for LSV can serve as a low-cost, simple, and rapid alternative to conventional detection assays. Copyright © 2016 Elsevier B.V. All rights reserved.

[Development and evaluation of a rapid PCR detection kit for Ophiocordyceps sinensis].

PubMed

Hou, Fei-Xia; Cao, Jing; Wang, Sha-Sha; Wang, Xi; Yuan, Yuan; Peng, Cheng; Wan, De-Guang; Guo, Jin-Lin

2017-03-01

Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage, expensive price and huge market demand, there are many adulterants of O. sinensis in markets. Therefore, it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis, this study developed a detection kit by optimizing the components and evaluated the specificity, detection limit, repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture, it could be detected successfully. Moreover, only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days, which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions, and in different batch under the same operation conditions gave the same result and accuracy, which showed good repeatability of the kit. It is simple, rapid and accurate to distinguish O. sinensis from its adulterants using the kit, and lays the foundation for commercialization of traditional Chinese medicine fast detection kit. Copyright© by the Chinese Pharmaceutical Association.

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

PubMed Central

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

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

PubMed

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

2015-11-01

Topical hydroquinone serves as a skin whitener and is usually available in cosmetics or on prescription based on the hydroquinone concentration. Quantification of hydroquinone content therefore becomes an important issue in topical agents. High-performance liquid chromatography (HPLC) is the commonest method for determining hydroquinone content in topical agents, but this method is time-consuming and uses many solvents that can become an environmental issue. We report a rapid method for quantifying hydroquinone content by chemiluminescent analysis. Hydroquinone induces the production of hydrogen peroxide in the presence of basic compounds. Hydrogen peroxide induced by hydroquinone oxidized light-emitting materials such as lucigenin, resulted in the production of ultra-weak chemiluminescence that was detected by a chemiluminescence analyzer. The intensity of the chemiluminescence was found to be proportional to the hydroquinone concentration. We suggest that the rapid (measurement time, 60 s) and virtually solvent-free (solvent volume, <2 mL) chemiluminescent method described here for quantifying hydroquinone content may be an alternative to HPLC analysis. Copyright © 2015 John Wiley & Sons, Ltd.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

PubMed

Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

2013-11-01

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25

PubMed Central

Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

2013-01-01

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg−1 GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25. PMID:24804053

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

PubMed

Sawamura, Kensuke; Hashimoto, Masahiko

2017-01-01

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

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

NASA Astrophysics Data System (ADS)

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

2007-05-01

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

Surface scattering plasmon resonance fibre sensors: demonstration of rapid influenza A virus detection

NASA Astrophysics Data System (ADS)

Franςois, A.; Boehm, J.; Oh, S. Y.; Kok, T.; Monro, T. M.

2011-06-01

The management of threats such as pandemics and explosives, and of health and the environment requires the rapid deployment of highly sensitive detection tools. Sensors based on Surface Plasmon Resonance (SPR) allow rapid, labelfree, highly sensitive detection, and indeed this phenomenon underpins the only label-free optical biosensing technology that is available commercially. In these sensors, the existence of surface plasmons is inferred indirectly from absorption features that correspond to the coupling of light to the surface plasmon. Although SPR is not intrinsically a radiative process, under certain conditions the surface plasmon can itself couple to the local photon states, and emit light as first described byKretschmann. Here we show that by collecting and characterising this re-emitted light, it is possible to realise new SPR sensing architectures that are more compact, versatile and robust than existing approaches. This approach addresses existing practical limitations associated with current SPR technologies, including bulk, cost and calibration. It is applicable to a range of SPR geometries, including optical fibres, planar waveguides and prism configurations, and is in principle capable of detecting multiple analytes simultaneously. Moreover, this technique allows to combine SPR sensing and fluorescence sensing into a single platform which has never been demonstrated before and consequently use these two methods for a more reliable diagnostic. As an example, this approach has been used to demonstrate the rapid detection of the seasonal influenza virus.

Rapid Microcystin Determination Using a Paper Spray Ionization Method with a Time-of-Flight Mass Spectrometry System.

PubMed

Zhu, Xiaoqiang; Huang, Zhengxu; Gao, Wei; Li, Xue; Li, Lei; Zhu, Hui; Mo, Ting; Huang, Bao; Zhou, Zhen

2016-07-13

The eutrophication of surface water sources and climate changes have resulted in an annual explosion of cyanobacterial blooms in many irrigating and drinking water resources. To decrease health risks to the public, a rapid real time method for the synchronous determination of two usually harmful microcystins (MC-RR and MC-LR) in environmental water samples was built by employing a paper spray ionization method coupled with a time-of-flight mass spectrometer system. With this approach, direct analysis of microcystin mixtures without sample preparation has been achieved. Rapid detection was performed, simulating the release process of microcystins in reservoir water samples, and the routine detection frequency was every three minutes. The identification time of microcystins was reduced from several hours to a few minutes. The limit of detection is 1 μg/L, and the limit of quantitation is 3 μg/L. This method displays the ability for carrying out rapid, direct, and high-throughput experiments for determination of microcystins, and it would be of significant interest for environmental and food safety applications.

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

PubMed

Bertol, Jéssica Wildgrube; Gatti, Maria Silvia Viccari

2016-08-01

Rapid, sensitive and specific methods are necessary to detect and quantify infectious viruses. Cultivating and detecting enteric viruses in cell culture are difficult, thus impairing the advancement of knowledge regarding virus-induced diarrhea. Rotavirus (RV) detection has been conducted by serological or molecular biology methods, which do not provide information regarding viral infectivity. Molecular beacons (MBs) have demonstrated efficacy for viral detection in cell culture. We propose a MB assay to detect human rotavirus group A (HuRVA) in cell culture. MA104 cells were mock-infected or infected with HuRVA strains (RotaTeq(®) vaccine and K8 strains), and a specific MB for the HuRVA VP6 gene was used for virus detection. Mock-infected cells showed basal fluorescence, while infected cells exhibited increased fluorescence emission. MB hybridization to the viral mRNA target of HuRVA was confirmed. Fluorescence increased according to the increase in the number of infectious viral particles per cell (MOI 0.5-MOI 1). This technique provides quick and efficient HuRVA detection in cell culture without a need for viral culture for several days or many times until cytopathic effects are visualized. This methodology could be applied in the selection of samples for developing RV vaccines. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid Detection of the Varicella Zoster Virus

NASA Technical Reports Server (NTRS)

Lewis, Michelle P.; Harding, Robert

2011-01-01

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

[Rapid detection of four antipertensive chemicals adulterated in traditional Chinese medicine for hypertension using TLC-SERS].

PubMed

Zhu, Qing-Xia; Cao, Yong-Bing; Cao, Ying-Ying; Lu, Feng

2014-04-01

A novel facile method for on-site detection of antipertensive chemicals (e. g. nicardipine hydrochloride, doxazosin mesylate, propranolol hydrochloride, and hydrochlorothiazide) adulterated in traditional Chinese medicine for hypertension using thin layer chromatography (TLC) combined with surface enhanced Raman spectroscopy (SERS) was reported in the present paper. Analytes and pharmaceutical matrices was separated by TLC, then SERS method was used to complete qualitative identification of trace substances on TLC plate. By optimizing colloidal silver concentration and developing solvent, as well as exploring the optimal limits of detection (LOD), the initially established TLC-SERS method was used to detect real hypertension Chinese pharmaceuticals. The results showed that this method had good specificity for the four chemicals and high sensitivity with a limit of detection as lower as to 0.005 microg. Finally, two of the ten antipertensive drugs were detected to be adulterated with chemicals. This simple and fast method can realize rapid detection of chemicals illegally for doping in antipertensive Chinese pharmaceuticals, and would have good prospects in on-site detection of chemicals for doping in Chinese pharmaceuticals.

Bioluminescence-based system for rapid detection of natural transformation.

PubMed

Santala, Ville; Karp, Matti; Santala, Suvi

2016-07-01

Horizontal gene transfer plays a significant role in bacterial evolution and has major clinical importance. Thus, it is vital to understand the mechanisms and kinetics of genetic transformations. Natural transformation is the driving mechanism for horizontal gene transfer in diverse genera of bacteria. Our study introduces a simple and rapid method for the investigation of natural transformation. This highly sensitive system allows the detection of a transformation event directly from a bacterial population without any separation step or selection of cells. The system is based on the bacterial luciferase operon from Photorhabdus luminescens The studied molecular tools consist of the functional modules luxCDE and luxAB, which involve a replicative plasmid and an integrative gene cassette. A well-established host for bacterial genetic investigations, Acinetobacter baylyi ADP1, is used as the model bacterium. We show that natural transformation followed by homologous recombination or plasmid recircularization can be readily detected in both actively growing and static biofilm-like cultures, including very rare transformation events. The system allows the detection of natural transformation within 1 h of introducing sample DNA into the culture. The introduced method provides a convenient means to study the kinetics of natural transformation under variable conditions and perturbations. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

PubMed

Ingianni, A; Floris, M; Palomba, P; Madeddu, M A; Quartuccio, M; Pompei, R

2001-10-01

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories. Copyright 2001 Academic Press.

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

PubMed Central

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

2013-01-01

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

Neer Award 2017: A rapid method for detecting Propionibacterium acnes in surgical biopsy specimens from the shoulder.

PubMed

Holmes, Scott; Pena Diaz, Ana M; Athwal, George S; Faber, Kenneth J; O'Gorman, David B

2017-02-01

Propionibacterium (P) acnes infection of the shoulder after arthroplasty is a common and serious complication. Current detection methods for P acnes involve anaerobic cultures that require prolonged incubation periods (typically 7-14 days). We have developed a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) approach that sensitively and specifically identifies P acnes in tissue specimens within a 24-hour period. Primers were designed to amplify a unique region of the 16S rRNA gene in P acnes that contained a unique HaeIII restriction enzyme site. PCR and RFLP analyses were optimized to detect P acnes DNA in in vitro cultures and in arthroscopic surgical biopsy specimens from patients with P acnes infections. A 564 base-pair PCR amplicon was derived from all of the known P acnes strains. HaeIII digests of the amplicon yielded a restriction fragment pattern that was unique to P acnes. P acnes-specific amplicons were detected in as few as 10 bacterial cells and in clinical biopsy specimens of infected shoulder tissues. This PCR-RFLP assay combines the sensitivity of PCR with the specificity of RFLP mapping to identify P acnes in surgical isolates. The assay is robust and rapid, and a P acnes-positive tissue specimen can be confirmed within 24 hours of sampling, facilitating treatment decision making, targeted antibiotic therapy, and monitoring to minimize implant failure and revision surgery. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2016-03-15

This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii (C. sakazakii), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti-C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti-C. sakazakii IgG to the magnetic nanoparticles was 86.23 ± 0.59%. The assay developed in this study detected as few as 3.3 × 10(3) CFUmL(-1) of C. sakazakii in pure culture within 2h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2 × 10(5) CFUmL(-1) of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10(3) CFUmL(-1) of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii. Copyright © 2015 Elsevier B.V. All rights reserved.

Advances in developing rapid, reliable and portable detection systems for alcohol.

PubMed

Thungon, Phurpa Dema; Kakoti, Ankana; Ngashangva, Lightson; Goswami, Pranab

2017-11-15

Development of portable, reliable, sensitive, simple, and inexpensive detection system for alcohol has been an instinctive demand not only in traditional brewing, pharmaceutical, food and clinical industries but also in rapidly growing alcohol based fuel industries. Highly sensitive, selective, and reliable alcohol detections are currently amenable typically through the sophisticated instrument based analyses confined mostly to the state-of-art analytical laboratory facilities. With the growing demand of rapid and reliable alcohol detection systems, an all-round attempt has been made over the past decade encompassing various disciplines from basic and engineering sciences. Of late, the research for developing small-scale portable alcohol detection system has been accelerated with the advent of emerging miniaturization techniques, advanced materials and sensing platforms such as lab-on-chip, lab-on-CD, lab-on-paper etc. With these new inter-disciplinary approaches along with the support from the parallel knowledge growth on rapid detection systems being pursued for various targets, the progress on translating the proof-of-concepts to commercially viable and environment friendly portable alcohol detection systems is gaining pace. Here, we summarize the progress made over the years on the alcohol detection systems, with a focus on recent advancement towards developing portable, simple and efficient alcohol sensors. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

USGS Publications Warehouse

Bushon, R.N.; Brady, A.M.; Likirdopulos, C.A.; Cireddu, J.V.

2009-01-01

Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

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

USDA-ARS?s Scientific Manuscript database

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

Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens.

PubMed

Sohn, Miryeong; Himmelsbach, David S; Barton, Franklin E; Fedorka-Cray, Paula J

2009-11-01

This study deals with the rapid detection and differentiation of Escherichia coli, Salmonella, and Campylobacter, which are the most commonly identified commensal and pathogenic bacteria in foods, using fluorescence spectroscopy and multivariate analysis. Each bacterial sample cultured under controlled conditions was diluted in physiologic saline for analysis. Fluorescence spectra were collected over a range of 200-700 nm with 0.5 nm intervals on the PerkinElmer Fluorescence Spectrometer. The synchronous scan technique was employed to find the optimum excitation (lambda(ex)) and emission (lambda(em)) wavelengths for individual bacteria with the wavelength interval (Deltalambda) being varied from 10 to 200 nm. The synchronous spectra and two-dimensional plots showed two maximum lambda(ex) values at 225 nm and 280 nm and one maximum lambda(em) at 335-345 nm (lambda(em) = lambda(ex) + Deltalambda), which correspond to the lambda(ex) = 225 nm, Deltalambda = 110-120 nm, and lambda(ex) = 280 nm, Deltalambda = 60-65 nm. For all three bacterial genera, the same synchronous scan results were obtained. The emission spectra from the three bacteria groups were very similar, creating difficulty in classification. However, the application of principal component analysis (PCA) to the fluorescence spectra resulted in successful classification of the bacteria by their genus as well as determining their concentration. The detection limit was approximately 10(3)-10(4) cells/mL for each bacterial sample. These results demonstrated that fluorescence spectroscopy, when coupled with PCA processing, has the potential to detect and to classify bacterial pathogens in liquids. The methodology is rapid (>10 min), inexpensive, and requires minimal sample preparation compared to standard analytical methods for bacterial detection.

Quartz crystal microbalance biosensor for rapid detection of aerosolized microorganisms

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

PubMed

Nakano, Ryuichi; Nakano, Akiyo; Ishii, Yoshikazu; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Kikuchi, Hirotoshi; Tansho-Nagakawa, Shigeru; Kamoshida, Go; Mu, Xiaoqin; Ono, Yasuo

2015-03-01

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Rapid Detection of Herpes Viruses for Clinical Applications

NASA Technical Reports Server (NTRS)

Pierson, Duane; Mehta, Satish

2013-01-01

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

Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips.

PubMed

Ma, Qinglin; Liu, Houming; Ye, Feidi; Xiang, Guangxin; Shan, Wanshui; Xing, Wanli

2017-12-01

To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing. Copyright © 2017 Elsevier Ltd. All rights reserved.

Innovative rapid construction/reconstruction methods.

DOT National Transportation Integrated Search

2005-07-01

Innovative construction and reconstruction methods provide the opportunity to significantly reduce the time of roadway projects while maintaining the necessary quality of workmanship. The need for these rapid methods stems from the increase in ...

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'

PubMed Central

Minguzzi, Stefano; Terlizzi, Federica; Lanzoni, Chiara; Poggi Pollini, Carlo; Ratti, Claudio

2016-01-01

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus. PMID:26742106

Rapid detection of irradiated frozen hamburgers

NASA Astrophysics Data System (ADS)

Delincée, Henry

2002-03-01

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

Early detection and rapid response

USGS Publications Warehouse

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

2011-01-01

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

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

PubMed

Wang, Min S; Nitin, Nitin

2014-10-01

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

Rapid Detection of Escherichia coli O157:H7 in Fresh Lettuce Based on Localized Surface Plasmon Resonance Combined with Immunomagnetic Separation.

PubMed

Lee, Nari; Choi, Sung-Wook; Chang, Hyun-Joo; Chun, Hyang Sook

2018-05-01

This study presents a method for rapid detection of Escherichia coli O157:H7 in fresh lettuce based on the properties of target separation and localized surface plasmon resonance of immunomagnetic nanoparticles. The multifunctional immunomagnetic nanoparticles enabling simultaneous separation and detection were prepared by synthesizing magnetic nanoparticles (ca. 10 nm in diameter) composed of an iron oxide (Fe 3 O 4 ) core and gold shell and then conjugating these nanoparticles with the anti- E. coli O157:H7 antibodies. The application of multifunctional immunomagnetic nanoparticles for detecting E. coli O157:H7 in a lettuce matrix allowed detection of the presence of <1 log CFU mL -1 without prior enrichment. In contrast, the detection limit of the conventional plating method was 2.74 log CFU mL -1 . The method, which requires no preenrichment, provides an alternative to conventional microbiological detection methods and can be used as a rapid screening tool for a large number of food samples.

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

PubMed Central

Settanni, Luca; van Sinderen, Douwe; Rossi, Jone; Corsetti, Aldo

2005-01-01

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies. PMID:15933001

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics

PubMed Central

Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung

2017-01-01

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388

A scoping review of rapid review methods.

PubMed

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

2015-09-16

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

Method for enhancing single-trial P300 detection by introducing the complexity degree of image information in rapid serial visual presentation tasks

PubMed Central

Lin, Zhimin; Zeng, Ying; Tong, Li; Zhang, Hangming; Zhang, Chi

2017-01-01

The application of electroencephalogram (EEG) generated by human viewing images is a new thrust in image retrieval technology. A P300 component in the EEG is induced when the subjects see their point of interest in a target image under the rapid serial visual presentation (RSVP) experimental paradigm. We detected the single-trial P300 component to determine whether a subject was interested in an image. In practice, the latency and amplitude of the P300 component may vary in relation to different experimental parameters, such as target probability and stimulus semantics. Thus, we proposed a novel method, Target Recognition using Image Complexity Priori (TRICP) algorithm, in which the image information is introduced in the calculation of the interest score in the RSVP paradigm. The method combines information from the image and EEG to enhance the accuracy of single-trial P300 detection on the basis of traditional single-trial P300 detection algorithm. We defined an image complexity parameter based on the features of the different layers of a convolution neural network (CNN). We used the TRICP algorithm to compute for the complexity of an image to quantify the effect of different complexity images on the P300 components and training specialty classifier according to the image complexity. We compared TRICP with the HDCA algorithm. Results show that TRICP is significantly higher than the HDCA algorithm (Wilcoxon Sign Rank Test, p<0.05). Thus, the proposed method can be used in other and visual task-related single-trial event-related potential detection. PMID:29283998

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

NASA Astrophysics Data System (ADS)

Tao, Feifei; Peng, Yankun; Xu, Tianfeng

2015-05-01

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

Rapid detection of food pathogens using RNA aptamers-immobilized slide.

PubMed

Maeng, Jin-Soo; Kim, Namsoo; Kim, Chong-Tai; Han, Seung Ryul; Lee, Young Ju; Lee, Seong-Wook; Lee, Myung-Hyun; Cho, Yong-Jin

2012-07-01

The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.

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

PubMed

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

2013-07-17

A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogens could be extracted directly from complex matrices such as raw milk using ASMNPs. The magnetically separated complexes of genomic DNA and ASMNPs were directly subjected to single PCR (S-PCR) or multiplex PCR (M-PCR) to detect single or multiple pathogens from raw milk samples. Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive) were used as model organisms to artificially contaminate raw milk samples. After magnetic separation and S-PCR, the detection sensitivities were 8 CFU mL(-1) and 13 CFU mL(-1) respectively for these two types of pathogens. Furthermore, this method was successfully used to detect multiple pathogens (S. Enteritidis and L. monocytogenes) from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU mL(-1) and 25 CFU mL(-1), respectively. This method has great potential to rapidly and sensitively detect pathogens in raw milk or other complex food matrices. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid and sensitive detection of malachite green in aquaculture water by electrochemical preconcentration and surface-enhanced Raman scattering.

PubMed

Xu, Kai-Xuan; Guo, Mei-Hong; Huang, Yu-Ping; Li, Xiao-Dong; Sun, Jian-Jun

2018-04-01

A highly sensitive and rapid method of in-situ surface-enhanced Raman spectroscopy (SERS) combining with electrochemical preconcentration (EP) in detecting malachite green (MG) in aquaculture water was established. Ag nanoparticles (AgNPs) were synthesized and spread onto the surface of gold electrodes after centrifuging to produce SERS-active substrates. After optimizing the pH values, preconcentration potentials and times, in-situ EP-SERS detection was carried out. A sensitive and rapid analysis of the low-concentration MG was accomplished within 200s and the limit of detection was 2.4 × 10 -16 M. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed Central

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

PubMed

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

2016-02-17

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 10(4) CFU mL(-1) or 10(5) 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 (R(2)) 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.

Comparison of two commercial rapid in-clinic serological tests for detection of antibodies against Leishmania spp. in dogs.

PubMed

Athanasiou, Labrini V; Petanides, Theodoros A; Chatzis, Manolis K; Kasabalis, Dimitrios; Apostolidis, Kosmas N; Saridomichelakis, Manolis N

2014-03-01

Antibodies against Leishmania spp. are detected in most dogs with clinical signs of leishmaniasis due to Leishmania infantum. Accurate, rapid in-clinic serological tests may permit immediate confirmation of the diagnosis and implementation of therapeutic measures. The aim of the current study was to evaluate the diagnostic accuracy of 2 commercial, rapid in-clinic serological tests for the detection of anti-Leishmania antibodies in sera of dogs, the Snap Canine Leishmania Antibody Test kit (IDEXX Laboratories Inc., Westbrook, Maine) and the ImmunoRun Antibody Detection kit (Biogal Galed Labs, Kibbutz Galed, Israel), using indirect fluorescent antibody test (IFAT) as the reference method. A total of 109 sera collected from 65 seropositive and 44 seronegative dogs were used. The sensitivities of the Snap and ImmunoRun kits were 89.23% (95% confidence interval: 79.05-95.54%) and 86.15% (95% confidence interval: 75.33-93.45%), respectively, and the specificity of both tests was 100%. A good agreement between each of the rapid in-clinic serological tests and IFAT and between the 2 rapid in-clinic serological tests was witnessed. Both rapid in-clinic serological tests showed an adequate diagnostic accuracy and can be used for the fast detection of antibodies against L. infantum in dogs.

Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor

PubMed Central

Baca, Justin T.; Severns, Virginia; Lovato, Debbie; Branch, Darren W.; Larson, Richard S.

2015-01-01

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 104 PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions. PMID:25875186

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Thai, Hong Thi Cam; Le, Mai Quynh; Vuong, Cuong Duc; Parida, Manmohan; Minekawa, Harumi; Notomi, Tsugunori; Hasebe, Futoshi; Morita, Kouichi

2004-01-01

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 102 PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries. PMID:15131154

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick.

PubMed

Wang, H; Sun, M; Xu, D; Podok, P; Xie, J; Jiang, Ys; Lu, Lq

2018-05-28

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited. © 2018 John Wiley & Sons Ltd.

Rapid and sensitive detection of hepatitis A virus in representative food matrices.

PubMed

Papafragkou, Efstathia; Plante, Michelle; Mattison, Kirsten; Bidawid, Sabah; Karthikeyan, Kalavethi; Farber, Jeffrey M; Jaykus, Lee-Ann

2008-01-01

Hepatitis A virus (HAV) is an important cause of foodborne disease worldwide. The detection of this virus in naturally contaminated food products is complicated by the absence of a reliable culture method, low levels of contamination, and the presence of matrix-associated compounds which inhibit molecular detection. In this study, we report a novel method to concentrate HAV from foods prior to the application of reverse transcription-PCR (RT-PCR) for detection. Specifically, we used cationically charged magnetic particles with an automated capture system (Pathatrix) to concentrate the virus from 25 g samples of artificially contaminated lettuce, strawberries, green onions, deli-turkey, oysters, and cake with frosting. Detection limits varied according to the product but in most cases, the virus could be consistently detected at input levels corresponding to 10(2)PFU/25 g food sample. For some products, detection was possible at levels as low as 10(-1)PFU/25 g. The assay was applied by a second independent laboratory and was also used to confirm viral contamination of produce items associated with a recent HAV outbreak. Parallel infectivity assays demonstrated that the cationically charged particles bound approximately 50% of the input virus. This is the first application of the automated magnetic capture technology to the concentration of viruses from foods, and it offers promise for facilitating the rapid detection of HAV from naturally contaminated products.

A novel kit for rapid detection of Vibrio cholerae O1.

PubMed

Hasan, J A; Huq, A; Tamplin, M L; Siebeling, R J; Colwell, R R

1994-01-01

We report on the development and testing of a novel, rapid, colorimetric immunodiagnostic kit, Cholera SMART, for direct detection of the presence of Vibrio cholerae O1 in clinical specimens. Unlike conventional culture methods requiring several days to complete, the Cholera SMART kit can be used directly in the field by untrained or minimally skilled personnel to detect V. cholerae O1 in less than 15 min, without cumbersome laboratory equipment. A total of 120 clinical and environmental bacterial strains, including both O1 and non-O1 serotypes of V. cholerae isolated from samples collected from a variety of geographical regions, were tested, and positive reactions were observed only with V. cholerae O1. Also, results of a field trial in Bangladesh, employing Cholera SMART, showed 100% specificity and 96% sensitivity compared with conventional culture methods. Another field trial, in Mexico, showed that Cholera SMART was 100% in agreement with a recently described coagglutination test when 108 stool specimens were tested.

Rapid, highly sensitive detection of Gram-negative bacteria with lipopolysaccharide based disposable aptasensor.

PubMed

Zhang, Jian; Oueslati, Rania; Cheng, Cheng; Zhao, Ling; Chen, Jiangang; Almeida, Raul; Wu, Jayne

2018-07-30

Gram-negative bacteria are one of the most common microorganisms in the environment. Their differential detection and recognition from Gram-positive bacteria has been attracting much attention over the years. Using Escherichia coli (E. coli) as a model, we demonstrated on-site detection of Gram-negative bacteria by an AC electrokinetics-based capacitive sensing method using commercial microelectrodes functionalized with an aptamer specific to lipopolysaccharides. Dielectrophoresis effect was utilized to enrich viable bacteria to the microelectrodes rapidly, achieving a detection limit of 10 2 cells/mL within a 30 s' response time. The sensor showed a negligible response to Staphylococcus aureus (S. aureus), a Gram-positive species. The developed sensor showed significant advantages in sensitivity, selectivity, cost, operation simplicity, and response time. Therefore, this sensing method has shown great application potential for environmental monitoring, food safety, and real-time diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

R Peak Detection Method Using Wavelet Transform and Modified Shannon Energy Envelope

PubMed Central

2017-01-01

Rapid automatic detection of the fiducial points—namely, the P wave, QRS complex, and T wave—is necessary for early detection of cardiovascular diseases (CVDs). In this paper, we present an R peak detection method using the wavelet transform (WT) and a modified Shannon energy envelope (SEE) for rapid ECG analysis. The proposed WTSEE algorithm performs a wavelet transform to reduce the size and noise of ECG signals and creates SEE after first-order differentiation and amplitude normalization. Subsequently, the peak energy envelope (PEE) is extracted from the SEE. Then, R peaks are estimated from the PEE, and the estimated peaks are adjusted from the input ECG. Finally, the algorithm generates the final R features by validating R-R intervals and updating the extracted R peaks. The proposed R peak detection method was validated using 48 first-channel ECG records of the MIT-BIH arrhythmia database with a sensitivity of 99.93%, positive predictability of 99.91%, detection error rate of 0.16%, and accuracy of 99.84%. Considering the high detection accuracy and fast processing speed due to the wavelet transform applied before calculating SEE, the proposed method is highly effective for real-time applications in early detection of CVDs. PMID:29065613

R Peak Detection Method Using Wavelet Transform and Modified Shannon Energy Envelope.

PubMed

Park, Jeong-Seon; Lee, Sang-Woong; Park, Unsang

2017-01-01

Rapid automatic detection of the fiducial points-namely, the P wave, QRS complex, and T wave-is necessary for early detection of cardiovascular diseases (CVDs). In this paper, we present an R peak detection method using the wavelet transform (WT) and a modified Shannon energy envelope (SEE) for rapid ECG analysis. The proposed WTSEE algorithm performs a wavelet transform to reduce the size and noise of ECG signals and creates SEE after first-order differentiation and amplitude normalization. Subsequently, the peak energy envelope (PEE) is extracted from the SEE. Then, R peaks are estimated from the PEE, and the estimated peaks are adjusted from the input ECG. Finally, the algorithm generates the final R features by validating R-R intervals and updating the extracted R peaks. The proposed R peak detection method was validated using 48 first-channel ECG records of the MIT-BIH arrhythmia database with a sensitivity of 99.93%, positive predictability of 99.91%, detection error rate of 0.16%, and accuracy of 99.84%. Considering the high detection accuracy and fast processing speed due to the wavelet transform applied before calculating SEE, the proposed method is highly effective for real-time applications in early detection of CVDs.

Gold Nanoparticles as a Direct and Rapid Sensor for Sensitive Analytical Detection of Biogenic Amines

NASA Astrophysics Data System (ADS)

El-Nour, K. M. A.; Salam, E. T. A.; Soliman, H. M.; Orabi, A. S.

2017-03-01

A new optical sensor was developed for rapid screening with high sensitivity for the existence of biogenic amines (BAs) in poultry meat samples. Gold nanoparticles (GNPs) with particle size 11-19 nm function as a fast and sensitive biosensor for detection of histamine resulting from bacterial decarboxylation of histidine as a spoilage marker for stored poultry meat. Upon reaction with histamine, the red color of the GNPs converted into deep blue. The appearance of blue color favorably coincides with the concentration of BAs that can induce symptoms of poisoning. This biosensor enables a semi-quantitative detection of analyte in real samples by eye-vision. Quality evaluation is carried out by measuring histamine and histidine using different analytical techniques such as UV-vis, FTIR, and fluorescence spectroscopy as well as TEM. A rapid quantitative readout of samples by UV-vis and fluorescence methods with standard instrumentation were proposed in a short time unlike chromatographic and electrophoretic methods. Sensitivity and limit of detection (LOD) of 6.59 × 10-4 and 0.6 μM, respectively, are determined for histamine as a spoilage marker with a correlation coefficient ( R 2) of 0.993.

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

NASA Astrophysics Data System (ADS)

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

2012-12-01

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

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

PubMed

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Recent developments and applications of hyperspectral imaging for rapid detection of mycotoxins and mycotoxigenic fungi in food products.

PubMed

Xing, Fuguo; Yao, Haibo; Liu, Yang; Dai, Xiaofeng; Brown, Robert L; Bhatnagar, Deepak

2017-08-28

Mycotoxins are the foremost naturally occurring contaminants of food products such as corn, peanuts, tree nuts, and wheat. As the secondary metabolites, mycotoxins are mainly synthesized by many species of the genera Aspergillus, Fusarium and Penicillium, and are considered highly toxic and carcinogenic to humans and animals. Most mycotoxins are detected and quantified by analytical chemistry-based methods. While mycotoxigenic fungi are usually identified and quantified by biological methods. However, these methods are time-consuming, laborious, costly, and inconsistent because of the variability of the grain-sampling process. It is desirable to develop rapid, non-destructive and efficient methods that objectively measure and evaluate mycotoxins and mycotoxigenic fungi in food. In recent years, some spectroscopy-based technologies such as hyperspectral imaging (HSI), Raman spectroscopy, and Fourier transform infrared spectroscopy have been extensively investigated for their potential use as tools for the detection, classification, and sorting of mycotoxins and toxigenic fungal contaminants in food. HSI integrates both spatial and spectral information for every pixel in an image, making it suitable for rapid detection of large quantities of samples and more heterogeneous samples and for in-line sorting in the food industry. In order to track the latest research developments in HSI, this paper gives a brief overview of the theories and fundamentals behind the technology and discusses its applications in the field of rapid detection and sorting of mycotoxins and toxigenic fungi in food products. Additionally, advantages and disadvantages of HSI are compared, and its potential use in commercial applications is reported.

BIOHAZ: Rapid On-Site Biological Detection for First Responders

DTIC Science & Technology

2001-09-01

numbers comprise the compilation report: ADP013371 thru ADP013468 UNCLASSIFIED 84. BIORAZ: Rapid On- Site Biological Detection for First Responders Randall... responders an integated capability to collect an environmental sample and to rapidly screen that sample on site for the presence of biological material. This...further analyzed on site with immunoassay tickets before being sent to a laboratory. This system provides the emergency responders with a capability that

A rapid method for preparation of the cerebrospinal fluid proteome.

PubMed

Larssen, Eivind; Brede, Cato; Hjelle, Anne Bjørnstad; Øysaed, Kjell Birger; Tjensvoll, Anne Bolette; Omdal, Roald; Ruoff, Peter

2015-01-01

The cerebrospinal fluid (CSF) proteome is of great interest for investigation of diseases and conditions involving the CNS. However, the presence of high-abundance proteins (HAPs) can interfere with the detection of low-abundance proteins, potentially hindering the discovery of new biomarkers. Therefore, an assessment of the CSF subproteome composition requires depletion strategies. Existing methods are time consuming, often involving multistep protocols. Here, we present a rapid, accurate, and reproducible method for preparing the CSF proteome, which allows the identification of a high number of proteins. This method involves acetonitrile (ACN) precipitation for depleting HAPs, followed by immediate trypsination. As an example, we demonstrate that this method allows discrimination between multiple sclerosis patients and healthy subjects. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Loop-mediated isothermal amplification assay targeting the mpb70 gene for rapid differential detection of Mycobacterium bovis.

PubMed

Zhang, Hui; Wang, Zhen; Cao, Xudong; Wang, Zhengrong; Sheng, Jinliang; Wang, Yong; Zhang, Jing; Li, Zhiqiang; Gu, Xinli; Chen, Chuangfu

2016-11-01

Loop-mediated isothermal amplification (LAMP) is a highly sensitive, rapid, cost-effective nucleic acid amplification method. Tuberculosis (TB) is widely popular in the world and it is difficult to cure. The fundamental treatment is to clear the types of TB pathogens such as Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M. tuberculosis). In order to detect and diagnose TB early, we constructed the differential diagnostic method of TB. In this study, we used LAMP for detection of M. bovis, based on amplification of the mpb70 gene which is a unique gene in M. bovis strain. The LAMP assay was able to detect only seven copies of the gene per reaction, whereas for the conventional PCR, it was 70 copies. The LAMP was evaluated for its specificity using six strains of five Mycobacterium species and 18 related non-Mycobacterium microorganism strains as controls. The target three Mycobacterium strains were all amplified, and no cross-reaction was found with 18 non-Mycobacterium microorganism strains. TB was detected by two methods, LAMP and conventional PCR (based on mpb70 gene); the positive rates of the two methods were 9.55 and 7.01 %, respectively. Our results indicate that the LAMP method should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of TB caused by M. bovis. Most importance is that the use of LAMP as diagnostic method in association with diagnostic tests based on mpb70 gene would allow the differentiation between M. bovis and other Mycobacterium in humans or animals. The LAMP method is actually in order to detect human TB, and it can be used for differential diagnosis in this paper.

Product ion filtering with rapid polarity switching for the detection of all fumonisins and AAL-toxins.

PubMed

Renaud, Justin B; Kelman, Megan J; Qi, Tianyu F; Seifert, Keith A; Sumarah, Mark W

2015-11-30

Fumonisins and AAL-toxins are structurally similar mycotoxins that contaminate agricultural crops and foodstuffs. Traditional analytical screening methods are designed to target the known compounds for which standards are available but there is clear evidence that many other derivatives exist and could be toxic. A fast, semi-targeted method for the detection of all known fumonisins, AAL-toxins and related emerging toxins is required. Strains of Fusarium verticillioides, Alternaria arborescens and Aspergillus welwitschiae were grown on their associated crops (maize, tomatoes, and grapes, respectively). Extracts were first analyzed in negative mode using product ion filtering to detect the tricarballylic ester product ion that is common to fumonisins and AAL-toxins (m/z 157.0142). During the same liquid chromatography (LC) run, rapid polarity switching was then used to collect positive mode tandem mass spectrometric (MS(2) ) data for characterization of the detected compounds. Fumonisin B1 , B2 , B3 and B4 were detected on Fusarium contaminated maize, AAL-toxins TA, TB, TD, TE were detected on Alternaria inoculated tomatoes and fumonisin B2 , B4 and B6 on Aspergillus contaminated grapes. Additionally, over 100 structurally related compounds possessing a tricarballylic ester were detected from the mould inoculated plant material. These included a hydroxyl-FB1 from F. verticillioides inoculated maize, keto derivatives of AAL-toxins from A. arborescens inoculated tomatoes, and two previously unreported classes of non-aminated fumonisins from Asp. welwitschiae contaminated grapes. A semi-targeted method for the detection of all fumonisins and AAL-toxins in foodstuffs was developed. The use of the distinctive tricarballylic ester product anion for detection combined with rapid polarity switching and positive mode MS(2) is an effective strategy for differentiating between known isomers such as FB1 and FB6 . This analytical tool is also effective for the identification of

A Novel Surface Plasmon Resonance Biosensor for the Rapid Detection of Botulinum Neurotoxins

PubMed Central

Patel, Kruti; Halevi, Shmuel; Melman, Paul; Schwartz, John; Cai, Shuowei; Singh, Bal Ram

2017-01-01

Botulinum neurotoxins (BoNTs) are Category A agents on the NIAID (National Institute of Allergy and Infectious Diseases) priority pathogen list owing to their extreme toxicity and the relative ease of production. These deadly toxins, in minute quantities (estimated human i.v. lethal dose LD50 of 1–2 ng/kg body weight), cause fatal flaccid paralysis by blocking neurotransmitter release. The current gold standard detection method, the mouse-bioassay, often takes days to confirm botulism. Furthermore, there are no effective antidotes known to reverse the symptoms of botulism, and as a result, patients with severe botulism often require meticulous care during the prolonged paralytic illness. To combat potential bio-terrorism incidents of botulinum neurotoxins, their rapid detection is paramount. Surface plasmon resonance (SPR) is a very sensitive technique to examine bio-molecular interactions. The label-free, real-time analysis, with high sensitivity and low sample consumption makes this technology particularly suitable for detection of the toxin. In this study, we demonstrated the feasibility in an assay with a newly designed SPR instrument for the rapid detection of botulinum neurotoxins. The LOD (limit of detection) of the Newton Photonics (NP) SPR based assay is 6.76 pg/mL for Botulinum Neurotoxin type A Light Chain (BoNT/A LC). We established that the detection sensitivity of the system is comparable to the traditional mouse LD50 bioassay in BoNT/A using this SPR technology. PMID:28783115

Rapid detection of Ebola virus with a reagent-free, point-of-care biosensor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baca, Justin T.; Severns, Virginia; Lovato, Debbie

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 10⁴ PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodologymore » has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.« less

Rapid detection of Ebola virus with a reagent-free, point-of-care biosensor

DOE PAGES

Baca, Justin T.; Severns, Virginia; Lovato, Debbie; ...

2015-04-14

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 10⁴ PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodologymore » has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.« less

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

PubMed

Butot, S; Putallaz, T; Sánchez, G

2007-01-01

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

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

DOEpatents

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

1995-01-01

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

Gold Nanorod-based Photo-PCR System for One-Step, Rapid Detection of Bacteria

PubMed Central

Kim, Jinjoo; Kim, Hansol; Park, Ji Ho; Jon, Sangyong

2017-01-01

The polymerase chain reaction (PCR) has been an essential tool for diagnosis of infectious diseases, but conventional PCR still has some limitations with respect to applications to point-of-care (POC) diagnostic systems that require rapid detection and miniaturization. Here we report a light-based PCR method, termed as photo-PCR, which enables rapid detection of bacteria in a single step. In the photo-PCR system, poly(enthylene glycol)-modified gold nanorods (PEG-GNRs), used as a heat generator, are added into the PCR mixture, which is subsequently periodically irradiated with a 808-nm laser to create thermal cycling. Photo-PCR was able to significantly reduce overall thermal cycling time by integrating bacterial cell lysis and DNA amplification into a single step. Furthermore, when combined with KAPA2G fast polymerase and cooling system, the entire process of bacterial genomic DNA extraction and amplification was further shortened, highlighting the potential of photo-PCR for use in a portable, POC diagnostic system. PMID:29071186

Evaluation of Anomaly Detection Method Based on Pattern Recognition

NASA Astrophysics Data System (ADS)

Fontugne, Romain; Himura, Yosuke; Fukuda, Kensuke

The number of threats on the Internet is rapidly increasing, and anomaly detection has become of increasing importance. High-speed backbone traffic is particularly degraded, but their analysis is a complicated task due to the amount of data, the lack of payload data, the asymmetric routing and the use of sampling techniques. Most anomaly detection schemes focus on the statistical properties of network traffic and highlight anomalous traffic through their singularities. In this paper, we concentrate on unusual traffic distributions, which are easily identifiable in temporal-spatial space (e.g., time/address or port). We present an anomaly detection method that uses a pattern recognition technique to identify anomalies in pictures representing traffic. The main advantage of this method is its ability to detect attacks involving mice flows. We evaluate the parameter set and the effectiveness of this approach by analyzing six years of Internet traffic collected from a trans-Pacific link. We show several examples of detected anomalies and compare our results with those of two other methods. The comparison indicates that the only anomalies detected by the pattern-recognition-based method are mainly malicious traffic with a few packets.

A review on detection methods used for foodborne pathogens

PubMed Central

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

2016-01-01

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

Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.

PubMed

van Gijlswijk, R P; Wiegant, J; Vervenne, R; Lasan, R; Tanke, H J; Raap, A K

1996-01-01

We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization. The method features full chemical synthesis of probes, strong FISH signals, and short processing periods, as well as multicolor capabilities.

A Bayesian method for detecting stellar flares

NASA Astrophysics Data System (ADS)

Pitkin, M.; Williams, D.; Fletcher, L.; Grant, S. D. T.

2014-12-01

We present a Bayesian-odds-ratio-based algorithm for detecting stellar flares in light-curve data. We assume flares are described by a model in which there is a rapid rise with a half-Gaussian profile, followed by an exponential decay. Our signal model also contains a polynomial background model required to fit underlying light-curve variations in the data, which could otherwise partially mimic a flare. We characterize the false alarm probability and efficiency of this method under the assumption that any unmodelled noise in the data is Gaussian, and compare it with a simpler thresholding method based on that used in Walkowicz et al. We find our method has a significant increase in detection efficiency for low signal-to-noise ratio (S/N) flares. For a conservative false alarm probability our method can detect 95 per cent of flares with S/N less than 20, as compared to S/N of 25 for the simpler method. We also test how well the assumption of Gaussian noise holds by applying the method to a selection of `quiet' Kepler stars. As an example we have applied our method to a selection of stars in Kepler Quarter 1 data. The method finds 687 flaring stars with a total of 1873 flares after vetos have been applied. For these flares we have made preliminary characterizations of their durations and and S/N.

Comparison of Three Biochemical Tests for Rapid Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae.

PubMed

Poirel, Laurent; Fernández, Javier; Nordmann, Patrice

2016-02-01

Enterobacterial isolates producing clavulanic-inhibited extended-spectrum β-lactamases (ESBLs) are increasingly spreading in the community and are often responsible for nosocomial infections. Rapid biochemical tests have been developed recently for their detection. Three tests, namely, the Rapid ESBL NDP test, the β-Lacta test, and the Rapid ESBL Screen, have been evaluated with a collection of 108 well-characterized strains, including wild-type strains, strains producing ESBLs, overexpressed cephalosporinases, and carbapenemases. The ESBL NDP test and the Rapid ESBL Screen (a copy of the ESBL NDP test) are aimed at detecting ESBL producers, while the β-Lacta test is aimed at detecting not only ESBL producers but also cephalosporinase and carbapenemase producers. The sensitivity and specificity for detecting ESBL producers (n = 60) were 95% and 100% for the Rapid ESBL NDP test, 80% and 87% (after 30 min) and 92% and 83% (after 2 h) for the Rapid ESBL Screen, and 88% and 71% for the β-Lacta test, respectively. Varied and time-consuming detection (up to 2 h) of ESBLs by the Rapid ESBL Screen and concomitant and varied detection of producers of AmpC and several types of carbapenemases correspond to significant shortcomings of using the Rapid Screen ESBL and β-Lacta tests, respectively. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

PubMed Central

Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

2013-01-01

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management. PMID:23811499

Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

PubMed Central

Ngui, Romano; Lim, Yvonne A. L.; Chua, Kek Heng

2012-01-01

Background Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species. PMID:22844538

Comparison of methods for the detection of coliphages in recreational water at two California, United States beaches.

PubMed

Rodríguez, Roberto A; Love, David C; Stewart, Jill R; Tajuba, Julianne; Knee, Jacqueline; Dickerson, Jerold W; Webster, Laura F; Sobsey, Mark D

2012-04-01

Methods for detection of two fecal indicator viruses, F+ and somatic coliphages, were evaluated for application to recreational marine water. Marine water samples were collected during the summer of 2007 in Southern California, United States from transects along Avalon Beach (n=186 samples) and Doheny Beach (n=101 samples). Coliphage detection methods included EPA method 1601 - two-step enrichment (ENR), EPA method 1602 - single agar layer (SAL), and variations of ENR. Variations included comparison of two incubation times (overnight and 5-h incubation) and two final detection steps (lysis zone assay and a rapid latex agglutination assay). A greater number of samples were positive for somatic and F+ coliphages by ENR than by SAL (p<0.01). The standard ENR with overnight incubation and detection by lysis zone assay was the most sensitive method for the detection of F+ and somatic coliphages from marine water, although the method takes up to three days to obtain results. A rapid 5-h enrichment version of ENR also performed well, with more positive samples than SAL, and could be performed in roughly 24h. Latex agglutination-based detection methods require the least amount of time to perform, although the sensitivity was less than lysis zone-based detection methods. Rapid culture-based enrichment of coliphages in marine water may be possible by further optimizing culture-based methods for saline water conditions to generate higher viral titers than currently available, as well as increasing the sensitivity of latex agglutination detection methods. Copyright © 2012 Elsevier B.V. All rights reserved.

Performance of three rapid screening methods in the detection of Schistosoma haematobium infection in school-age children in Southeastern Nigeria.

PubMed

Okeke, Ogochukwu Caroline; Ubachukwu, Patience Obiageli

2014-03-01

A cross-sectional study of primary school children was conducted to evaluate and compare the performance of some rapid screening methods in the detection of Schistosoma haematobium infection in Nigeria Cement Factory (NigerCem) and Nike Lake areas of Southeastern Nigeria. Urine samples of school children were examined for macro-haematuria and tested for micro-haematuria and proteinuria using reagent strips followed by egg microscopy. Self-reported haematuria was assessed using simple questionnaire. The performances of these rapid diagnoses singly and in combination were calculated using egg microscopy as gold standard. The prevalence of the infection was 26·6% in NigerCem and 5·1% in Nike Lake area, classifying these areas as moderate- and low-prevalence areas (MPA and LPA); while in the subsample used for self-reported haematuria, the prevalence was 27·2 and 4·2% in MPA and LPA, respectively. The positive predictive value (PPV) of micro-haematuria was comparable in MPA (55·26%) and LPA (57·89%). Overall PPV of macro-haematuria was 87·50% in MPA and 66·70% in LPA while in the detection of heavy infection; PPV was higher in LPA (75%) than in MPA (66·67%). In LPA and MPA, combination of micro-haematuria and proteinuria, and concomitant presence of macro-haematuria, micro-haematuria, and proteinuria had PPV of 83·33 and 63·16%, and 100 versus 66·67%, respectively. Generally, the rapid screening tests had lower negative predictive values (NPVs) in MPA than in LPA. The use of simple questionnaire increased the PPV of heavy infection in MPA (77·78%). This was further increased to 80% when self-reported haematuria was combined with micro-haematuria. The result suggests that in MPA with chronic infections, combination of self-reported haematuria and micro-haematuria may reduce the chance of missing those who should be treated.

A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

PubMed

Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

2014-12-01

Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

Rapid, in situ detection of cocaine residues based on paper spray ionization coupled with ion mobility spectrometry.

PubMed

Li, Ming; Zhang, Jingjing; Jiang, Jie; Zhang, Jing; Gao, Jing; Qiao, Xiaolin

2014-04-07

In this paper, a novel approach based on paper spray ionization coupled with ion mobility spectrometry (PSI-IMS) was developed for rapid, in situ detection of cocaine residues in liquid samples and on various surfaces (e.g. glass, marble, skin, wood, fingernails), without tedious sample pretreatment. The obvious advantages of PSI are its low cost, easy operation and simple configuration without using nebulizing gas or discharge gas. Compared with mass spectrometry, ion mobility spectrometry (IMS) takes advantage of its low cost, easy operation, and simple configuration without requiring a vacuum system. Therefore, IMS is a more congruous detection method for PSI in the case of rapid, in situ analysis. For the analysis of cocaine residues in liquid samples, dynamic responses from 5 μg mL(-1) to 200 μg mL(-1) with a linear coefficient (R(2)) of 0.992 were obtained. In this case, the limit of detection (LOD) was calculated to be 2 μg mL(-1) as signal to noise (S/N) was 3 with a relative standard deviation (RSD) of 6.5% for 11 measurements (n = 11). Cocaine residues on various surfaces such as metal, glass, marble, wood, skin, and fingernails were also directly analyzed before wiping the surfaces with a piece of paper. The LOD was calculated to be as low as 5 ng (S/N = 3, RSD = 6.3%, n = 11). This demonstrates the capability of the PSI-IMS method for direct detection of cocaine residues at scenes of cocaine administration. Our results show that PSI-IMS is a simple, sensitive, rapid and economical method for in situ detection of this illicit drug, which could help governments to combat drug abuse.

Label-Free Nanopore Biosensor for Rapid and Highly Sensitive Cocaine Detection in Complex Biological Fluids.

PubMed

Rauf, Sana; Zhang, Ling; Ali, Asghar; Liu, Yang; Li, Jinghong

2017-02-24

Detection of very low amounts of illicit drugs such as cocaine in clinical fluids like serum continues to be important for many areas in the fight against drug trafficking. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of cocaine in human serum and saliva samples based on target-induced strand release strategy. In this bioassay, an aptamer for cocaine was prehybridized with a short complementary DNA. Owing to cocaine specific binding with aptamer, the short DNA strand was displaced from aptamer and translocation of this output DNA through α-hemolysin nanopore generated distinct spike-like current blockages. When plotted in double-logarithmic scale, a linear relationship between target cocaine concentration and output DNA event frequency was obtained in a wide concentration range from 50 nM to 100 μM of cocaine, with the limit of detection down to 50 nM. In addition, this aptamer-based sensor method was successfully applied for cocaine detection in complex biological fluids like human saliva and serum samples with great selectivity. Simple preparation, low cost, rapid, label-free, and real sample detection are the motivating factors for practical application of the proposed biosensor.

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

PubMed

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

2016-08-01

Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein-protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10(4) pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner. Copyright © 2016 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Y.; Dunn, J.; Gao, S.

2008-10-31

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

Rapid detection of malignant bio-species using digital holographic pattern recognition and nano-photonics

NASA Astrophysics Data System (ADS)

Sarkisov, Sergey S.; Kukhtareva, Tatiana; Kukhtarev, Nickolai V.; Curley, Michael J.; Edwards, Vernessa; Creer, Marylyn

2013-03-01

There is a great need for rapid detection of bio-hazardous species particularly in applications to food safety and biodefense. It has been recently demonstrated that the colonies of various bio-species could be rapidly detected using culture-specific and reproducible patterns generated by scattered non-coherent light. However, the method heavily relies on a digital pattern recognition algorithm, which is rather complex, requires substantial computational power and is prone to ambiguities due to shift, scale, or orientation mismatch between the analyzed pattern and the reference from the library. The improvement could be made, if, in addition to the intensity of the scattered optical wave, its phase would be also simultaneously recorded and used for the digital holographic pattern recognition. In this feasibility study the research team recorded digital Gabor-type (in-line) holograms of colonies of micro-organisms, such as Salmonella with a laser diode as a low-coherence light source and a lensless high-resolution (2.0x2.0 micron pixel pitch) digital image sensor. The colonies were grown in conventional Petri dishes using standard methods. The digitally recorded holograms were used for computational reconstruction of the amplitude and phase information of the optical wave diffracted on the colonies. Besides, the pattern recognition of the colony fragments using the cross-correlation between the digital hologram was also implemented. The colonies of mold fungi Altenaria sp, Rhizophus, sp, and Aspergillus sp have been also generating nano-colloidal silver during their growth in specially prepared matrices. The silver-specific plasmonic optical extinction peak at 410-nm was also used for rapid detection and growth monitoring of the fungi colonies.

Evaluation of nutrient agar for the culture of Mycobacterium tuberculosis using the microcolony detection method.

PubMed

Satti, L; Abbasi, S; Faiz, U

2012-07-01

We evaluated nutrient agar using the microcolony detection method for the recovery of Mycobacterium tuberculosis on 37 acid-fast bacilli (AFB) positive sputum specimens, and compared it with conventional Löwenstein-Jensen (LJ) medium. Nutrient agar detected 35 isolates compared to 34 on LJ medium. The mean time to detection of mycobacteria on nutrient agar and LJ medium was respectively 9.6 and 21.4 days. The contamination rate on nutrient agar and LJ medium was respectively 5.4% and 2.7%. Nutrient agar detects M. tuberculosis more rapidly than LJ medium, and could be an economical, rapid culture method in resource-poor settings, provided our findings are confirmed by further studies.

A rapid method to visualize von willebrand factor multimers by using agarose gel electrophoresis, immunolocalization and luminographic detection.

PubMed

Krizek, D R; Rick, M E

2000-03-15

A highly sensitive and rapid clinical method for the visualization of the multimeric structure of von Willebrand Factor in plasma and platelets is described. The method utilizes submerged horizontal agarose gel electrophoresis, followed by transfer of the von Willebrand Factor onto a polyvinylidine fluoride membrane, and immunolocalization and luminographic visualization of the von Willebrand Factor multimeric pattern. This method distinguishes type 1 from types 2A and 2B von Willebrand disease, allowing timely evaluation and classification of von Willebrand Factor in patient plasma. It also allows visualization of the unusually high molecular weight multimers present in platelets. There are several major advantages to this method including rapid processing, simplicity of gel preparation, high sensitivity to low concentrations of von Willebrand Factor, and elimination of radioactivity.

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

PubMed Central

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

2015-01-01

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

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

DOEpatents

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

1995-04-11

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

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

PubMed Central

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

2010-01-01

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

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

PubMed

Patel, Pranav; Landt, Olfert; Kaiser, Marco; Faye, Oumar; Koppe, Tanja; Lass, Ulrich; Sall, Amadou A; Niedrig, Matthias

2013-02-14

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

Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry

PubMed Central

Waller, David F.; Hew, Brian E.; Holdaway, Charlie; Jen, Michael; Peckham, Gabriel D.

2016-01-01

Portable detection and quantitation methods for Bacillus anthracis (anthrax) spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min) result in short sample-to-signal times (less than an hour). With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field. PMID:27999382

Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

PubMed

Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

2015-06-02

The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid detection of meticillin-resistant Staphylococcus aureus bacteraemia using combined three-hour short-incubation matrix-assisted laser desorption/ionization time-of-flight MS identification and Alere Culture Colony PBP2a detection test.

PubMed

Delport, Johannes Andries; Mohorovic, Ivor; Burn, Sandi; McCormick, John Kenneth; Schaus, David; Lannigan, Robert; John, Michael

2016-07-01

Meticillin-resistant Staphylococcus aureus (MRSA) bloodstream infection is responsible for significant morbidity, with mortality rates as high as 60 % if not treated appropriately. We describe a rapid method to detect MRSA in blood cultures using a combined three-hour short-incubation BRUKER matrix-assisted laser desorption/ionization time-of-flight MS BioTyper protocol and a qualitative immunochromatographic assay, the Alere Culture Colony Test PBP2a detection test. We compared this combined method with a molecular method detecting the nuc and mecA genes currently performed in our laboratory. One hundred and seventeen S. aureus blood cultures were tested of which 35 were MRSA and 82 were meticillin-sensitive S. aureus (MSSA). The rapid combined test correctly identified 100 % (82/82) of the MSSA and 85.7 % (30/35) of the MRSA after 3 h. There were five false negative results where the isolates were correctly identified as S. aureus, but PBP2a was not detected by the Culture Colony Test. The combined method has a sensitivity of 87.5 %, specificity of 100 %, a positive predictive value of 100 % and a negative predictive value of 94.3 % with the prevalence of MRSA in our S. aureus blood cultures. The combined rapid method offers a significant benefit to early detection of MRSA in positive blood cultures.

Multicentre evaluations of two new rapid IgG4 tests (WB rapid and panLF rapid) for detection of lymphatic filariasis

PubMed Central

Noordin, Rahmah; Itoh, Makoto; Kimura, Eisaku; Abdul Rahman, Rohana; Ravindran, Balachandran; Mahmud, Rohela; Supali, Taniawati; Weerasooriya, Mirani

2007-01-01

In the global effort to eliminate lymphatic filariasis (LF), rapid field-applicable tests are useful tools that will allow on-site testing to be performed in remote places and the results to be obtained rapidly. Exclusive reliance on the few existing tests may jeopardize the progress of the LF elimination program, thus the introduction of other rapid tests would be useful to address this issue. Two new rapid immunochromatographic IgG4 cassette tests have been produced, namely WB rapid and panLF rapid, for detection of bancroftian filariasis and all three species of lymphatic filaria respectively. WB rapid was developed using BmSXP recombinant antigen, while PanLF rapid was developed using BmR1 and BmSXP recombinant antigens. A total of 165 WB rapid and 276 panLF rapid tests respectively were evaluated at USM and the rest were couriered to another university in Malaysia (98 WB rapid, 129 panLF rapid) and to universities in Indonesia (56 WB rapid, 62 panLF rapid), Japan (152 of each test) and India (18 of each test) where each of the tests underwent independent evaluations in a blinded manner. The average sensitivities of WB rapid and panLF rapid were found to be 97.6% (94%–100%) and 96.5% (94%–100%) respectively; while their average specificities were both 99.6% (99%–100%). Thus this study demonstrated that both the IgG4 rapid tests were highly sensitive and specific, and would be useful additional tests to facilitate the global drive to eliminate this disease. PMID:17961262

Rapid Identification of Vancomycin Resistant Enterococcus Faecalis Clinical Isolates using a Sugar Fermentation Method

PubMed Central

Raeisi, Javad; Saifi, Mahnaz; Pourshafie, Mohammad Reza; Habibi, Mehri; Mohajerani, Hamid Reza; Akbari, Neda

2017-01-01

Introduction Vancomycin Resistant Enterococci (VRE) can be found all over the world. Thus, rapid detection of the isolates could be of high importance in the treatment or prevention of the associated disease. Aim To measure the turanose fermentation in Enterococcus faecalis clinical isolates for rapid differentiation of VRE and Vancomycin-Susceptible E. faecalis (VSE) isolates. Materials and Methods Forty E. faecalis samples were isolated from 200 clinical samples in Tehran Medical Center, Iran, from October 2012 to December 2012. These isolates were detected according to the standard microbial and biochemical tests. Detection of VRE isolates was originally performed by disk diffusion using 1 μg vancomycin disk, followed by Polymerase Chain Reaction (PCR) amplification of the vanA gene. Finally, the turanose consumption in 1%, 0.7% and 0.5% dilutions was detected by a phenotypic method. Results Among the 40 E. faecalis isolates, 20 vancomycin-susceptible and 20 vancomycin-resistant E. faecalis were isolated according to the disk diffusion and PCR of the vanA gene. There was a considerable difference between VRE and VSE isolates in 0.7% dilution of turanose. However, there was no significant difference between VRE and VSE in 1% and 0.5% dilutions of turanose. Conclusion Since detection of VRE isolates is of high importance, especially in nosocomial infections, phenotypic methods may be highly useful for this purpose. In conclusion, our data indicate that VRE isolated from clinical samples could be distinguished from VSE isolates by turanose fermentation at dilution 0.7%. PMID:28511382

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

ERIC Educational Resources Information Center

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

2010-01-01

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

Efficient synthesis of highly fluorescent carbon dots by microreactor method and their application in Fe3+ ion detection.

PubMed

Rao, Longshi; Tang, Yong; Li, Zongtao; Ding, Xinrui; Liang, Guanwei; Lu, Hanguang; Yan, Caiman; Tang, Kairui; Yu, Binhai

2017-12-01

Rapidly obtaining strong photoluminescence (PL) of carbon dots with high stability is crucial in all practical applications of carbon dots, such as cell imaging and biological detection. In this study, we proposed a rapid, continuous carbon dots synthesis technique by using a microreactor method. By taking advantage of the microreactor, we were able to rapidly synthesized CDs at a large scale in less than 5min, and a high quantum yield of 60.1% was achieved. This method is faster and more efficient than most of the previously reported methods. To explore the relationship between the microreactor structure and CDs PL properties, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were carried out. The results show the surface functional groups and element contents influence the PL emission. Subsequent ion detection experiments indicated that CDs are very suitable for use as nanoprobes for Fe 3+ ion detection, and the lowest detection limit for Fe 3+ is 0.239μM, which is superior to many other research studies. This rapid and simple synthesis method will not only aid the development of the quantum dots industrialization but also provide a powerful and portable tool for the rapid and continuous online synthesis of quantum dots supporting their application in cell imaging and safety detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid detection of the neonicotinoid insecticide imidacloprid using a quenchbody assay.

PubMed

Zhao, Shitao; Dong, Jinhua; Jeong, Hee-Jin; Okumura, Koichi; Ueda, Hiroshi

2018-04-28

Contamination of the land and water by neonicotinoid insecticide residues is currently a severe environmental problem. However, the traditional methods for pesticide residue analysis are time consuming and laborious. To tackle this problem, here we describe a novel quenchbody (Q-body) immunoassay reagent that allows the rapid and sensitive detection of imidacloprid, one of the most frequently used neonicotinoid pesticides, in aqueous solution. A Q-body comprises an antibody Fab fragment that is site-specifically labeled with a fluorescent dye. The Fab fragment quenches the dye with its internal tryptophan residues via photoinduced electron transfer. The subsequent addition of imidacloprid stabilizes the antibody structure and displaces the quenched dye to the outside of the protein, resulting in increased fluorescence. The constructed Q-body assay exhibited a high dynamic range and a low limit of detection (10 ng mL -1 ), and the entire assay procedure could be completed in a few minutes. The assay showed a low cross-reactivity with possible interfering analogous compounds, indicating that it has a good selectivity. Hence, the developed Q-body assay has excellent potential as a universal technology for monitoring neonicotinoid residues in environmental and food samples. Graphical abstract A novel quenchbody (Q-body) immunoassay reagent that allows the rapid and sensitive detection of imidacloprid, one of the most frequently used neonicotinoid pesticides, in aqueous solution was developed. The addition of imidacloprid stabilizes the Q-body structure and displaces the quenched dye to the outside of the protein, resulting in increased fluorescence. The constructed Q-body assay exhibited a high dynamic range and a low limit of detection (10 ng mL -1 ), and completed in a few minutes.

Microfluidics for rapid detection of isocitrate dehydrogenase 1 mutation for intraoperative application.

PubMed

Aibaidula, Abudumijiti; Zhao, Wang; Wu, Jin-Song; Chen, Hong; Shi, Zhi-Feng; Zheng, Lu-Lu; Mao, Ying; Zhou, Liang-Fu; Sui, Guo-Dong

2016-06-01

OBJECT Conventional methods for isocitrate dehydrogenase 1 (IDH1) detection, such as DNA sequencing and immunohistochemistry, are time- and labor-consuming and cannot be applied for intraoperative analysis. To develop a new approach for rapid analysis of IDH1 mutation from tiny tumor samples, this study used microfluidics as a method for IDH1 mutation detection. METHODS Forty-seven glioma tumor samples were used; IDH1 mutation status was investigated by immunohistochemistry and DNA sequencing. The microfluidic device was fabricated from polydimethylsiloxane following standard soft lithography. The immunoanalysis was conducted in the microfluidic chip. Fluorescence images of the on-chip microcolumn taken by the charge-coupled device camera were collected as the analytical results readout. Fluorescence signals were analyzed by NIS-Elements software to gather detailed information about the IDH1 concentration in the tissue samples. RESULTS DNA sequencing identified IDH1 R132H mutation in 33 of 47 tumor samples. The fluorescence signal for IDH1-mutant samples was 5.49 ± 1.87 compared with 3.90 ± 1.33 for wild type (p = 0.005). Thus, microfluidics was capable of distinguishing IDH1-mutant tumor samples from wild-type samples. When the cutoff value was 4.11, the sensitivity of microfluidics was 87.9% and the specificity was 64.3%. CONCLUSIONS This new approach was capable of analyzing IDH1 mutation status of tiny tissue samples within 30 minutes using intraoperative microsampling. This approach might also be applied for rapid pathological diagnosis of diffuse gliomas, thus guiding personalized resection.

PCR-Based Method for the Detection of Toxic Mushrooms Causing Food-Poisoning Incidents.

PubMed

Nomura, Chie; Masayama, Atsushi; Yamaguchi, Mizuka; Sakuma, Daisuke; Kajimura, Keiji

2017-01-01

In this study, species-specific identification of five toxic mushrooms, Chlorophyllum molybdites, Gymnopilus junonius, Hypholoma fasciculare, Pleurocybella porrigens, and Tricholoma ustale, which have been involved in food-poisoning incidents in Japan, was investigated. Specific primer pairs targeting internal transcribed spacer (ITS) regions were designed for PCR detection. The specific amplicons were obtained from fresh, cooked, and simulated gastric fluid (SGF)-treated samples. No amplicons were detected from other mushrooms with similar morphology. Our method using one-step extraction of mushrooms allows rapid detection within 2.5 hr. It could be utilized for rapid identification or screening of toxic mushrooms.

ETV Tech Brief: Rapid Fungi and Bacteria Detection Technologies

EPA Science Inventory

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

Rapid pretreatment and detection of trace aflatoxin B1 in traditional soybean sauce.

PubMed

Xie, Fang; Lai, WeiHua; Saini, Jasdeep; Shan, Shan; Cui, Xi; Liu, DaoFeng

2014-05-01

Soybean sauce, a traditional fermented food in China, has different levels of aflatoxin B1 pollution. Two kinds of direct and indirect immunomagnetic bead methods for the pretreatment of aflatoxin B1 were evaluated in this work. A method was established to detect aflatoxin B1 in soybean sauce using an immunomagnetic bead system for pretreatment and ELISA for quantification. The pretreatment method of immunomagnetic beads performed better compared with the conventional extraction and immunoaffinity column method. ELISA exhibited a good linear relationship at an aflatoxin B1 concentration of 0.05-0.3μg/kg (r(2)=0.9842). The average recoveries across spike levels varied from 0.5 to 7μg/kg were 83.6-104% with a relative standard deviation between 4.2% and 11.7%. With the advantages of rapid detection, easy operation, simple equipment, sensitivity, accuracy, and high recovery; this method can be well applied in the trace determination of aflatoxin B1 in soybean sauce samples. Copyright © 2013 Elsevier Ltd. All rights reserved.

A method of rapidly evaluating image quality of NED optical system

NASA Astrophysics Data System (ADS)

Sun, Qi; Qiu, Chuankai; Yang, Huan

2014-11-01

In recent years, with the development of technology of micro-display, advanced optics and the software and hardware, near-to-eye display ( NED) optical system will have a wide range of potential applications in the fields of amusement and virtual reality. However, research on the evaluating image quality of this kind optical system is comparatively lagging behind. Although now there are some methods and equipment for evaluation, they can't be applied in commercial production because of their complex operation and inaccuracy. In this paper, an academic method is proposed and a Rapid Evaluation System (RES) is designed to evaluate the image of optical system rapidly and exactly. Firstly, a set of parameters that eyes are sensitive to and also express the quality of system should be extracted and quantized to be criterion, so the evaluation standards can be established. Then, some parameters can be detected by RES consisted of micro-display, CCD camera and computer and so on. By process of scaling, the measuring results of the RES are exact and creditable, relationship between object measurement, subjective evaluation and the RES will be established. After that, image quality of optical system can be evaluated just by detecting parameters of that. The RES is simple and the results of evaluation are exact and keeping with human vision. So the method can be used not only for optimizing design of optical system, but also for evaluation in commercial production.

Development of a rapid diagnostic method for identification of Staphylococcus aureus and antimicrobial resistance in positive blood culture bottles using a PCR-DNA-chromatography method.

PubMed

Ohshiro, Takeya; Miyagi, Chihiro; Tamaki, Yoshikazu; Mizuno, Takuya; Ezaki, Takayuki

2016-06-01

Blood culturing and the rapid reporting of results are essential for infectious disease clinics to obtain bacterial information that can affect patient prognosis. When gram-positive coccoid cells are observed in blood culture bottles, it is important to determine whether the strain is Staphylococcus aureus and whether the strain has resistance genes, such as mecA and blaZ, for proper antibiotic selection. Previous work led to the development of a PCR method that is useful for rapid identification of bacterial species and antimicrobial susceptibility. However, that method has not yet been adopted in community hospitals due to the high cost and methodological complexity. We report here the development of a quick PCR and DNA-chromatography test, based on single-tag hybridization chromatography, that permits detection of S. aureus and the mecA and blaZ genes; results can be obtained within 1 h for positive blood culture bottles. We evaluated this method using 42 clinical isolates. Detection of S. aureus and the resistance genes by the PCR-DNA-chromatography method was compared with that obtained via the conventional identification method and actual antimicrobial susceptibility testing. Our method had a sensitivity of 97.0% and a specificity of 100% for the identification of the bacterial species. For the detection of the mecA gene of S. aureus, the sensitivity was 100% and the specificity was 95.2%. For the detection of the blaZ gene of S. aureus, the sensitivity was 100% and the specificity was 88.9%. The speed and simplicity of this PCR-DNA-chromatography method suggest that our method will facilitate rapid diagnoses. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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 Detection of Visually Provocative Animals by Preschool Children and Adults

ERIC Educational Resources Information Center

Penkunas, Michael J.; Coss, Richard G.

2013-01-01

The ability to detect dangerous animals rapidly in complex landscapes has been historically important during human evolution. Previous research has shown that snake images are more readily detected than images of benign animals. To provide a stringent test of superior snake detection in preschool children and adults, Experiment 1 consisted of two…

Rapid detection of TiO2 (E171) in table sugar using Raman spectroscopy.

PubMed

Tan, Chen; Zhao, Bin; Zhang, Zhiyun; He, Lili

2017-02-01

The potential toxic effects of titanium dioxide (TiO 2 ) to humans remain debatable despite its broad application as a food additive. Thus, confirmation of the existence of TiO 2 particles in food matrices and subsequently quantifying them are becoming increasingly critical. This study developed a facile, rapid (< 30 min) and highly reliable method to detect and quantify TiO 2 particles (E171) from food products (e.g., table sugar) by Raman spectroscopy. To detect TiO 2 particles from sugar solution, sequential centrifugation and washing procedures were effectively applied to separate and recover 97% of TiO 2 particles from the sugar solution. The peak intensity of TiO 2 sensitively responded to the concentration of TiO 2 with a limit of detection (LOD) of 0.073 mg kg -1 . In the case of sugar granules, a mapping technique was applied to directly estimate the level of TiO 2 , which can be potentially used for rapid online monitoring. The plot of averaged intensity to TiO 2 concentration in the sugar granules exhibited a good linear relationship in the wide range of 5-2000 mg kg -1 , with an LOD of 8.46 mg kg -1 . Additionally, we applied Raman spectroscopy to prove the presence of TiO 2 in sugar-coated doughnuts. This study begins to fill in the analytical gaps that exist regarding the rapid detection and quantification of TiO 2 in food, which facilitate the risk assessment of TiO 2 through food exposure.

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

PubMed

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

2012-01-07

In this study, a new method combining magnetic separation (MS) and surface-enhanced Raman scattering (SERS) was developed to detect genetically modified organisms (GMOs). An oligonucleotide probe which is specific for 35 S DNA target was immobilized onto gold coated magnetic nanospheres to form oligonucleotide-coated nanoparticles. A self assembled monolayer was formed on gold nanorods using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and the second probe of the 35 S DNA target was immobilized on the activated nanorod surfaces. Probes on the nanoparticles were hybridized with the target oligonucleotide. Optimization parameters for hybridization were investigated by high performance liquid chromatography. Optimum hybridization parameters were determined as: 4 μM probe concentration, 20 min immobilization time, 30 min hybridization time, 55 °C hybridization temperature, 750 mM buffer salt concentration and pH: 7.4. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. The correlation between the target concentration and the SERS signal was found to be linear within the range of 25-100 nM. The analyses were performed with only one hybridization step in 40 min. Real sample analysis was conducted using Bt-176 maize sample. The results showed that the developed MS-SERS assay is capable of detecting GMOs in a rapid and selective manner. This journal is © The Royal Society of Chemistry 2012

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

PubMed

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

2012-12-01

In this work, we present a new MR spectroscopy approach for directly observing nuclear spins that undergo exchange, metabolic conversion, or, generally, any frequency shift during a mixing time. Unlike conventional approaches to observe these processes, such as exchange spectroscopy (EXSY), this rapid approach requires only a single encoding step and thus is readily applicable to hyperpolarized MR in which the magnetization is not replenished after T(1) decay and RF excitations. This method is based on stimulated-echoes and uses phase-sensitive detection in conjunction with precisely chosen echo times in order to separate spins generated during the mixing time from those present prior to mixing. We are calling the method Metabolic Activity Decomposition Stimulated-echo Acquisition Mode or MAD-STEAM. We have validated this approach as well as applied it in vivo to normal mice and a transgenic prostate cancer mouse model for observing pyruvate-lactate conversion, which has been shown to be elevated in numerous tumor types. In this application, it provides an improved measure of cellular metabolism by separating [1-(13)C]-lactate produced in tissue by metabolic conversion from [1-(13)C]-lactate that has flowed into the tissue or is in the blood. Generally, MAD-STEAM can be applied to any system in which spins undergo a frequency shift. Copyright © 2012 Elsevier Inc. All rights reserved.

Rapid and sensitive microRNA detection with laminar flow-assisted dendritic amplification on power-free microfluidic chip.

PubMed

Arata, Hideyuki; Komatsu, Hiroshi; Hosokawa, Kazuo; Maeda, Mizuo

2012-01-01

Detection of microRNAs, small noncoding single-stranded RNAs, is one of the key topics in the new generation of cancer research because cancer in the human body can be detected or even classified by microRNA detection. This report shows rapid and sensitive microRNA detection using a power-free microfluidic device, which is driven by degassed poly(dimethylsiloxane), thus eliminating the need for an external power supply. MicroRNA is detected by sandwich hybridization, and the signal is amplified by laminar flow-assisted dendritic amplification. This method allows us to detect microRNA of specific sequences at a limit of detection of 0.5 pM from a 0.5 µL sample solution with a detection time of 20 min. Together with the advantages of self-reliance of this device, this method might contribute substantially to future point-of-care early-stage cancer diagnosis.

Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

PubMed

Valverde, Estefanía J; Cano, Irene; Castro, Dolores; Paley, Richard K; Borrego, Juan J

2017-03-01

Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.

A Rapid Growth-Independent Antibiotic Resistance Detection Test by SYBR Green/Propidium Iodide Viability Assay.

PubMed

Feng, Jie; Yee, Rebecca; Zhang, Shuo; Tian, Lili; Shi, Wanliang; Zhang, Wen-Hong; Zhang, Ying

2018-01-01

Antibiotic-resistant bacteria have caused huge concerns and demand innovative approaches for their prompt detection. Current antimicrobial susceptibility tests (AST) rely on the growth of the organisms which takes 1-2 days for fast-growing organisms and several weeks for slow growing organisms. Here, we show for the first time the utility of the SYBR Green I/propidium iodide (PI) viability assay for rapidly identifying antibiotic resistance in less than 30 min for major, antibiotic-resistant, fast-growing bacteria, such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae , and Acinetobacter baumannii for bactericidal and bacteriostatic agents and in 16 h for extremely rapid detection of drug resistance for isoniazid and pyrazinamide in slow-growing Mycobacterium tuberculosis . The SYBR Green I/PI assay generated rapid and robust results in concordance with traditional AST methods. This novel growth-independent methodology changes the concept of the current growth-based AST and may revolutionize current drug susceptibility testing for all cells of prokaryotic and eukaryotic origin and, subject to further clinical validation, may play a major role in saving lives and improving patient outcomes.

A novel, colorimetric method for biogenic amine detection based on arylalkylamine N-acetyltransferase.

PubMed

Leng, Pei-Qiang; Zhao, Feng-Lan; Yin, Bin-Cheng; Ye, Bang-Ce

2015-05-21

We developed a novel colorimetric method for rapid detection of biogenic amines based on arylalkylamine N-acetyltransferase (aaNAT). The proposed method offers distinct advantages including simple handling, high speed, low cost, good sensitivity and selectivity.

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

PubMed Central

Bai, Jaewoo; Kim, You-Tae; Ryu, Sangryeol; Lee, Ju-Hoon

2016-01-01

Bacteriophages have been suggested as natural food preservatives as well as rapid detection materials for food-borne pathogens in various foods. Since Listeria monocytogenes-targeting phage cocktail (ListShield) was approved for applications in foods, numerous phages have been screened and experimentally characterized for phage applications in foods. A single phage and phage cocktail treatments to various foods contaminated with food-borne pathogens including E. coli O157:H7, Salmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Cronobacter sakazakii, and Vibrio spp. revealed that they have great potential to control various food-borne pathogens and may be alternative for conventional food preservatives. In addition, phage-derived endolysins with high host specificity and host lysis activities may be preferred to food applications rather than phages. For rapid detection of food-borne pathogens, cell-wall binding domains (CBDs) from endolysins have been suggested due to their high host-specific binding. Fluorescence-tagged CBDs have been successfully evaluated and suggested to be alternative materials of expensive antibodies for various detection applications. Most recently, reporter phage systems have been developed and tested to confirm their usability and accuracy for specific detection. These systems revealed some advantages like rapid detection of only viable pathogenic cells without interference by food components in a very short reaction time, suggesting that these systems may be suitable for monitoring of pathogens in foods. Consequently, phage is the next-generation biocontrol agent as well as rapid detection tool to confirm and even identify the food-borne pathogens present in various foods. PMID:27092128

Simple Approach for the Rapid Detection of Alternariol in Pear Fruit by Surface-Enhanced Raman Scattering with Pyridine-Modified Silver Nanoparticles.

PubMed

Pan, Ting-Tiao; Sun, Da-Wen; Pu, Hongbin; Wei, Qingyi

2018-03-07

A simple method based on surface-enhanced Raman scattering (SERS) was developed for the rapid determination of alternariol (AOH) in pear fruits using an easily prepared silver-nanoparticle (AgNP) substrate. The AgNP substrate was modified by pyridine to circumvent the weak affinity of the AOH molecules to the silver surface and to improve the sensitivity of detection. Quantitative analysis was performed in AOH solutions at concentrations ranging from 3.16 to 316.0 μg/L, and the limit of detection was 1.30 μg/L. The novel method was also applied to the detection of AOH residues in pear fruits purchased from the market and in pear fruits that were artificially inoculated with Alternaria alternata. AOH was not found in any of the fresh fruit, whereas it resided in the rotten and inoculated fruits. Finally, the SERS method was cross validated against HPLC. It was revealed that the SERS method has great potential utility in the rapid detection of AOH in pear fruits and other agricultural products.

Microbial detection method based on sensing molecular hydrogen

NASA Technical Reports Server (NTRS)

Wilkins, J. R.; Stoner, G. E.; Boykin, E. H.

1974-01-01

A simple method for detecting bacteria, based on the time of hydrogen evolution, was developed and tested against various members of the Enterobacteriaceae group. The test system consisted of (1) two electrodes, platinum and a reference electrode, (2) a buffer amplifier, and (3) a strip-chart recorder. Hydrogen evolution was measured by an increase in voltage in the negative (cathodic) direction. A linear relationship was established between inoculum size and the time hydrogen was detected (lag period). Lag times ranged from 1 h for 1 million cells/ml to 7 h for 1 cell/ml. For each 10-fold decrease in inoculum, length of the lag period increased 60 to 70 min. Based on the linear relationship between inoculum and lag period, these results indicate the potential application of the hydrogen-sensing method for rapidly detecting coliforms and other gas-producing microorganisms in a variety of clinical, food, and other samples.

Microfluidic Capillaric Circuit for Rapid and Facile Bacteria Detection.

PubMed

Olanrewaju, Ayokunle Oluwafemi; Ng, Andy; DeCorwin-Martin, Philippe; Robillard, Alessandra; Juncker, David

2017-06-20

Urinary tract infections (UTI) are one of the most common bacterial infections and would greatly benefit from a rapid point-of-care diagnostic test. Although significant progress has been made in developing microfluidic systems for nucleic acid and whole bacteria immunoassay tests, their practical application is limited by complex protocols, bulky peripherals, and slow operation. Here we present a microfluidic capillaric circuit (CC) optimized for rapid and automated detection of bacteria in urine. Molds for CCs were constructed using previously established design rules, then 3D-printed and replicated into poly(dimethylsiloxane). CCs autonomously and sequentially performed all liquid delivery steps required for the assay. For efficient bacteria capture, on-the-spot packing of antibody-functionalized microbeads was completed in <20 s followed by autonomous sequential delivery of 100 μL of bacteria sample, biotinylated detection antibodies, fluorescent streptavidin conjugate, and wash buffer for a total volume ≈115 μL. The assay was completed in <7 min. Fluorescence images of the microbead column revealed captured bacteria as bright spots that were easily counted manually or using an automated script for user-independent assay readout. The limit of detection of E. coli in synthetic urine was 1.2 × 10 2 colony-forming-units per mL (CFU/mL), which is well below the clinical diagnostic criterion (>10 5 CFU/mL) for UTI. The self-powered, peripheral-free CC presented here has potential for use in rapid point-of-care UTI screening.

Rapid shape detection signals in area V4

PubMed Central

Weiner, Katherine F.; Ghose, Geoffrey M.

2014-01-01

Vision in foveate animals is an active process that requires rapid and constant decision-making. For example, when a new object appears in the visual field, we can quickly decide to inspect it by directing our eyes to the object's location. We studied the contribution of primate area V4 to these types of rapid foveation decisions. Animals performed a reaction time task that required them to report when any shape appeared within a peripherally-located noisy stimulus by making a saccade to the stimulus location. We found that about half of the randomly sampled V4 neurons not only rapidly and precisely represented the appearance of this shape, but they were also predictive of the animal's saccades. A neuron's ability to predict the animal's saccades was not related to the specificity with which the cell represented a single type of shape but rather to its ability to signal whether any shape was present. This relationship between sensory sensitivity and behavioral predictiveness was not due to global effects such as alertness, as it was equally likely to be observed for cells with increases and decreases in firing rate. Careful analysis of the timescales of reliability in these neurons implies that they reflect both feedforward and feedback shape detecting processes. In approximately 7% of our recorded sample, individual neurons were able to predict both the delay and precision of the animal's shape detection performance. This suggests that a subset of V4 neurons may have been directly and causally contributing to task performance and that area V4 likely plays a critical role in guiding rapid, form-based foveation decisions. PMID:25278828

Impact Of Rapid Incident Detection On Freeway Accident Fatalities

DOT National Transportation Integrated Search

1996-06-01

Heavily congested urban highways in the United States have spurred the development of Freeway Management Systems (FMS). Among the goals of these systems is the improvement of traffic management and the facilitation of more rapid incident detection an...

Simultaneous detection of perchlorate and bromate using rapid high-performance ion exchange chromatography-tandem mass spectrometry and perchlorate removal in drinking water.

PubMed

West, Danielle M; Mu, Ruipu; Gamagedara, Sanjeewa; Ma, Yinfa; Adams, Craig; Eichholz, Todd; Burken, Joel G; Shi, Honglan

2015-06-01

Perchlorate and bromate occurrence in drinking water causes health concerns due to their effects on thyroid function and carcinogenicity, respectively. The purpose of this study was threefold: (1) to advance a sensitive method for simultaneous rapid detection of perchlorate and bromate in drinking water system, (2) to systematically study the occurrence of these two contaminants in Missouri drinking water treatment systems, and (3) to examine effective sorbents for minimizing perchlorate in drinking water. A rapid high-performance ion exchange chromatography-tandem mass spectrometry (HPIC-MS/MS) method was advanced for simultaneous detection of perchlorate and bromate in drinking water. The HPIC-MS/MS method was rapid, required no preconcentration of the water samples, and had detection limits for perchlorate and bromate of 0.04 and 0.01 μg/L, respectively. The method was applied to determine perchlorate and bromate concentrations in total of 23 selected Missouri drinking water treatment systems during differing seasons. The water systems selected include different source waters: groundwater, lake water, river water, and groundwater influenced by surface water. The concentrations of perchlorate and bromate were lower than or near to method detection limits in most of the drinking water samples monitored. The removal of perchlorate by various adsorbents was studied. A cationic organoclay (TC-99) exhibited effective removal of perchlorate from drinking water matrices.

Nucleic acid detection system and method for detecting influenza

DOEpatents

Cai, Hong; Song, Jian

2015-03-17

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

Black phosphorus nanosheets for rapid microRNA detection.

PubMed

Zhou, Jie; Li, Zhongjun; Ying, Ming; Liu, Maixian; Wang, Xiaomei; Wang, Xianyou; Cao, Liwei; Zhang, Han; Xu, Gaixia

2018-03-15

Herein, for the first time, a sensitive sensing platform for rapid detection of microRNA was developed by employing black phosphorus nanosheets as the fluorescence quenching material. The biosensor displayed a good linear response to microRNA ranging from 10 nM to 1000 nM. Moreover, the biosensor could distinguish triple nucleotide polymorphism.

Application of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cow Components Adulterated in Buffalo Milk/Meat.

PubMed

Deb, Rajib; Sengar, Gyanendra Singh; Singh, Umesh; Kumar, Sushil; Alyethodi, R R; Alex, Rani; Raja, T V; Das, A K; Prakash, B

2016-12-01

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for amplification of DNA with rapid and minimal equipment requirement. In the present study, we applied the LAMP assay for rapid detection of cow components adulteration in buffalo milk/meat samples. The test can be completed within around 1 h 40 min starting from DNA extraction and can be performed in water bath without requirement of thermocycler. The cow DNA in buffalo samples were identified in the developed LAMP assay by either visualizing with SYBR Green I/HNB dyes or observing the typical ladder pattern on gel electrophoresis. The test can detect up to 5 % level of cow milk/meat mixed in buffalo counterparts. Due to the simplicity and specificity, the developed LAMP test can be easily adapted in any laboratory for rapid detection of cow species identification in livestock by products.

Indigenous people's detection of rapid ecological change.

PubMed

Aswani, Shankar; Lauer, Matthew

2014-06-01

When sudden catastrophic events occur, it becomes critical for coastal communities to detect and respond to environmental transformations because failure to do so may undermine overall ecosystem resilience and threaten people's livelihoods. We therefore asked how capable of detecting rapid ecological change following massive environmental disruptions local, indigenous people are. We assessed the direction and periodicity of experimental learning of people in the Western Solomon Islands after a tsunami in 2007. We compared the results of marine science surveys with local ecological knowledge of the benthos across 3 affected villages and 3 periods before and after the tsunami. We sought to determine how people recognize biophysical changes in the environment before and after catastrophic events such as earthquakes and tsunamis and whether people have the ability to detect ecological changes over short time scales or need longer time scales to recognize changes. Indigenous people were able to detect changes in the benthos over time. Detection levels differed between marine science surveys and local ecological knowledge sources over time, but overall patterns of statistically significant detection of change were evident for various habitats. Our findings have implications for marine conservation, coastal management policies, and disaster-relief efforts because when people are able to detect ecological changes, this, in turn, affects how they exploit and manage their marine resources. © 2014 Society for Conservation Biology.

A new strategy for fast radiofrequency CW EPR imaging: Direct detection with rapid scan and rotating gradients

PubMed Central

Subramanian, Sankaran; Koscielniak, Janusz W.; Devasahayam, Nallathamby; Pursley, Randall H.; Pohida, Thomas J.; Krishna, Murali C.

2007-01-01

Rapid field scan on the order of T/s using high frequency sinusoidal or triangular sweep fields superimposed on the main Zeeman field, was used for direct detection of signals without low-frequency field modulation. Simultaneous application of space-encoding rotating field gradients have been employed to perform fast CW EPR imaging using direct detection that could, in principle, approach the speed of pulsed FT EPR imaging. The method takes advantage of the well-known rapid-scan strategy in CW NMR and EPR that allows arbitrarily fast field sweep and the simultaneous application of spinning gradients that allows fast spatial encoding. This leads to fast functional EPR imaging and, depending on the spin concentration, spectrometer sensitivity and detection band width, can provide improved temporal resolution that is important to interrogate dynamics of spin perfusion, pharmacokinetics, spectral spatial imaging, dynamic oxymetry, etc. PMID:17350865

Rapid virus detection procedure for molecular tracing of shellfish associated with disease outbreaks.

PubMed

de Roda Husman, Ana Maria; Lodder-Verschoor, Froukje; van den Berg, Harold H J L; Le Guyader, Françoise S; van Pelt, Hilde; van der Poel, Wim H M; Rutjes, Saskia A

2007-04-01

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III-spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.

3D metal-organic framework as highly efficient biosensing platform for ultrasensitive and rapid detection of bisphenol A.

PubMed

Wang, Xue; Lu, Xianbo; Wu, Lidong; Chen, Jiping

2015-03-15

As is well known, bisphenol A (BPA), usually exists in daily plastic products, is one of the most important endocrine disrupting chemicals. In this work, copper-centered metal-organic framework (Cu-MOF) was synthesized, which was characterized by SEM, TEM, XRD, FTIR and electrochemical method. The resultant Cu-MOF was explored as a robust electrochemical biosensing platform by choosing tyrosinase (Tyr) as a model enzyme for ultrasensitive and rapid detection of BPA. The Cu-MOF provided a 3D structure with a large specific surface area, which was beneficial for enzyme and BPA absorption, and thus improved the sensitivity of the biosensor. Furthermore, Cu-MOF as a novel sorbent could increase the available BPA concentration to react with tyrosinase through π-π stacking interactions between BPA and Cu-MOF. The Tyr biosensor exhibited a high sensitivity of 0.2242A M(-1) for BPA, a wide linear range from 5.0×10(-8) to 3.0×10-6moll(-1), and a low detection limit of 13nmoll(-1). The response time for detection of BPA is less than 11s. The proposed method was successfully applied to rapid and selective detection of BPA in plastic products with satisfactory results. The recoveries are in the range of 94.0-101.6% for practical applications. With those remarkable advantages, MOFs-based 3D structures show great prospect as robust biosensing platform for ultrasensitive and rapid detection of BPA. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Ultrasensitive and rapid detection of β-conglutin combining aptamers and isothermal recombinase polymerase amplification.

PubMed

Jauset-Rubio, Miriam; Sabaté Del Río, Jonathan; Mairal, Teresa; Svobodová, Markéta; El-Shahawi, Mohammad S; Bashammakh, Abdulaziz S; Alyoubi, Abdulrahman O; O'Sullivan, Ciara K

2017-01-01

Lupin is increasingly being used in a variety of food products due to its nutritional, functional and nutraceutical properties. However, several examples of severe and even fatal food-associated anaphylaxis due to lupin inhalation or ingestion have been reported, resulting in the lupin subunit β-conglutin, being defined as the Lup an 1 allergen by the International Union of Immunological Societies (IUIS) in 2008. Here, we report an innovative method termed aptamer-recombinase polymerase amplification (Apta-RPA) exploiting the affinity and specificity of a DNA aptamer selected against the anaphylactic β-conglutin allergen termed β-conglutin binding aptamer II (β-CBA II), facilitating ultrasensitive detection via isothermal amplification. Combining magnetic beads as the solid phase with Apta-RPA detection, the total assay time was reduced from 210 min to just 25 min, with a limit of detection of 3.5 × 10 -11  M, demonstrating a rapid and ultrasensitive generic methodology that can be used with any aptamer. Future work will focus on further simplification of the assay to a lateral flow format. Graphical Abstract Schematic representation of the rapid and novel bead-based Apta-RPA assay.

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

PubMed

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

2015-12-15

Fumonisin B1 (FB1) is the most common and highest toxic of fumonisins species, exists frequently in corn and corn-based foods, leading to several animal and human diseases. Furthermore, FB1 was reported that it was associated with the human esophageal cancer. In view of the harmful of FB1, it is urgent to develop a feasible and accuracy method for rapid detection of FB1. In this study, a competitive immunoassay for FB1 detection was developed based on colloidal gold-antibody conjugate. The FB1-keyhole limpet hemoeyanin (FB1-KLH) conjugate was embedded in the test line, and goat anti-mouse IgG antibody embedded in the control line. The color density of the test line correlated with the concentration of FB1 in the range from 2.5 to 10 ng/mL, and the visual limit detection of test for FB1 was 2.5 ng/mL. The results indicated that the test strip is specific for FB1, and no cross-reactivity to other toxins. The quantitative detection for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time of visual evaluation was less than 5 min. Hence, the developed colloidal gold-antibody assay can be used as a feasible method for FB1 rapid and quantitative detection in corn samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-08-19

As a contaminant in drinking water, N-nitrosodimethylamine (NDMA) is of great concern because of its carcinogenicity; it has been limited to levels of ng/L by regulatory bodies worldwide. Consequently, a rapid and sensitive method for monitoring NDMA in drinking water is urgently required. In this study, we report an improvement of our previously proposed HPLC-based system for NDMA determination. The approach consists of the HPLC separation of NDMA, followed by NDMA photolysis to form peroxynitrite and detection with a luminol chemiluminescence reaction. The detection limit for the improved HPLC method was 0.2ng/L, which is 10 times more sensitive than our previously reported system. For tap water measurements, only the addition of an ascorbic acid solution to eliminate residual chlorine and passage through an Oasis MAX solid-phase extraction cartridge are needed. The proposed NDMA determination method requires a sample volume of less than 2mL and a complete analysis time of less than 15min per sample. The method was utilized for the long-term monitoring of NDMA in tap water. The NDMA level measured in the municipal water survey was 4.9ng/L, and a seasonal change of the NDMA concentration in tap water was confirmed. The proposed method should constitute a useful NDMA monitoring method for protecting drinking water quality. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid determination of gizzerosine in fish meals using microchip capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Xiao, Meng-Wei; Bai, Xiao-Lin; Xu, Pei-Li; Zhao, Yan; Yang, Li; Liu, Yi-Ming; Liao, Xun

2017-05-01

Sensitive detection of gizzerosine, a causative agent for deadly gizzard erosion in chicken feeds, is very important to the poultry industry. In this work, a new method was developed based on microchip capillary electrophoresis (MCE) with laser-induced fluorescence (LIF) detection for rapid analysis of gizzerosine, a biogenic amine in fish meals. The MCE separation was performed on a glass microchip using sodium dodecyl sulfate (SDS) as dynamic coating modifier. Separation conditions, including running buffer pH and concentration, SDS concentration, and the separation voltage were investigated to achieve fast and sensitive quantification of gizzerosine. The assay proposed was very quick and could be completed within 65 s. A linear calibration curve was obtained in the range from 0.04 to 1.8 μg ml -1 gizzerosine. The detection limit was 0.025 μg ml -1 (0.025 mg kg -1 ), which was far more sensitive than those previously reported. Gizzerosine was well separated from other endogenous components in fish meal samples. Recovery of gizzerosine from this sample matrix (n = 3) was determined to be 97.2-102.8%. The results from analysing fish meal samples indicated that the present MCE-LIF method might hold the potential for rapid detection of gizzerosine in poultry feeds.

A rapid and convenient method for detecting a broad spectrum of malignant cells from malignant pleuroperitoneal effusion of patients using a multifunctional NIR heptamethine dye.

PubMed

Tian, Ying; Sun, Jing; Yan, Huaijiang; Teng, Zhaogang; Zeng, Leyong; Liu, Ying; Li, Yanjun; Wang, Jiandong; Wang, Shouju; Lu, Guangming

2015-02-07

Detection of malignant cells from malignant effusion is crucial to establish or adjust therapies of patients with cancer. The conventional qualitative detection in malignant pleuroperitoneal effusion is cytological analysis, which is time-consuming and complicated. Therefore, a faster and more convenient detection strategy is urgently needed. In this study, we report a rapid method to detect malignant cells from malignant pleuroperitoneal effusion (hydrothorax and ascites) of patients using IR-808, a tumor-targeted near-infrared (NIR) fluorescent heptamethine dye (tNRI dye), which exhibited superior labeling efficacy without specific conjugation to biomarkers. The targeted imaging performance toward malignant cells using IR-808 was confirmed by comparing with normal cells, and the fluorescence stability assay of IR-808 in malignant effusion was performed from 1 h to 48 h. In order to save time and dose, the incubation time and concentration were optimized to 10 min and 5 μM, which were used to detect malignant cells from 28 clinical samples of malignant pleuroperitoneal effusion. The results revealed that IR-808 could be internalized selectively by malignant cells of samples, and these malignant cells could be easily distinguished from normal cells under a fluorescence microscope. The positive rates between cytological analysis and the IR-808 staining method were 86% (24/28) and 79% (22/28), respectively. An excellent concordance level (Kappa = 0.752, P < 0.001) was observed between the two methods. Our results indicated that IR-808, a new NIR fluorescent heptamethine dye with unique optical imaging and tumor targeting properties, could provide a fast and simple way to detect a broad spectrum of malignant cells from malignant pleuroperitoneal effusion in patients.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Li, Rong; Yue, Qiao-Yun; Liu, Guo-Hua; Bai, Jian-Shan; Deng, Yan; Qiu, De-Yi; Zhu, Xing-Quan

2014-01-01

Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.

Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum.

PubMed

Sun, Kui; Xing, Weiwei; Yu, Xinling; Fu, Wenliang; Wang, Yuanyuan; Zou, Minji; Luo, Zhihong; Xu, Donggang

2016-08-31

With the continuous decline in prevalence and intensity of Schistosoma japonicum infection in China, more accurate and sensitive methods suitable for field detection become much needed for schistosomiasis control. Here, a novel rapid and visual detection method based on the combination of recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) was developed to detect S. japonicum DNA in fecal samples. The LFD-RPA assay targeting SjR2 could detect 5 fg S. japonicum DNA, which was identical to qPCR and real-time RPA assay, and showed no cross-reaction with other parasites. The detection could be finished within 15-20 min at a wide temperature range (25-45 °C), and the results could be visualized by naked eye. The diagnostic validity of LFD-RPA assay was further assessed with 14 fecal samples of infected patients diagnosed by Kato-Katz method and 31 fecal samples of healthy persons, and compared with that of Enzyme-linked immunosorbent assay (ELSIA) and Indirect Hemagglutination Assay (IHA). The LFD-RPA assay showed 92.68 % sensitivity, 100 % specificity and excellent diagnostic agreement with the gold standard Kato-Katz test (k = 0.947, Z = 6.36, P < 0.001), whereas ELISA showed 85.71 % sensitivity, 93.55 % specificity, and substantial diagnostic agreement (k = 0.793, Z = 5.31, P < 0.001), and IHA showed 78.57 % sensitivity, 83.87 % specificity, and moderate diagnostic agreement (k = 0.600, Z = 4.05, P < 0.001), indicating that the LFD-RPA was much better than the traditional methods. The LFD-RPA assay established by us is a sensitive, specific, rapid and convenient method for the diagnosis of schistosomiasis, and shows a great potency in field application.

Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing technology.

PubMed

Koontz, Deborah A; Huckins, Jacqueline J; Spencer, Antonina; Gallagher, Margaret L

2009-08-24

Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1-2 are derived from CYP2A7, and exons 3-9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology. A single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow. Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics. This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

PubMed

Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

2016-10-01

Eucalyptus dieback disease, caused by Cylindrocladium scoparium , has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium . The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

PubMed Central

Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

2016-01-01

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus.

PubMed

Wang, Jianchang; Wang, Jinfeng; Geng, Yunyun; Yuan, Wanzhe

2017-10-01

A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 10 2 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection.

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus

PubMed Central

Wang, Jianchang; Wang, Jinfeng; Geng, Yunyun; Yuan, Wanzhe

2017-01-01

A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 102 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection. PMID:29081590

Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays.

PubMed

Hou, Peili; Wang, Hongmei; Zhao, Guimin; He, Chengqiang; He, Hongbin

2017-12-13

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV. Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens. RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%. IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.

A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR.

PubMed

Nikolaou, Anastasios; Saxami, Georgia; Kourkoutas, Yiannis; Galanis, Alex

2011-02-01

In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry. Copyright © 2010 Elsevier B.V. All rights reserved.

Rapid detection of microbial cell abundance in aquatic systems

DOE PAGES

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

2016-06-01

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

Rapid detection of microbial cell abundance in aquatic systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

An intelligent detection method for high-field asymmetric waveform ion mobility spectrometry.

PubMed

Li, Yue; Yu, Jianwen; Ruan, Zhiming; Chen, Chilai; Chen, Ran; Wang, Han; Liu, Youjiang; Wang, Xiaozhi; Li, Shan

2018-04-01

In conventional high-field asymmetric waveform ion mobility spectrometry signal acquisition, multi-cycle detection is time consuming and limits somewhat the technique's scope for rapid field detection. In this study, a novel intelligent detection approach has been developed in which a threshold was set on the relative error of α parameters, which can eliminate unnecessary time spent on detection. In this method, two full-spectrum scans were made in advance to obtain the estimated compensation voltage at different dispersion voltages, resulting in a narrowing down of the whole scan area to just the peak area(s) of interest. This intelligent detection method can reduce the detection time to 5-10% of that of the original full-spectrum scan in a single cycle.

Enhanced Microbial Detection Capabilities by a Rapid Portable Instrument

NASA Technical Reports Server (NTRS)

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

2010-01-01

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

Rapid detection of microorganisms based on active and passive modes of QCM.

PubMed

Farka, Zdeněk; Kovář, David; Skládal, Petr

2014-12-23

Label-free immunosensors are well suited for detection of microorganisms because of their fast response and reasonable sensitivity comparable to infection doses of common pathogens. Active (lever oscillator and frequency counter) and passive (impedance analyzer) modes of quartz crystal microbalance (QCM) were used and compared for rapid detection of three strains of E. coli. Different approaches for antibody immobilization were compared, the immobilization of reduced antibody using Sulfo-SMCC was most effective achieving the limit of detection (LOD) 8 × 104 CFU·mL-1 in 10 min. For the passive mode, software evaluating impedance characteristics in real-time was developed and used. Almost the same results were achieved using both active and passive modes confirming that the sensor properties are not limited by the frequency evaluation method but mainly by affinity of the antibody. Furthermore, reference measurements were done using surface plasmon resonance. Effect of condition of cells on signal was observed showing that cells ruptured by ultrasonication provided slightly higher signal changes than intact microbes.

Rapid and specific detection of Asian- and African-lineage Zika viruses.

PubMed

Chotiwan, Nunya; Brewster, Connie D; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M; Fauver, Joseph R; Andre, Barb; Gray, Meg; Black, William C; Kading, Rebekah C; Ebel, Gregory D; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T A; Brault, Aaron C; Harris, Eva; Foy, Brian D; Quackenbush, Sandra L; Perera, Rushika; Rovnak, Joel

2017-05-03

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. Copyright © 2017, American Association for the Advancement of Science.

Rapid and specific detection of Asian- and African-lineage Zika viruses

PubMed Central

Chotiwan, Nunya; Brewster, Connie D.; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K.; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M.; Fauver, Joseph R.; Andre, Barb; Gray, Meg; Black, William C.; Kading, Rebekah C.; Ebel, Gregory D.; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T. A.; Brault, Aaron C.; Harris, Eva; Foy, Brian D.; Quackenbush, Sandra L.; Perera, Rushika; Rovnak, Joel

2017-01-01

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. PMID:28469032

Performance of Traditional and Molecular Methods for Detecting Biological Agents in Drinking Water

USGS Publications Warehouse

Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Bertke, Erin E.; Kephart, Christopher M.; Likirdopulos, Christina A.; Mailot, Brian E.; Schaefer, Frank W.; Lindquist, H.D. Alan

2009-01-01

To reduce the impact from a possible bioterrorist attack on drinking-water supplies, analytical methods are needed to rapidly detect the presence of biological agents in water. To this end, 13 drinking-water samples were collected at 9 water-treatment plants in Ohio to assess the performance of a molecular method in comparison to traditional analytical methods that take longer to perform. Two 100-liter samples were collected at each site during each sampling event; one was seeded in the laboratory with six biological agents - Bacillus anthracis (B. anthracis), Burkholderia cepacia (as a surrogate for Bu. pseudomallei), Francisella tularensis (F. tularensis), Salmonella Typhi (S. Typhi), Vibrio cholerae (V. cholerae), and Cryptospordium parvum (C. parvum). The seeded and unseeded samples were processed by ultrafiltration and analyzed by use of quantiative polymerase chain reaction (qPCR), a molecular method, and culture methods for bacterial agents or the immunomagnetic separation/fluorescent antibody (IMS/FA) method for C. parvum as traditional methods. Six replicate seeded samples were also processed and analyzed. For traditional methods, recoveries were highly variable between samples and even between some replicate samples, ranging from below detection to greater than 100 percent. Recoveries were significantly related to water pH, specific conductance, and dissolved organic carbon (DOC) for all bacteria combined by culture methods, but none of the water-quality characteristics tested were related to recoveries of C. parvum by IMS/FA. Recoveries were not determined by qPCR because of problems in quantifying organisms by qPCR in the composite seed. Instead, qPCR results were reported as detected, not detected (no qPCR signal), or +/- detected (Cycle Threshold or 'Ct' values were greater than 40). Several sample results by qPCR were omitted from the dataset because of possible problems with qPCR reagents, primers, and probes. For the remaining 14 qPCR results