[Isolation and identification methods of enterobacteria group and its technological advancement].

PubMed

Furuta, Itaru

2007-08-01

In the last half-century, isolation and identification methods of enterobacteria groups have markedly improved by technological advancement. Clinical microbiology tests have changed overtime from tube methods to commercial identification kits and automated identification. Tube methods are the original method for the identification of enterobacteria groups, that is, a basically essential method to recognize bacterial fermentation and biochemical principles. In this paper, traditional tube tests are discussed, such as the utilization of carbohydrates, indole, methyl red, and citrate and urease tests. Commercial identification kits and automated instruments by computer based analysis as current methods are also discussed, and those methods provide rapidity and accuracy. Nonculture techniques of nucleic acid typing methods using PCR analysis, and immunochemical methods using monoclonal antibodies can be further developed.

[Microbiology--laboratory examinations for bacterias].

PubMed

Hen, Renjun; Imafuku, Yuji; Yoshida, Hiroshi

2002-11-01

As it has been required to identify pathogenic microbes in shorter times, simple and rapid methods have been developed and used. Here, we summarized the present situation of rapid diagnostic testing in clinical microbiology in Japan, and also presented our results on PBP2' detection. The rapid test kits available in Japan for E. coli, Helicobacter pylori, Salmonella, Streptococcus and Staphylococcus aureus were described. Rapid examination methods are based mainly on immunologic reactions, which included slide agglutination using latex particle, immunochromatography and ELISA. Times required for the identification are 10 to 15 minutes. Moreover, rapid test kits employing PCR are also marketed. Further, we evaluated MRSA-LA "Seiken" which is a rapid detection kit for PBP2' produced by MRSA. The test was shown to be highly sensitive and specific. For the rapid identification of pathogenic microbes, simple and rapid test kits described here will be used more in clinical diagnosis.

[Special application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbiological diagnostics].

PubMed

Nagy, Erzsébet; Abrók, Marianna; Bartha, Noémi; Bereczki, László; Juhász, Emese; Kardos, Gábor; Kristóf, Katalin; Miszti, Cecilia; Urbán, Edit

2014-09-21

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a new possibility for rapid identification of bacteria and fungi revolutionized the clinical microbiological diagnostics. It has an extreme importance in the routine microbiological laboratories, as identification of the pathogenic species rapidly will influence antibiotic selection before the final determination of antibiotic resistance of the isolate. The classical methods for identification of bacteria or fungi, based on biochemical tests, are influenced by many environmental factors. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a rapid method which is able to identify a great variety of the isolated bacteria and fungi based on the composition of conserved ribosomal proteins. Recently several other applications of the method have also been investigated such as direct identification of pathogens from the positive blood cultures. There are possibilities to identify bacteria from the urine samples in urinary tract infection or from other sterile body fluids. Using selective enrichment broth Salmonella sp from the stool samples can be identified more rapidly, too. The extended spectrum beta-lactamase or carbapenemase production of the isolated bacteria can be also detected by this method helping the antibiotic selection in some cases. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry based methods are suitable to investigate changes in deoxyribonucleic acid or ribonucleic acid, to carry out rapid antibiotic resistance determination or other proteomic analysis. The aim of this paper is to give an overview about present possibilities of using this technique in the clinical microbiological routine procedures.

Comparison among four proposed direct blood culture microbial identification methods using MALDI-TOF MS.

PubMed

Bazzi, Ali M; Rabaan, Ali A; El Edaily, Zeyad; John, Susan; Fawarah, Mahmoud M; Al-Tawfiq, Jaffar A

Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry facilitates rapid and accurate identification of pathogens, which is critical for sepsis patients. In this study, we assessed the accuracy in identification of both Gram-negative and Gram-positive bacteria, except for Streptococcus viridans, using four rapid blood culture methods with Vitek MALDI-TOF-MS. We compared our proposed lysis centrifugation followed by washing and 30% acetic acid treatment method (method 2) with two other lysis centrifugation methods (washing and 30% formic acid treatment (method 1); 100% ethanol treatment (method 3)), and picking colonies from 90 to 180min subculture plates (method 4). Methods 1 and 2 identified all organisms down to species level with 100% accuracy, except for Streptococcus viridans, Streptococcus pyogenes, Enterobacter cloacae and Proteus vulgaris. The latter two were identified to genus level with 100% accuracy. Each method exhibited excellent accuracy and precision in terms of identification to genus level with certain limitations. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

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.

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...

Identification of insecticide residues with a conducting-polymer electronic nose

Treesearch

A.D. Wilson

2014-01-01

The identification of insecticide residues on crop foliage is needed to make periodic pest management decisions. Electronic-nose (e-nose) methods were developed and tested as a means of acquiring rapid identifications of insecticide residue types at relatively low cost by detection of headspace volatiles released from inert surfaces in vitro. Detection methods were...

Use of the BioMerieux ID 32C yeast identification system for identification of aerobic actinomycetes of medical importance.

PubMed Central

Muir, D B; Pritchard, R C

1997-01-01

The BioMerieux ID 32C Yeast Identification System was examined to determine its usefulness as a rapid method for the identification of medically important aerobic actinomycetes. More than 290 strains were tested by this method and the results were compared to those obtained by conventional methods. It was found that aerobic actinomycetes could be differentiated to species level in 7 days by the ID 32C system. PMID:9399526

In situ Identification of Labile Precursor Compounds and their Short-lived Intermediates in Plants using in vivo Nanospray High-resolution Mass Spectrometry.

PubMed

Chang, Qing; Peng, Yue'e; Shi, Bin; Dan, Conghui; Yang, Yijun; Shuai, Qin

2016-05-01

Many secondary metabolites in plants are labile compounds which under environmental stress, are difficult to detect and track due to the lack of rapid in situ identification techniques, making plant metabolomics research difficult. Therefore, developing a reliable analytical method for rapid in situ identification of labile compounds and their short-lived intermediates in plants is of great importance. To develop under atmospheric pressure, a rapid in situ method for effective identification of labile compounds and their short-lived intermediates in fresh plants. An in vivo nanospray high-resolution mass spectrometry (HR-MS) method was used for rapid capture of labile compounds and their short-lived intermediates in plants. A quartz capillary was partially inserted into fresh plant tissues, and the liquid flowed out through the capillary tube owing to the capillary effect. A high direct current (d.c.) voltage was applied to the plant to generate a spray of charged droplets from the tip of the capillary carrying bioactive molecules toward the inlet of mass spectrometer for full-scan and MS/MS analysis. Many labile compounds and short-lived intermediates were identified via this method: including glucosinolates and their short-lived intermediates (existing for only 10 s) in Raphanus sativus roots, alliin and its conversion intermediate (existing for 20 s) in Allium sativum and labile precursor compound chlorogenic acid in Malus pumila Mill. The method is an effective approach for in situ identification of internal labile compounds and their short-lived intermediates in fresh plants and it can be used as an auxiliary tool to explore the degradation mechanisms of new labile plant compounds. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

PubMed

Page, Brent T; Shields, Christine E; Merz, William G; Kurtzman, Cletus P

2006-09-01

This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

Comparison of Nested PCR and RFLP for Identification and Classification of Malassezia Yeasts from Healthy Human Skin

PubMed Central

Oh, Byung Ho; Song, Young Chan; Choe, Yong Beom; Ahn, Kyu Joong

2009-01-01

Background Malassezia yeasts are normal flora of the skin found in 75~98% of healthy subjects. The accurate identification of the Malassezia species is important for determining the pathogenesis of the Malassezia yeasts with regard to various skin diseases such as Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis. Objective This research was conducted to determine a more accurate and rapid molecular test for the identification and classification of Malassezia yeasts. Methods We compared the accuracy and efficacy of restriction fragment length polymorphism (RFLP) and the nested polymerase chain reaction (PCR) for the identification of Malassezia yeasts. Results Although both methods demonstrated rapid and reliable results with regard to identification, the nested PCR method was faster. However, 7 different Malassezia species (1.2%) were identified by the nested PCR compared to the RFLP method. Conclusion Our results show that RFLP method was relatively more accurate and reliable for the detection of various Malassezia species compared to the nested PCR. But, in the aspect of simplicity and time saving, the latter method has its own advantages. In addition, the 26S rDNA, which was targeted in this study, contains highly conserved base sequences and enough sequence variation for inter-species identification of Malassezia yeasts. PMID:20523823

Prosthodontics an “arsenal” in forensic dentistry

PubMed Central

Bathala, Lakshmana Rao; Rachuri, Narendra Kumar; Rayapati, Srinivas Rao; Kondaka, Sudheer

2016-01-01

After major disasters such as earthquakes, fires, floods, tsunami, bomb blasts or terrorist attacks, accurate, and early identification of the dead and injured becomes an utmost importance. Restorations, cariesteeth, missingteeth and/or prostheses are most useful aids for the dental identification. At times, only identifiable remains are a victim's partial or complete dentures. The central principle of dental identification is that postmortem dental remains can be compared with antemortem dental records which include, studycasts, radiographs, etc., to confirm the identity of the victims. Marking/labeling dentures have been considered an important aid in forensic dentistry. Other than finger printing, when compared with all the methods, the marking/labeling of dentures is an accurate and rapid method to identify the unknown victims. There are no standardized methods to follow, but dental practitioners needs to maintain some dental records of their patients. This may include documentation of the “marking of dentures.” The preparedness is the key to success in mass disaster identification. The aim of this review article is to discuss the methods of denture identification, advantages of denture labeling for the rapid identification during major disasters/accidents and the importance of maintaining the patient records. PMID:28123274

Functional recognition imaging using artificial neural networks: applications to rapid cellular identification via broadband electromechanical response

NASA Astrophysics Data System (ADS)

Nikiforov, M. P.; Reukov, V. V.; Thompson, G. L.; Vertegel, A. A.; Guo, S.; Kalinin, S. V.; Jesse, S.

2009-10-01

Functional recognition imaging in scanning probe microscopy (SPM) using artificial neural network identification is demonstrated. This approach utilizes statistical analysis of complex SPM responses at a single spatial location to identify the target behavior, which is reminiscent of associative thinking in the human brain, obviating the need for analytical models. We demonstrate, as an example of recognition imaging, rapid identification of cellular organisms using the difference in electromechanical activity over a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat

PubMed Central

Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

2014-01-01

Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

MALDI-TOF MS Versus VITEK®2: Comparison of Systems for the Identification of Microorganisms Responsible for Bacteremia.

PubMed

Febbraro, Filomena; Rodio, Donatella Maria; Puggioni, Gianluca; Antonelli, Guido; Pietropaolo, Valeria; Trancassini, Maria

2016-12-01

We evaluated the reliability and accuracy of the combined use of MALDI-TOF MS and classical ID VITEK 2 to identify monomicrobial infection in blood culture bottles. In total, 70 consecutive positive blood cultures were included in this study. Positive blood culture bottles were subjected to Gram staining and subcultured on solid media. Isolates grown from such culture media were used for classical ID using VITEK 2 system. In parallel, an aliquot was subjected to a lysing-centrifugation method and used for the identification with the MALDI-TOF system. Results evidenced the correct genus and species identification of 91.4 % of microorganisms responsible for bacteremia with an agreement to the species and the genus level. If compared with the standard method VITEK 2 , our simple and cost-effective sample preparation method would be very useful for rapid identification of microorganisms using blood culture bottles. In fact, the direct method showed rapid and reliable results, especially for the gram-negative group.

Direct identification of pathogens from positive blood cultures using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

PubMed

Rodríguez-Sánchez, B; Sánchez-Carrillo, C; Ruiz, A; Marín, M; Cercenado, E; Rodríguez-Créixems, M; Bouza, E

2014-07-01

In recent years, matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has proved a rapid and reliable method for the identification of bacteria and yeasts that have already been isolated. The objective of this study was to evaluate this technology as a routine method for the identification of microorganisms directly from blood culture bottles (BCBs), before isolation, in a large collection of samples. For this purpose, 1000 positive BCBs containing 1085 microorganisms have been analysed by conventional phenotypic methods and by MALDI-TOF MS. Discrepancies have been resolved using molecular methods: the amplification and sequencing of the 16S rRNA gene or the Superoxide Dismutase gene (sodA) for streptococcal isolates. MALDI-TOF predicted a species- or genus-level identification of 81.4% of the analysed microorganisms. The analysis by episode yielded a complete identification of 814 out of 1000 analysed episodes (81.4%). MALDI-TOF identification is available for clinicians within hours of a working shift, as oppose to 18 h later when conventional identification methods are performed. Moreover, although further improvement of sample preparation for polymicrobial BCBs is required, the identification of more than one pathogen in the same BCB provides a valuable indication of unexpected pathogens when their presence may remain undetected in Gram staining. Implementation of MALDI-TOF identification directly from the BCB provides a rapid and reliable identification of the causal pathogen within hours. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Direct identification of microorganisms from positive blood cultures by MALDI-TOF MS using an in-house saponin method.

PubMed

Yonetani, Shota; Ohnishi, Hiroaki; Ohkusu, Kiyofumi; Matsumoto, Tetsuya; Watanabe, Takashi

2016-11-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria. A MALDI Sepsityper kit is generally used to prepare samples obtained directly from culture bottles. However, the relatively high cost of this kit is a major obstacle to introducing this method into routine clinical use. In this study, the accuracies of three different preparation methods for rapid direct identification of bacteria from positive blood culture bottles by MALDI-TOF MS analysis were compared. In total, 195 positive bottles were included in this study. Overall, 78.5%, 68.7%, and 76.4% of bacteria were correctly identified to the genus level (score ≥1.7) directly from positive blood cultures using the Sepsityper, centrifugation, and saponin methods, respectively. The identification rates using the Sepsityper and saponin methods were significantly higher than that using the centrifugation method (Sepsityper vs. centrifugation, p<0.001; saponin vs. centrifugation, p=0.003). These results suggest that the saponin method is superior to the centrifugation method and comparable to the Sepsityper method in the accuracy of rapid bacterial identification directly from blood culture bottles, and could be a less expensive alternative to the Sepsityper method. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Evaluation of a Semiquantitative Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Method for Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures.

PubMed

Jung, Jette S; Hamacher, Christina; Gross, Birgit; Sparbier, Katrin; Lange, Christoph; Kostrzewa, Markus; Schubert, Sören

2016-11-01

With the increasing prevalence of multidrug-resistant Gram-negative bacteria, rapid identification of the pathogen and its individual antibiotic resistance is crucial to ensure adequate antiinfective treatment at the earliest time point. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for the identification of bacteria directly from the blood culture bottle has been widely established; however, there is still an urgent need for new methods that permit rapid resistance testing. Recently, a semiquantitative MALDI-TOF mass spectrometry-based method for the prediction of antibiotic resistance was described. We evaluated this method for detecting nonsusceptibility against two β-lactam and two non-β-lactam antibiotics. A collection of 30 spiked blood cultures was tested for nonsusceptibility against gentamicin and ciprofloxacin. Furthermore, 99 patient-derived blood cultures were tested for nonsusceptibility against cefotaxime, piperacillin-tazobactam, and ciprofloxacin in parallel with MALDI-TOF mass spectrometry identification from the blood culture fluid. The assay correctly classified all isolates tested for nonsusceptibility against gentamicin and cefotaxime. One misclassification for ciprofloxacin nonsusceptibility and five misclassifications for piperacillin-tazobactam nonsusceptibility occurred. Identification of the bacterium and prediction of nonsusceptibility was possible within approximately 4 h. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?

PubMed

Antonov, Valery A; Tkachenko, Galina A; Altukhova, Viktoriya V; Savchenko, Sergey S; Zinchenko, Olga V; Viktorov, Dmitry V; Zamaraev, Valery S; Ilyukhin, Vladimir I; Alekseev, Vladimir V

2008-12-01

Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains.

Rapid Identification and Classification of Listeria spp. and Serotype Assignment of Listeria monocytogenes Using Fourier Transform-Infrared Spectroscopy and Artificial Neural Network Analysis

PubMed Central

Romanolo, K. F.; Gorski, L.; Wang, S.; Lauzon, C. R.

2015-01-01

The use of Fourier Transform-Infrared Spectroscopy (FT-IR) in conjunction with Artificial Neural Network software NeuroDeveloper™ was examined for the rapid identification and classification of Listeria species and serotyping of Listeria monocytogenes. A spectral library was created for 245 strains of Listeria spp. to give a biochemical fingerprint from which identification of unknown samples were made. This technology was able to accurately distinguish the Listeria species with 99.03% accuracy. Eleven serotypes of Listeria monocytogenes including 1/2a, 1/2b, and 4b were identified with 96.58% accuracy. In addition, motile and non-motile forms of Listeria were used to create a more robust model for identification. FT-IR coupled with NeuroDeveloper™ appear to be a more accurate and economic choice for rapid identification of pathogenic Listeria spp. than current methods. PMID:26600423

Rapid label-free identification of Klebsiella pneumoniae antibiotic resistant strains by the drop-coating deposition surface-enhanced Raman scattering method

NASA Astrophysics Data System (ADS)

Cheong, Youjin; Kim, Young Jin; Kang, Heeyoon; Choi, Samjin; Lee, Hee Joo

2017-08-01

Although many methodologies have been developed to identify unknown bacteria, bacterial identification in clinical microbiology remains a complex and time-consuming procedure. To address this problem, we developed a label-free method for rapidly identifying clinically relevant multilocus sequencing typing-verified quinolone-resistant Klebsiella pneumoniae strains. We also applied the method to identify three strains from colony samples, ATCC70063 (control), ST11 and ST15; these are the prevalent quinolone-resistant K. pneumoniae strains in East Asia. The colonies were identified using a drop-coating deposition surface-enhanced Raman scattering (DCD-SERS) procedure coupled with a multivariate statistical method. Our workflow exhibited an enhancement factor of 11.3 × 106 to Raman intensities, high reproducibility (relative standard deviation of 7.4%), and a sensitive limit of detection (100 pM rhodamine 6G), with a correlation coefficient of 0.98. All quinolone-resistant K. pneumoniae strains showed similar spectral Raman shifts (high correlations) regardless of bacterial type, as well as different Raman vibrational modes compared to Escherichia coli strains. Our proposed DCD-SERS procedure coupled with the multivariate statistics-based identification method achieved excellent performance in discriminating similar microbes from one another and also in subtyping of K. pneumoniae strains. Therefore, our label-free DCD-SERS procedure coupled with the computational decision supporting method is a potentially useful method for the rapid identification of clinically relevant K. pneumoniae strains.

Reducing time to identification of aerobic bacteria and fastidious micro-organisms in positive blood cultures.

PubMed

Intra, J; Sala, M R; Falbo, R; Cappellini, F; Brambilla, P

2016-12-01

Rapid and early identification of micro-organisms in blood has a key role in the diagnosis of a febrile patient, in particular, in guiding the clinician to define the correct antibiotic therapy. This study presents a simple and very fast method with high performances for identifying bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after only 4 h of incubation. We used early bacterial growth on PolyViteX chocolate agar plates inoculated with five drops of blood-broth medium deposited in the same point and spread with a sterile loop, followed by a direct transfer procedure on MALDI-TOF MS target slides without additional modification. Ninety-nine percentage of aerobic bacteria were correctly identified from 600 monomicrobial-positive blood cultures. This procedure allowed obtaining the correct identification of fastidious pathogens, such as Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae that need complex nutritional and environmental requirements in order to grow. Compared to the traditional pathogen identification from blood cultures that takes over 24 h, the reliability of results, rapid performance and suitability of this protocol allowed a more rapid administration of optimal antimicrobial treatment in the patients. Bloodstream infections are serious conditions with a high mortality and morbidity rate. Rapid identification of pathogens and appropriate antimicrobial therapy have a key role for successful patient outcome. In this work, we developed a rapid, simplified, accurate, and efficient method, reaching 99 % identification of aerobic bacteria from monomicrobial-positive blood cultures by using early growth on enriched medium, direct transfer to target plate without additional procedures, matrix-assisted laser desorption ionization-time of flight mass spectrometry and SARAMIS database. The application of this protocol allows to anticipate appropriate antibiotic therapy. © 2016 The Society for Applied Microbiology.

Improved bacterial identification directly from urine samples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Kitagawa, Koichi; Shigemura, Katsumi; Onuma, Ken-Ichiro; Nishida, Masako; Fujiwara, Mayu; Kobayashi, Saori; Yamasaki, Mika; Nakamura, Tatsuya; Yamamichi, Fukashi; Shirakawa, Toshiro; Tokimatsu, Issei; Fujisawa, Masato

2018-03-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) contributes to rapid identification of pathogens in the clinic but has not yet performed especially well for Gram-positive cocci (GPC) causing complicated urinary tract infection (UTI). The goal of this study was to investigate the possible clinical use of MALDI-TOF MS as a rapid method for bacterial identification directly from urine in complicated UTI. MALDI-TOF MS was applied to urine samples gathered from 142 suspected complicated UTI patients in 2015-2017. We modified the standard procedure (Method 1) for sample preparation by adding an initial 10 minutes of ultrasonication followed by centrifugation at 500 g for 1 minutes to remove debris such as epithelial cells and leukocytes from the urine (Method 2). In 133 urine culture-positive bacteria, the rate of corresponded with urine culture in GPC by MALDI-TOF MS in urine with standard sample preparation (Method 1) was 16.7%, but the modified sample preparation (Method 2) significantly improved that rate to 52.2% (P=.045). Method 2 also improved the identification accuracy for Gram-negative rods (GNR) from 77.1% to 94.2% (P=.022). The modified Method 2 significantly improved the average MALDI score from 1.408±0.153 to 2.166±0.045 (P=.000) for GPC and slightly improved the score from 2.107±0.061 to 2.164±0.037 for GNR. The modified sample preparation for MALDI-TOF MS can improve identification accuracy for complicated UTI causative bacteria. This simple modification offers a rapid and accurate routine diagnosis for UTI, and may possibly be a substitute for urine cultures. © 2017 Wiley Periodicals, Inc.

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.

Evaluation of MALDI-TOF MS (Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry) for routine identification of anaerobic bacteria.

PubMed

Rodríguez-Sánchez, Belén; Alcalá, Luis; Marín, Mercedes; Ruiz, Adrián; Alonso, Elena; Bouza, Emilio

2016-12-01

Information regarding the use of MALDI-TOF MS as an alternative to conventional laboratory methods for the rapid and reliable identification of bacterial isolates is still limited. In this study, MALDI-TOF MS was evaluated on 295 anaerobic isolates previously identified by 16S rRNA gene sequencing and with biochemical tests (Rapid ID 32A system, BioMérieux). In total, 85.8% of the isolates were identified by MALDI-TOF MS at the species level vs 49.8% using the Rapid ID 32A system (p < 0.0001). None of the isolates was discordantly identified at the genus level using MALDI-TOF MS and only 9 of them could not be identified using the method. Thus, our results show that MALDI-TOF MS is a robust and reliable tool for the identification of anaerobic isolates in the microbiology laboratory. Its implementation will reduce the turnaround time for a final identification and the number of isolates that require 16S rRNA sequencing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Research of mine water source identification based on LIF technology

NASA Astrophysics Data System (ADS)

Zhou, Mengran; Yan, Pengcheng

2016-09-01

According to the problem that traditional chemical methods to the mine water source identification takes a long time, put forward a method for rapid source identification system of mine water inrush based on the technology of laser induced fluorescence (LIF). Emphatically analyzes the basic principle of LIF technology. The hardware composition of LIF system are analyzed and the related modules were selected. Through the fluorescence experiment with the water samples of coal mine in the LIF system, fluorescence spectra of water samples are got. Traditional water source identification mainly according to the ion concentration representative of the water, but it is hard to analysis the ion concentration of the water from the fluorescence spectra. This paper proposes a simple and practical method of rapid identification of water by fluorescence spectrum, which measure the space distance between unknown water samples and standard samples, and then based on the clustering analysis, the category of the unknown water sample can be get. Water source identification for unknown samples verified the reliability of the LIF system, and solve the problem that the current coal mine can't have a better real-time and online monitoring on water inrush, which is of great significance for coal mine safety in production.

Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

PubMed Central

Pérez-Osorio, Ailyn C.; Boyle, David S.; Ingham, Zachary K.; Ostash, Alla; Gautom, Romesh K.; Colombel, Craig; Houze, Yolanda

2012-01-01

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampinr), isoniazidr, and pyrazinamider TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampinr TB identification; 60.6% and 100% for isoniazidr TB identification; and 75.0% and 98.1% for pyrazinamider TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced. PMID:22162548

Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

PubMed Central

Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

PubMed

Ramkissoon, Kevin R; Miller, Jennifer K; Ojha, Sunil; Watson, Douglas S; Bomar, Martha G; Galande, Amit K; Shearer, Alexander G

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

Application of MALDI-TOF MS for the Identification of Food Borne Bacteria

PubMed Central

Pavlovic, Melanie; Huber, Ingrid; Konrad, Regina; Busch, Ulrich

2013-01-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS based identification of bacteria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality. This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed. PMID:24358065

Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses

USDA-ARS?s Scientific Manuscript database

Several species and hyphal anastomosis groups (AG) of Rhizoctonia solani (sensu lato) cause brown patch diseases of turfgrasses. Conventional methods of identification of Rhizoctonia pathogens are time consuming and often inaccurate. A rapid identification assay for Waitea circinata (anamorph: Rhizo...

Method for genetic identification of unknown organisms

DOEpatents

Colston, Jr., Billy W.; Fitch, Joseph P.; Hindson, Benjamin J.; Carter, Chance J.; Beer, Neil Reginald

2016-08-23

A method of rapid, genome and proteome based identification of unknown pathogenic or non-pathogenic organisms in a complex sample. The entire sample is analyzed by creating millions of emulsion encapsulated microdroplets, each containing a single pathogenic or non-pathogenic organism sized particle and appropriate reagents for amplification. Following amplification, the amplified product is analyzed.

Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

PubMed Central

Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

2014-01-01

Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

Comparison of Vitek Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Versus Conventional Methods in Candida Identification.

PubMed

Keçeli, Sema Aşkın; Dündar, Devrim; Tamer, Gülden Sönmez

2016-02-01

Candida species are generally identified by conventional methods such as germ tube or morphological appearance on corn meal agar, biochemical methods using API kits and molecular biological methods. Alternative to these methods, rapid and accurate identification methods of microorganisms called matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDİ-TOF MS) has recently been described. In this study, Candida identification results by API Candida kit, API 20C AUX kit and identifications on corn meal agar (CMA) are compared with the results obtained on Vitek-MS. All results were confirmed by sequencing internal transcribed spacer (ITS) regions of rDNA. Totally, 97 Candida strains were identified by germ tube test, CMA, API and Vitek-MS. Vitek-MS results were compatible with 74.2 % of API 20C AUX and 81.4 % of CMA results. The difference between the results of API Candida and API 20C AUX was detected. The ratio of discrepancy between Vitek-MS and API 20C AUX was 25.8 %. Candida species mostly identified as C. famata or C. tropicalis by and not compatible with API kits were identified as C. albicans by Vitek-MS. Sixteen Candida species having discrepant results with Vitek-MS, API or CMA were randomly chosen, and ITS sequence analysis was performed. The results of sequencing were compatible 56.2 % with API 20C AUX, 50 % with CMA and 93.7 % with Vitek-MS. When compared with conventional identification methods, MS results are more reliable and rapid for Candida identification. MS system may be used as routine identification method in clinical microbiology laboratories.

Identification of Microorganisms by Modern Analytical Techniques.

PubMed

Buszewski, Bogusław; Rogowska, Agnieszka; Pomastowski, Paweł; Złoch, Michał; Railean-Plugaru, Viorica

2017-11-01

Rapid detection and identification of microorganisms is a challenging and important aspect in a wide range of fields, from medical to industrial, affecting human lives. Unfortunately, classical methods of microorganism identification are based on time-consuming and labor-intensive approaches. Screening techniques require the rapid and cheap grouping of bacterial isolates; however, modern bioanalytics demand comprehensive bacterial studies at a molecular level. Modern approaches for the rapid identification of bacteria use molecular techniques, such as 16S ribosomal RNA gene sequencing based on polymerase chain reaction or electromigration, especially capillary zone electrophoresis and capillary isoelectric focusing. However, there are still several challenges with the analysis of microbial complexes using electromigration technology, such as uncontrolled aggregation and/or adhesion to the capillary surface. Thus, an approach using capillary electrophoresis of microbial aggregates with UV and matrix-assisted laser desorption ionization time-of-flight MS detection is presented.

MALDI-TOF MS Profiling-Advances in Species Identification of Pests, Parasites, and Vectors.

PubMed

Murugaiyan, Jayaseelan; Roesler, Uwe

2017-01-01

Invertebrate pests and parasites of humans, animals, and plants continue to cause serious diseases and remain as a high treat to agricultural productivity and storage. The rapid and accurate species identification of the pests and parasites are needed for understanding epidemiology, monitoring outbreaks, and designing control measures. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as a rapid, cost effective, and high throughput technique of microbial species identification in modern diagnostic laboratories. The development of soft ionization techniques and the release of commercial pattern matching software platforms has resulted in the exponential growth of applications in higher organisms including parasitology. The present review discusses the proof-of-principle experiments and various methods of MALDI MS profiling in rapid species identification of both laboratory and field isolates of pests, parasites and vectors.

MALDI-TOF MS Profiling-Advances in Species Identification of Pests, Parasites, and Vectors

PubMed Central

Murugaiyan, Jayaseelan; Roesler, Uwe

2017-01-01

Invertebrate pests and parasites of humans, animals, and plants continue to cause serious diseases and remain as a high treat to agricultural productivity and storage. The rapid and accurate species identification of the pests and parasites are needed for understanding epidemiology, monitoring outbreaks, and designing control measures. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as a rapid, cost effective, and high throughput technique of microbial species identification in modern diagnostic laboratories. The development of soft ionization techniques and the release of commercial pattern matching software platforms has resulted in the exponential growth of applications in higher organisms including parasitology. The present review discusses the proof-of-principle experiments and various methods of MALDI MS profiling in rapid species identification of both laboratory and field isolates of pests, parasites and vectors. PMID:28555175

Comparison of Real-Time PCR and Conventional Biochemical Methods for Identification of Staphylococcus lugdunensis▿

PubMed Central

Pinsky, Benjamin A.; Samson, Divinia; Ghafghaichi, Laleh; Baron, Ellen J.; Banaei, Niaz

2009-01-01

Staphylococcus lugdunensis is an aggressive, virulent member of the coagulase-negative staphylococci (CoNS) that is responsible for severe, rapidly progressive skin and soft tissue infections and native valve endocarditis. To facilitate prompt identification and appropriate therapy, we describe here a rapid and robust multiplex real-time PCR assay that is able to definitively distinguish S. lugdunensis from other staphylococci. Using melting curve analysis, the assay also identifies Staphylococcus aureus and CoNS other than S. lugdunensis and determines MecA-dependent resistance to methicillin (meticillin). When applied to a panel of well-characterized staphylococcal reference strains, as well as 165 clinical isolates previously identified by conventional methods, the assay was both sensitive and specific for S. lugdunensis, correctly identifying the reference strain and all 47 S. lugdunensis isolates without inappropriate amplification of other staphylococci. Furthermore, rapid biochemical identification using the WEE-TAB system to detect ornithine decarboxylase activity was found to be unsuitable as an alternative to PCR identification, displaying just 31% sensitivity and 77% specificity when tested on a subset (90 isolates) of the clinical strains. We therefore propose that this simple, accurate PCR approach will allow for the routine and timely identification of S. lugdunensis in the clinical microbiology laboratory. PMID:19741081

A Decision Mixture Model-Based Method for Inshore Ship Detection Using High-Resolution Remote Sensing Images

PubMed Central

Bi, Fukun; Chen, Jing; Zhuang, Yin; Bian, Mingming; Zhang, Qingjun

2017-01-01

With the rapid development of optical remote sensing satellites, ship detection and identification based on large-scale remote sensing images has become a significant maritime research topic. Compared with traditional ocean-going vessel detection, inshore ship detection has received increasing attention in harbor dynamic surveillance and maritime management. However, because the harbor environment is complex, gray information and texture features between docked ships and their connected dock regions are indistinguishable, most of the popular detection methods are limited by their calculation efficiency and detection accuracy. In this paper, a novel hierarchical method that combines an efficient candidate scanning strategy and an accurate candidate identification mixture model is presented for inshore ship detection in complex harbor areas. First, in the candidate region extraction phase, an omnidirectional intersected two-dimension scanning (OITDS) strategy is designed to rapidly extract candidate regions from the land-water segmented images. In the candidate region identification phase, a decision mixture model (DMM) is proposed to identify real ships from candidate objects. Specifically, to improve the robustness regarding the diversity of ships, a deformable part model (DPM) was employed to train a key part sub-model and a whole ship sub-model. Furthermore, to improve the identification accuracy, a surrounding correlation context sub-model is built. Finally, to increase the accuracy of candidate region identification, these three sub-models are integrated into the proposed DMM. Experiments were performed on numerous large-scale harbor remote sensing images, and the results showed that the proposed method has high detection accuracy and rapid computational efficiency. PMID:28640236

A Decision Mixture Model-Based Method for Inshore Ship Detection Using High-Resolution Remote Sensing Images.

PubMed

Bi, Fukun; Chen, Jing; Zhuang, Yin; Bian, Mingming; Zhang, Qingjun

2017-06-22

With the rapid development of optical remote sensing satellites, ship detection and identification based on large-scale remote sensing images has become a significant maritime research topic. Compared with traditional ocean-going vessel detection, inshore ship detection has received increasing attention in harbor dynamic surveillance and maritime management. However, because the harbor environment is complex, gray information and texture features between docked ships and their connected dock regions are indistinguishable, most of the popular detection methods are limited by their calculation efficiency and detection accuracy. In this paper, a novel hierarchical method that combines an efficient candidate scanning strategy and an accurate candidate identification mixture model is presented for inshore ship detection in complex harbor areas. First, in the candidate region extraction phase, an omnidirectional intersected two-dimension scanning (OITDS) strategy is designed to rapidly extract candidate regions from the land-water segmented images. In the candidate region identification phase, a decision mixture model (DMM) is proposed to identify real ships from candidate objects. Specifically, to improve the robustness regarding the diversity of ships, a deformable part model (DPM) was employed to train a key part sub-model and a whole ship sub-model. Furthermore, to improve the identification accuracy, a surrounding correlation context sub-model is built. Finally, to increase the accuracy of candidate region identification, these three sub-models are integrated into the proposed DMM. Experiments were performed on numerous large-scale harbor remote sensing images, and the results showed that the proposed method has high detection accuracy and rapid computational efficiency.

Methods for a Rapid Systematic Review and Metaanalysis in Evaluating Selective Serotonin Reuptake Inhibitors for Premature Ejaculation

ERIC Educational Resources Information Center

Martyn-St. James, Marrissa; Cooper, Katy; Kaltenthaler, Eva

2017-01-01

The aim of this study was to evaluate a rapid systematic review method in which randomised controlled trial (RCT) data was extracted from existing reviews and subsequent RCTs. The method enabled: identification of RCTs not included by existing reviews; cross-checking RCT data for consistency where there was more than one review; double data…

Rapid in situ identification of bioactive compounds in plants by in vivo nanospray high-resolution mass spectrometry.

PubMed

Chang, Qing; Peng, Yue'e; Dan, Conghui; Shuai, Qin; Hu, Shenghong

2015-03-25

A method for the rapid in situ identification of bioactive compounds in fresh plants has been developed using in vivo nanospray coupled to high-resolution mass spectrometry (HR-MS). Using a homemade in vivo nanospray ion source, the plant liquid was drawn out from a target region and ionized in situ. The ionized bioactive compounds were then identified using Q-Orbitrap HR-MS. The accurate mass measurements of these bioactive compounds were performed by full-scan or selected ion monitoring (SIM), and tandem mass spectrometry (MS/MS) was used in the structural elucidation. Without sample pretreatment, 12 bioactive compounds in 7 different plant species were identified, namely, isoalliin in onion; butylphthalide in celery; N-methylpelletierine, pelletierine, and pseudopelletierine in pomegranate; chlorogenic acid in crabapple; solamargine, solasonine, and solasodine in nightshade; aloin and aloe-emodin in aloe; and menthone in mint. This work demonstrates that in vivo nanospray HR-MS is a good method for rapid in situ identification of bioactive compounds in plants.

Rapid screening, identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC-ESI-TOF-MS combined with semipreparative HPLC.

PubMed

Zhang, Minmin; Zhao, Hengqiang; Zhao, Zhiguo; Yan, Huijiao; Lv, Ruimin; Cui, Li; Yuan, Jinpeng; Wang, Daijie; Geng, Yanling; Liu, Daicheng; Wang, Xiao

2016-06-01

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti-influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti-influenza components from Zicao. Semipreparative high-performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high-performance liquid chromatography with the purity over 98% for all of them by high-performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β-dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti-influenza active ingredients from complex Chinese herbal medicines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid Identification of Legionella Pathogenicity by Surface-Enhanced Raman Spectroscopy.

PubMed

Li, Jing; Qin, Tian; Jia, Xiao Xiao; Deng, Ai Hua; Zhang, Xu; Fan, Wen Hui; Huo, Shuai Dong; Wen, Ting Yi; Liu, Wen Jun

2015-06-01

To establish Surface-enhanced Raman Spectroscopy (SERS) can be used as a rapid and reliable method to distinguish virulent strain and mild strain of L. pneumophila. Mortality data were collected from company departments through administrative documents, death certificates, etc. Trend analyses of cancer mortality were performed on the basis of 925 cancer deaths between 2001 and 2010. Our results indicated that the peaks of high virulence strains reached ⋝4000. This criterion was verified by subsequent cell experiments. In addition, we also conducted SERS rapid identification on the virulence of several collected clinical strains and obtained accurate results. The present study indicates that the established SERS protocol can be used as a rapid and reliable method to distinguish virulent and mildly virulent strains of L. pneumophila, which can be further used in clinical samples. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Comparison of Three Commercial Systems for Identification of Yeasts Commonly Isolated in the Clinical Microbiology Laboratory

PubMed Central

Wadlin, Jill K.; Hanko, Gayle; Stewart, Rebecca; Pape, John; Nachamkin, Irving

1999-01-01

We evaluated three commercial systems (RapID Yeast Plus System; Innovative Diagnostic Systems, Norcross, Ga.; API 20C Aux; bioMerieux-Vitek, Hazelwood, Mo.; and Vitek Yeast Biochemical Card, bioMerieux-Vitek) against an auxinographic and microscopic morphologic reference method for the ability to identify yeasts commonly isolated in our clinical microbiology laboratory. Two-hundred one yeast isolates were compared in the study. The RapID Yeast Plus System was significantly better than either API 20C Aux (193 versus 167 correct identifications; P < 0.0001) or the Vitek Yeast Biochemical Card (193 versus 173 correct identifications; P = 0.003) for obtaining correct identifications to the species level without additional testing. There was no significant difference between results obtained with API 20C Aux and the Vitek Yeast Biochemical Card system (P = 0.39). The API 20C Aux system did not correctly identify any of the Candida krusei isolates (n = 23) without supplemental testing and accounted for the major differences between the API 20C Aux and RapID Yeast Plus systems. Overall, the RapID Yeast Plus System was easy to use and is a good system for the routine identification of clinically relevant yeasts. PMID:10325356

Identification and determination of the saikosaponins in Radix bupleuri by accelerated solvent extraction combined with rapid-resolution LC-MS.

PubMed

Yang, Yun-Yun; Tang, You-Zhi; Fan, Chun-Lin; Luo, Hui-Tai; Guo, Peng-Ran; Chen, Jian-Xin

2010-07-01

A method based on accelerated solvent extraction combined with rapid-resolution LC-MS for efficient extraction, rapid separation, online identification and accurate determination of the saikosaponins (SSs) in Radix bupleuri (RB) was developed. The RB samples were extracted by accelerated solvent extraction using 70% aqueous ethanol v/v as solvent, at a temperature of 120 degrees C and pressure of 100 bar, with 10 min of static extraction time and three extraction cycles. Rapid-resolution LC separation was performed by using a C(18) column at gradient elution of water (containing 0.5% formic acid) and acetonitrile, and the major constituents were well separated within 20 min. A TOF-MS and an IT-MS were used for online identification of the major constituents, and 27 SSs were identified or tentatively identified. Five major bioactive SSs (SSa, SSc, SSd, 6''-O-acetyl-SSa and 6''-O-acetyl-SSd) with obvious peak areas and good resolution were chosen as benchmark substances, and a triple quadrupole MS operating in multiple-reaction monitoring mode was used for their quantitative analysis. A total of 16 RB samples from different regions of China were analyzed. The results indicated that the method was rapid, efficient, accurate and suitable for use in the quality control of RB.

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

PubMed Central

Lagier, Jean-Christophe; Hugon, Perrine; Khelaifia, Saber; Fournier, Pierre-Edouard; La Scola, Bernard

2015-01-01

SUMMARY Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers. Metagenomics studies have since been applied to clinical microbiology; however, a “dark matter” of prokaryotes, which corresponds to a hole in our knowledge and includes minority bacterial populations, is not elucidated by these studies. By replicating the natural environment, environmental microbiologists were the first to reduce the “great plate count anomaly,” which corresponds to the difference between microscopic and culture counts. The revolution in bacterial identification also allowed rapid progress. 16S rRNA bacterial identification allowed the accurate identification of new species. Mass spectrometry allowed the high-throughput identification of rare species and the detection of new species. By using these methods and by increasing the number of culture conditions, culturomics allowed the extension of the known human gut repertoire to levels equivalent to those of pyrosequencing. Finally, taxonogenomics strategies became an emerging method for describing new species, associating the genome sequence of the bacteria systematically. We provide a comprehensive review on these topics, demonstrating that both empirical and hypothesis-driven approaches will enable a rapid increase in the identification of the human prokaryote repertoire. PMID:25567229

Rapid identification of oral Actinomyces species cultivated from subgingival biofilm by MALDI-TOF-MS

PubMed Central

Stingu, Catalina S.; Borgmann, Toralf; Rodloff, Arne C.; Vielkind, Paul; Jentsch, Holger; Schellenberger, Wolfgang; Eschrich, Klaus

2015-01-01

Background Actinomyces are a common part of the residential flora of the human intestinal tract, genitourinary system and skin. Isolation and identification of Actinomyces by conventional methods is often difficult and time consuming. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a rapid and simple method to identify bacteria. Objective The present study evaluated a new in-house algorithm using MALDI-TOF-MS for rapid identification of different species of oral Actinomyces cultivated from subgingival biofilm. Design Eleven reference strains and 674 clinical strains were used in this study. All the strains were preliminarily identified using biochemical methods and then subjected to MALDI-TOF-MS analysis using both similarity-based analysis and classification methods (support vector machine [SVM]). The genotype of the reference strains and of 232 clinical strains was identified by sequence analysis of the 16S ribosomal RNA (rRNA). Results The sequence analysis of the 16S rRNA gene of all references strains confirmed their previous identification. The MALDI-TOF-MS spectra obtained from the reference strains and the other clinical strains undoubtedly identified as Actinomyces by 16S rRNA sequencing were used to create the mass spectra reference database. Already a visual inspection of the mass spectra of different species reveals both similarities and differences. However, the differences between them are not large enough to allow a reliable differentiation by similarity analysis. Therefore, classification methods were applied as an alternative approach for differentiation and identification of Actinomyces at the species level. A cross-validation of the reference database representing 14 Actinomyces species yielded correct results for all species which were represented by more than two strains in the database. Conclusions Our results suggest that a combination of MALDI-TOF-MS with powerful classification algorithms, such as SVMs, provide a useful tool for the differentiation and identification of oral Actinomyces. PMID:25597306

Identification of Bodies by Unique Serial Numbers on Implanted Medical Devices.

PubMed

Blessing, Melissa M; Lin, Peter T

2018-05-01

Visual identification is the most common identification method used by medical examiners but is not always possible. Alternative methods include X-ray, fingerprint, or DNA comparison, but these methods require additional resources. Comparison of serial numbers on implanted medical devices is a rapid and definitive method of identification. To assess the practicality of using this method, we reviewed 608 consecutive forensic autopsies performed at a regional medical examiner office. Of these, 56 cases required an alternative method of identification due to decomposition (n = 35), gunshot wound (n = 9), blunt trauma (n = 6), or charring (n = 6). Of these 56 cases, eight (14.3%) were known to have an implanted medical device. Of these eight cases, five (63%) could be positively identified by comparing serial numbers. If an implanted medical device is known to be present, and medical records are available, identification by medical device serial number should be a first-line method. © 2017 American Academy of Forensic Sciences.

Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

PubMed Central

Nakanishi, Tsuyoshi; Ando, Eiji; Furuta, Masaru; Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru; Tsunasawa, Susumu; Nishimura, Osamu

2007-01-01

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3. PMID:18166671

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry (MALDI-TOF MS) Based Microbial Identifications: Challenges and Scopes for Microbial Ecologists

PubMed Central

Rahi, Praveen; Prakash, Om; Shouche, Yogesh S.

2016-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based biotyping is an emerging technique for high-throughput and rapid microbial identification. Due to its relatively higher accuracy, comprehensive database of clinically important microorganisms and low-cost compared to other microbial identification methods, MALDI-TOF MS has started replacing existing practices prevalent in clinical diagnosis. However, applicability of MALDI-TOF MS in the area of microbial ecology research is still limited mainly due to the lack of data on non-clinical microorganisms. Intense research activities on cultivation of microbial diversity by conventional as well as by innovative and high-throughput methods has substantially increased the number of microbial species known today. This important area of research is in urgent need of rapid and reliable method(s) for characterization and de-replication of microorganisms from various ecosystems. MALDI-TOF MS based characterization, in our opinion, appears to be the most suitable technique for such studies. Reliability of MALDI-TOF MS based identification method depends mainly on accuracy and width of reference databases, which need continuous expansion and improvement. In this review, we propose a common strategy to generate MALDI-TOF MS spectral database and advocated its sharing, and also discuss the role of MALDI-TOF MS based high-throughput microbial identification in microbial ecology studies. PMID:27625644

Determination of new retention indices for quick identification of essential oils compounds.

PubMed

Hérent, Marie-France; De Bie, Véronique; Tilquin, Bernard

2007-02-19

The classical methods of chromatographic identification of compounds were based on calculation of retention indices by using different stationary phases. The aim of the work was to differentiate essential oils extracted from different plant species by identification of some of their major compounds. The method of identification was based on the calculation of new retention indices of essential oils compounds fractionated on a polar chromatographic column with temperature programming system. Similar chromatograms have been obtained on the same column for one plant family with two different temperature gradients allowing the rapid identification of essential oils of different species, sub-species or chemotypes of Citrus, Mentha and Thymus.

Nuclear Magnetic Resonance Spectroscopy-Based Identification of Yeast.

PubMed

Himmelreich, Uwe; Sorrell, Tania C; Daniel, Heide-Marie

2017-01-01

Rapid and robust high-throughput identification of environmental, industrial, or clinical yeast isolates is important whenever relatively large numbers of samples need to be processed in a cost-efficient way. Nuclear magnetic resonance (NMR) spectroscopy generates complex data based on metabolite profiles, chemical composition and possibly on medium consumption, which can not only be used for the assessment of metabolic pathways but also for accurate identification of yeast down to the subspecies level. Initial results on NMR based yeast identification where comparable with conventional and DNA-based identification. Potential advantages of NMR spectroscopy in mycological laboratories include not only accurate identification but also the potential of automated sample delivery, automated analysis using computer-based methods, rapid turnaround time, high throughput, and low running costs.We describe here the sample preparation, data acquisition and analysis for NMR-based yeast identification. In addition, a roadmap for the development of classification strategies is given that will result in the acquisition of a database and analysis algorithms for yeast identification in different environments.

Use of Enzyme Tests in Characterization and Identification of Aerobic and Facultatively Anaerobic Gram-Positive Cocci

PubMed Central

Bascomb, Shoshana; Manafi, Mammad

1998-01-01

The contribution of enzyme tests to the accurate and rapid routine identification of gram-positive cocci is introduced. The current taxonomy of the genera of aerobic and facultatively anaerobic cocci based on genotypic and phenotypic characterization is reviewed. The clinical and economic importance of members of these taxa is briefly summarized. Tables summarizing test schemes and kits available for the identification of staphylococci, enterococci, and streptococci on the basis of general requirements, number of tests, number of taxa, test classes, and completion times are discussed. Enzyme tests included in each scheme are compared on the basis of their synthetic moiety. The current understanding of the activity of enzymes important for classification and identification of the major groups, methods of testing, and relevance to the ease and speed of identification are reviewed. Publications describing the use of different identification kits are listed, and overall identification successes and problems are discussed. The relationships between the results of conventional biochemical and rapid enzyme tests are described and considered. The use of synthetic substrates for the detection of glycosidases and peptidases is reviewed, and the advantages of fluorogenic synthetic moieties are discussed. The relevance of enzyme tests to accurate and meaningful rapid routine identification is discussed. PMID:9564566

Potential Impact of Rapid Blood Culture Testing for Gram-Positive Bacteremia in Japan with the Verigene Gram-Positive Blood Culture Test

PubMed Central

Matsuda, Mari; Iguchi, Shigekazu; Mizutani, Tomonori; Hiramatsu, Keiichi; Tega-Ishii, Michiru; Sansaka, Kaori; Negishi, Kenta; Shimada, Kimie; Umemura, Jun; Notake, Shigeyuki; Yanagisawa, Hideji; Yabusaki, Reiko; Araoka, Hideki; Yoneyama, Akiko

2017-01-01

Background. Early detection of Gram-positive bacteremia and timely appropriate antimicrobial therapy are required for decreasing patient mortality. The purpose of our study was to evaluate the performance of the Verigene Gram-positive blood culture assay (BC-GP) in two special healthcare settings and determine the potential impact of rapid blood culture testing for Gram-positive bacteremia within the Japanese healthcare delivery system. Furthermore, the study included simulated blood cultures, which included a library of well-characterized methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) isolates reflecting different geographical regions in Japan. Methods. A total 347 BC-GP assays were performed on clinical and simulated blood cultures. BC-GP results were compared to results obtained by reference methods for genus/species identification and detection of resistance genes using molecular and MALDI-TOF MS methodologies. Results. For identification and detection of resistance genes at two clinical sites and simulated blood cultures, overall concordance of BC-GP with reference methods was 327/347 (94%). The time for identification and antimicrobial resistance detection by BC-GP was significantly shorter compared to routine testing especially at the cardiology hospital, which does not offer clinical microbiology services on weekends and holidays. Conclusion. BC-GP generated accurate identification and detection of resistance markers compared with routine laboratory methods for Gram-positive organisms in specialized clinical settings providing more rapid results than current routine testing. PMID:28316631

Bioassay selection, experimental design and quality control/assurance for use in effluent assessment and control.

PubMed

Johnson, Ian; Hutchings, Matt; Benstead, Rachel; Thain, John; Whitehouse, Paul

2004-07-01

In the UK Direct Toxicity Assessment Programme, carried out in 1998-2000, a series of internationally recognised short-term toxicity test methods for algae, invertebrates and fishes, and rapid methods (ECLOX and Microtox) were used extensively. Abbreviated versions of conventional tests (algal growth inhibition tests, Daphnia magna immobilisation test and the oyster embryo-larval development test) were valuable for toxicity screening of effluent discharges and the identification of causes and sources of toxicity. Rapid methods based on chemiluminescence and bioluminescence were not generally useful in this programme, but may have a role where the rapid test has been shown to be an acceptable surrogate for a standardised test method. A range of quality assurance and control measures were identified. Requirements for quality control/assurance are most stringent when deriving data for characterising the toxic hazards of effluents and monitoring compliance against a toxicity reduction target. Lower quality control/assurance requirements can be applied to discharge screening and the identification of causes and sources of toxicity.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of fungal rhinosinusitis pathogens.

PubMed

Huang, Yanfei; Wang, Jinglin; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Lu, Xinxin

2017-03-01

Filamentous fungi are among the most important pathogens, causing fungal rhinosinusitis (FRS). Current laboratory diagnosis of FRS pathogens mainly relies on phenotypic identification by culture and microscopic examination, which is time consuming and expertise dependent. Although matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has been employed to identify various fungi, its efficacy in the identification of FRS fungi is less clear. A total of 153 FRS isolates obtained from patients were analysed at the Clinical Laboratory at the Beijing Tongren Hospital affiliated to the Capital Medical University, between January 2014 and December 2015. They were identified by traditional phenotypic methods and Bruker MALDI-TOF MS (Bruker, Biotyper version 3.1), respectively. Discrepancies between the two methods were further validated by sequencing. Among the 153 isolates, 151 had correct species identification using MALDI-TOF MS (Bruker, Biot 3.1, score ≥2.0 or 2.3). MALDI-TOF MS enabled identification of some very closely related species that were indistinguishable by conventional phenotypic methods, including 1/10 Aspergillus versicolor, 3/20 Aspergillus flavus, 2/30 Aspergillus fumigatus and 1/20 Aspergillus terreus, which were misidentified by conventional phenotypic methods as Aspergillus nidulans, Aspergillus oryzae, Aspergillus japonicus and Aspergillus nidulans, respectively. In addition, 2/2 Rhizopus oryzae and 1/1 Rhizopus stolonifer that were identified only to the genus level by the phenotypic method were correctly identified by MALDI-TOF MS. MALDI-TOF MS is a rapid and accurate technique, and could replace the conventional phenotypic method for routine identification of FRS fungi in clinical microbiology laboratories.

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.

Rapid and easy identification of Illicium verum Hook. f. and its adulterant Illicium anisatum Linn. by fluorescent microscopy and gas chromatography.

PubMed

Joshi, Vaishali C; Srinivas, Pullela V; Khan, Ikhlas A

2005-01-01

Illicium verum Hook. f. is used as an herbal tea to treat colic pain in infants. Reports suggest that Star anise herbal tea may be adulterated with Illicium anisatum Linn. A short and rapid method using microscopy and gas chromatography (GC) was developed to detect I. anisatum Linn., an adulterant in the powdered mixture of I. verum. Anatomical differences in the epicarp cells of I. verum and I. anisatum fruits were clearly defined as examined under fluorescent microscopy and scanning electron microscopy. A GC method was developed for quick identification of possible I. anisatum adulteration with I. verum.

Rapid detection of bacterial pathogens using flourescence spectroscopy and chemometrics

USDA-ARS?s Scientific Manuscript database

This work presents the development of a method for rapid bacterial identification based on the fluorescence spectroscopy combined with multivariate analysis. Fluorescence spectra of pure three different genera of bacteria (Escherichia coli, Salmonella, and Campylobacter) were collected from 200...

Accurate, safe, and rapid method of intraoperative tumor identification for totally laparoscopic distal gastrectomy: injection of mixed fluid of sodium hyaluronate and patent blue.

PubMed

Nakagawa, Masatoshi; Ehara, Kazuhisa; Ueno, Masaki; Tanaka, Tsuyoshi; Kaida, Sachiko; Udagawa, Harushi

2014-04-01

In totally laparoscopic distal gastrectomy, determining the resection line with safe proximal margins is often difficult, particularly for tumors located in a relatively upper area. This is because, in contrast to open surgery, identifying lesions by palpating or opening the stomach is essentially impossible. This study introduces a useful method of tumor identification that is accurate, safe, and rapid. On the operation day, after inducing general anesthesia, a mixture of sodium hyaluronate and patent blue is injected into the submucosal layer of the proximal margin. When resecting stomach, all marker spots should be on the resected side. In all cases, the proximal margin is examined histologically by using frozen sections during the operation. From October 2009 to September 2011, a prospective study that evaluated this method was performed. A total of 34 patients who underwent totally laparoscopic distal gastrectomy were enrolled in this study. Approximately 5 min was required to complete the procedure. Proximal margins were negative in all cases, and the mean ± standard deviation length of the proximal margin was 23.5 ± 12.8 mm. No side effects, such as allergy, were encountered. As a method of tumor identification for totally laparoscopic distal gastrectomy, this procedure appears accurate, safe, and rapid.

Direct identification of bacteria from positive BacT/ALERT blood culture bottles using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

PubMed

Mestas, Javier; Felsenstein, Susanna; Bard, Jennifer Dien

2014-11-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and robust method for the identification of bacteria. In this study, we evaluate the performance of a laboratory-developed lysis method (LDT) for the rapid identification of bacteria from positive BacT/ALERT blood culture bottles. Of the 168 positive bottles tested, 159 were monomicrobial, the majority of which were Gram-positive organisms (61.0% versus 39.0%). Using a cut-off score of ≥1.7, 80.4% of the organisms were correctly identified to the species level, and the identification rate of Gram-negative organisms (90.3%) was found to be significantly greater than that of Gram-positive organisms (78.4%). The simplicity and cost-effectiveness of the LDT enable it to be fully integrated into the routine workflow of the clinical microbiology laboratory, allowing for rapid identification of Gram-positive and Gram-negative bacteria within an hour of blood culture positivity. Copyright © 2014 Elsevier Inc. All rights reserved.

Rapid microbiochemical identification of Corynebacterium diphtheriae and other medically important corynebacteria.

PubMed Central

Thompson, J S; Gates-Davis, D R; Yong, D C

1983-01-01

A rapid biochemical method based on the fermentation of carbohydrates, the hydrolysis of urea, and the reduction of nitrate was used to identify Corynebacterium diphtheriae, C. ulcerans, C. pseudodiphtheriticum, C. haemolyticum, C. pseudotuberculosis, C. pyogenes, C. ovis, the Centers for Disease Control JK group, and Rhodococcus (Corynebacterium) equi. With this procedure identification was confirmed for 133 stock cultures and clinical isolates of corynebacteria. Most were identified within 1 h and all were identified within 4 h after inoculation into the test substrates. PMID:6355166

The use of newly developed real-time PCR for the rapid identification of bacteria in culture-negative osteomyelitis.

PubMed

Kobayashi, Naomi; Bauer, Thomas W; Sakai, Hiroshige; Togawa, Daisuke; Lieberman, Isador H; Fujishiro, Takaaki; Procop, Gary W

2006-12-01

We report a case of a culture-negative osteomyelitis in which our newly developed real-time polymerase chain reaction (PCR) could differentiate Staphylococcus aureus from Staphylococcus epidermidis. This is the first report that described the application of this novel assay to an orthopedics clinical sample. This assay may be useful for other clinical culture-negative cases in a combination with a broad-spectrum assay as a rapid microorganism identification method.

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Panda, A; Kurapati, S; Samantaray, J C; Myneedu, V P; Verma, A; Srinivasan, A; Ahmad, H; Behera, D; Singh, U B

2013-01-01

The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

A brief review of machine vision in the context of automated wood identification systems

Treesearch

John C. Hermanson; Alex C. Wiedenhoeft

2011-01-01

The need for accurate and rapid field identification of wood to combat illegal logging around the world is outpacing the ability to train personnel to perform this task. Despite increased interest in non-anatomical (DNA, spectroscopic, chemical) methods for wood identification, anatomical characteristics are the least labile data that can be extracted from solid wood...

Fourier-Transform Infrared Microspectroscopy, a Novel and Rapid Tool for Identification of Yeasts

PubMed Central

Wenning, Mareike; Seiler, Herbert; Scherer, Siegfried

2002-01-01

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 μm in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level. PMID:12324312

A NOVEL TECHNIQUE FOR THE RAPID IDENTIFICATION OF ALPHA EMITTERS RELEASED DURING A RADIOLOGICAL INCIDENT.

EPA Science Inventory

Currently there are no standard radioanalytical methods applicable to the initial phase of a radiological emergency, for the early identification and quantification of alpha emitting radionuclides. Of particular interest are determinations of the presence and concentration of is...

Rapid Identification of Candida Species by Using Nuclear Magnetic Resonance Spectroscopy and a Statistical Classification Strategy

PubMed Central

Himmelreich, Uwe; Somorjai, Ray L.; Dolenko, Brion; Lee, Ok Cha; Daniel, Heide-Marie; Murray, Ronan; Mountford, Carolyn E.; Sorrell, Tania C.

2003-01-01

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates of C. parapsilosis and C. glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms. PMID:12902244

Rapid molecular identification and characteristics of Lactobacillus strains.

PubMed

Markiewicz, L H; Biedrzycka, E; Wasilewska, E; Bielecka, M

2010-09-01

Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S-23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)(5) primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2-3 d (in comparison to the standard methods).

A new rapid method for direct antimicrobial susceptibility testing of bacteria from positive blood cultures.

PubMed

Barnini, Simona; Brucculeri, Veronica; Morici, Paola; Ghelardi, Emilia; Florio, Walter; Lupetti, Antonella

2016-08-12

Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections can lead to prompt appropriate antimicrobial therapy. To shorten species identification, in this study bacteria were recovered from monomicrobial blood cultures by serum separator tubes and spotted onto the target plate for direct MALDI-TOF MS identification. Proper antibiotics were selected for direct AST based on species identification. In order to obtain rapid AST results, bacteria were recovered from positive blood cultures by two different protocols: by serum separator tubes (further referred to as PR1), or after a short-term subculture in liquid medium (further referred to as PR2). The results were compared with those obtained by the method currently used in our laboratory consisting in identification by MALDI-TOF and AST by Vitek 2 or Sensititre on isolated colonies. The direct MALDI-TOF method concordantly identified with the current method 97.5 % of the Gram-negative bacteria and 96.1 % of the Gram-positive cocci contained in monomicrobial blood cultures. The direct AST by PR1 and PR2 for all isolate/antimicrobial agent combinations was concordant/correct with the current method for 87.8 and 90.5 % of Gram-negative bacteria and for 93.1 and 93.8 % of Gram-positive cocci, respectively. In particular, 100 % categorical agreement was found with levofloxacin for Enterobacteriaceae by both PR1 and PR2, and 99.0 and 100 % categorical agreement was observed with linezolid for Gram-positive cocci by PR1 and PR2, respectively. There was no significant difference in accuracy between PR1 and PR2 for Gram-negative bacteria and Gram-positive cocci. This newly described method seems promising for providing accurate AST results. Most importantly, these results would be available in a few hours from blood culture positivity, which would help clinicians to promptly confirm or streamline an effective antibiotic therapy in patients with bloodstream infections.

Rapid Identification of Microorganisms from Positive Blood Culture by MALDI-TOF MS After Short-Term Incubation on Solid Medium.

PubMed

Curtoni, Antonio; Cipriani, Raffaella; Marra, Elisa Simona; Barbui, Anna Maria; Cavallo, Rossana; Costa, Cristina

2017-01-01

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a useful tool for rapid identification of microorganisms. Unfortunately, its direct application to positive blood culture is still lacking standardized procedures. In this study, we evaluated an easy- and rapid-to-perform protocol for MALDI-TOF MS direct identification of microorganisms from positive blood culture after a short-term incubation on solid medium. This protocol was used to evaluate direct identification of microorganisms from 162 positive monomicrobial blood cultures; at different incubation times (3, 5, 24 h), MALDI-TOF MS assay was performed from the growing microorganism patina. Overall, MALDI-TOF MS concordance with conventional methods at species level was 60.5, 80.2, and 93.8% at 3, 5, and 24 h, respectively. Considering only bacteria, the identification performances at species level were 64.1, 85.0, and 94.1% at 3, 5, and 24 h, respectively. This protocol applied to a commercially available MS typing system may represent, a fast and powerful diagnostic tool for pathogen direct identification and for a promptly and pathogen-driven antimicrobial therapy in selected cases.

A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: application to a field survey.

PubMed

Nkouawa, Agathe; Sako, Yasuhito; Li, Tiaoying; Chen, Xingwang; Nakao, Minoru; Yanagida, Tetsuya; Okamoto, Munehiro; Giraudoux, Patrick; Raoul, Francis; Nakaya, Kazuhiro; Xiao, Ning; Qiu, Jiamin; Qiu, Dongchuan; Craig, Philip S; Ito, Akira

2012-12-01

In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Rapid identification of Lonicerae japonicae Flos and Lonicerae Flos by Fourier transform infrared (FT-IR) spectroscopy and two-dimensional correlation analysis

NASA Astrophysics Data System (ADS)

Yan, Rui; Chen, Jian-bo; Sun, Su-qin; Guo, Bao-lin

2016-11-01

Lonicerae japonicae Flos (LJF) and Lonicerae Flos (LF) are widely-used herbs derived from several plants of the genus Lonicera with similar appearances. LF are usually misused or counterfeited as LJF for economically motivated adulteration. However, the saponins in LF may cause serious side-effects. In this research, the infrared spectroscopic tri-step identification approach is used to develop a simple and rapid method to discriminate LJF and LF to ensure the safety and efficacy of these herbal drugs. In the primary identification by Fourier transform infrared spectra, LJF and LF show different peaks near 1534, 1404, and 781 cm-1. In the secondary identification by the second derivative infrared spectra, LJF and LF show more different peaks near 1078, 1050, 988, 923, 855, 815, and 781 cm-1. In the tertiary identification by the two-dimensional correlation infrared spectra, the differences between LJF and LF are shown more remarkably and convincingly. The results show the potential of the infrared spectroscopic tri-step identification approach in the rapid identification of LJF and LF when the samples are too few to build a statistical recognition rule. This should be very helpful to ensure the quality, safety, and efficacy of LJF and LF for clinical applications.

Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry System for Identification of Clinically Relevant Filamentous Fungi.

PubMed

McMullen, Allison R; Wallace, Meghan A; Pincus, David H; Wilkey, Kathy; Burnham, C A

2016-08-01

Invasive fungal infections have a high rate of morbidity and mortality, and accurate identification is necessary to guide appropriate antifungal therapy. With the increasing incidence of invasive disease attributed to filamentous fungi, rapid and accurate species-level identification of these pathogens is necessary. Traditional methods for identification of filamentous fungi can be slow and may lack resolution. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and accurate method for identification of bacteria and yeasts, but a paucity of data exists on the performance characteristics of this method for identification of filamentous fungi. The objective of our study was to evaluate the accuracy of the Vitek MS for mold identification. A total of 319 mold isolates representing 43 genera recovered from clinical specimens were evaluated. Of these isolates, 213 (66.8%) were correctly identified using the Vitek MS Knowledge Base, version 3.0 database. When a modified SARAMIS (Spectral Archive and Microbial Identification System) database was used to augment the version 3.0 Knowledge Base, 245 (76.8%) isolates were correctly identified. Unidentified isolates were subcultured for repeat testing; 71/319 (22.3%) remained unidentified. Of the unidentified isolates, 69 were not in the database. Only 3 (0.9%) isolates were misidentified by MALDI-TOF MS (including Aspergillus amoenus [n = 2] and Aspergillus calidoustus [n = 1]) although 10 (3.1%) of the original phenotypic identifications were not correct. In addition, this methodology was able to accurately identify 133/144 (93.6%) Aspergillus sp. isolates to the species level. MALDI-TOF MS has the potential to expedite mold identification, and misidentifications are rare. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

How to: identify non-tuberculous Mycobacterium species using MALDI-TOF mass spectrometry.

PubMed

Alcaide, F; Amlerová, J; Bou, G; Ceyssens, P J; Coll, P; Corcoran, D; Fangous, M-S; González-Álvarez, I; Gorton, R; Greub, G; Hery-Arnaud, G; Hrábak, J; Ingebretsen, A; Lucey, B; Marekoviċ, I; Mediavilla-Gradolph, C; Monté, M R; O'Connor, J; O'Mahony, J; Opota, O; O'Reilly, B; Orth-Höller, D; Oviaño, M; Palacios, J J; Palop, B; Pranada, A B; Quiroga, L; Rodríguez-Temporal, D; Ruiz-Serrano, M J; Tudó, G; Van den Bossche, A; van Ingen, J; Rodriguez-Sanchez, B

2018-06-01

The implementation of MALDI-TOF MS for microorganism identification has changed the routine of the microbiology laboratories as we knew it. Most microorganisms can now be reliably identified within minutes using this inexpensive, user-friendly methodology. However, its application in the identification of mycobacteria isolates has been hampered by the structure of their cell wall. Improvements in the sample processing method and in the available database have proved key factors for the rapid and reliable identification of non-tuberculous mycobacteria isolates using MALDI-TOF MS. The main objective is to provide information about the proceedings for the identification of non-tuberculous isolates using MALDI-TOF MS and to review different sample processing methods, available databases, and the interpretation of the results. Results from relevant studies on the use of the available MALDI-TOF MS instruments, the implementation of innovative sample processing methods, or the implementation of improved databases are discussed. Insight about the methodology required for reliable identification of non-tuberculous mycobacteria and its implementation in the microbiology laboratory routine is provided. Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Rapid diagnostic tests apply for pediatric infections at outpatient clinic setting.

PubMed

Ushijima, Hiroshi; Thongprachum, Aksara; Tran, Dinh Nguyen; Fujimoto, Tsuguto; Hanaoka, Nozomu; Okitsu, Shoko; Takanashi, Sayaka; Mizuguchi, Masashi; Hayakawa, Satoshi

2015-01-01

Early identification of the etiology of infection is beneficial. Most infections are treated as outpatients. However, facilities for rapid diagnosis are not available in clinic settings. We applied Immunochromatography (IC) and Loop-mediated Isothermal Amplification (LAMP) methods to rapidly diagnose pathogens among 31 children with respiratory infection and 12 with gastroenteritis at a clinic in Saitama prefecture, Japan. Pathogens were then screened by multiplex conventional and real-time PCRs and bacterial culture. Respiratory pathogens were found in 64.5%. Despite the narrow spectrum, rapid tests identified pathogens in 28.6% of cases with a high agreement rate of 89.3% with PCR. Gastroenteritis pathogens were found in 66.7%. E. coli was positive in 3 cases and all were negative for verotoxin by LAMP. The agreement rate of IC and PCR assay was high, 100%. IC and LAMP are reliable and suitable methods in limited-resource settings for early pathogenic identification, which will help appropriate management, avoid unnecessary intervention, and cost saving.

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

PubMed

Irinyi, Laszlo; Serena, Carolina; Garcia-Hermoso, Dea; Arabatzis, Michael; Desnos-Ollivier, Marie; Vu, Duong; Cardinali, Gianluigi; Arthur, Ian; Normand, Anne-Cécile; Giraldo, Alejandra; da Cunha, Keith Cassia; Sandoval-Denis, Marcelo; Hendrickx, Marijke; Nishikaku, Angela Satie; de Azevedo Melo, Analy Salles; Merseguel, Karina Bellinghausen; Khan, Aziza; Parente Rocha, Juliana Alves; Sampaio, Paula; da Silva Briones, Marcelo Ribeiro; e Ferreira, Renata Carmona; de Medeiros Muniz, Mauro; Castañón-Olivares, Laura Rosio; Estrada-Barcenas, Daniel; Cassagne, Carole; Mary, Charles; Duan, Shu Yao; Kong, Fanrong; Sun, Annie Ying; Zeng, Xianyu; Zhao, Zuotao; Gantois, Nausicaa; Botterel, Françoise; Robbertse, Barbara; Schoch, Conrad; Gams, Walter; Ellis, David; Halliday, Catriona; Chen, Sharon; Sorrell, Tania C; Piarroux, Renaud; Colombo, Arnaldo L; Pais, Célia; de Hoog, Sybren; Zancopé-Oliveira, Rosely Maria; Taylor, Maria Lucia; Toriello, Conchita; de Almeida Soares, Célia Maria; Delhaes, Laurence; Stubbe, Dirk; Dromer, Françoise; Ranque, Stéphane; Guarro, Josep; Cano-Lira, Jose F; Robert, Vincent; Velegraki, Aristea; Meyer, Wieland

2015-05-01

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification of Sildenafil (Viagra) and Its Metabolite (UK 103,320) in Six Aviation Fatalities

DTIC Science & Technology

2006-02-01

Identification of Sildenafil ( Viagra ®) and Its Metabolite (UK-103,320) in Six Aviation Fatalities Robert D. Johnson Russell J. Lewis Civil...DOT/FAA/AM-06/3 4. Title and Subtitle 5. Report Date February 2006 Identification of Sildenafil ( Viagra ®) and Its Metabolite (UK-103,320...report presents a rapid and reliable method for the identification and quantitation of sildenafil ( Viagra ®) and its active metabolite, UK-103,320. This

Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria.

PubMed

Mishra, Prashant K; Fox, Roland T V; Culham, Alastair

2003-01-28

Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis.

Identification of gender in yellow perch Perca flavescens using external morphology

USDA-ARS?s Scientific Manuscript database

A non-lethal and rapid method for reliable identification of gender in yellow perch has been developed. On average, yellow perch females grow faster than males and undergo sexual maturity at an earlier age. Such size discrepancies in mixed culture situations pose difficulties with aquaculture produc...

A near-infrared spectroscopy routine for unambiguous identification of cryptic ant species

USDA-ARS?s Scientific Manuscript database

The identification of species – of importance for most biological disciplines – is not always straightforward as cryptic species present a hurdle for traditional species discrimination. Fibre-optic near-infrared spectroscopy (NIRS) is a rapid and cheap method for a wide range of different applicatio...

Convenient Detection of the Citrus Greening (Huanglongbing) Bacterium ‘Candidatus Liberibacter asiaticus’ by Direct PCR from the Midrib Extract

PubMed Central

Fujikawa, Takashi; Miyata, Shin-Ichi; Iwanami, Toru

2013-01-01

A phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing. PMID:23437295

Rapid identification of Prototheca species by the API 20C system.

PubMed Central

Padhye, A A; Baker, J G; D'Amato, R F

1979-01-01

The conventional auxanographic method of testing for the assimilation of carbohydrates and alcohols by the various species of Prototheca requires at least 2 weeks of incubation at 25 to 30 degrees C before definitive results are obtained. Even though Prototheca spp., in culture as well as in fixed tissues, can be identified more rapidly by fluorescent-antibody techniques in which species-specific reagents are used, such diagnostic facilities and reagents are not available in most diagnostic laboratories. The API 20C clinical yeast identification system, a commercially available ready-to-use micromethod, was found to permit the definitive identification of P. stagnora, P. wickerhamii, and P. zopfii within 4 days. Images PMID:393722

Using DNA chips for identification of tephritid pest species.

PubMed

Chen, Yen-Hou; Liu, Lu-Yan; Tsai, Wei-Huang; Haymer, David S; Lu, Kuang-Hui

2014-08-01

The ability correctly to identify species in a rapid and reliable manner is critical in many situations. For insects in particular, the primary tools for such identification rely on adult-stage morphological characters. For a number of reasons, however, there is a clear need for alternatives. This paper reports on the development of a new method employing DNA biochip technology for the identification of pest species within the family Tephritidae. The DNA biochip developed and tested here quickly and efficiently identifies and discriminates between several tephritid species, except for some that are members of a complex of closely related taxa and that may in fact not represent distinct biological species. The use of these chips offers a number of potential advantages over current methods. Results can be obtained in less than 5 h using material from any stage of the life cycle and with greater sensitivity than other methods currently available. This technology provides a novel tool for the rapid and reliable identification of several major pest species that may be intercepted in imported fruits or other commodities. The existing chips can also easily be expanded to incorporate additional markers and species as needed. © 2013 Society of Chemical Industry.

Mass spectrometry applied to the identification of Mycobacterium tuberculosis and biomarker discovery.

PubMed

López-Hernández, Y; Patiño-Rodríguez, O; García-Orta, S T; Pinos-Rodríguez, J M

2016-12-01

An adequate and effective tuberculosis (TB) diagnosis system has been identified by the World Health Organization as a priority in the fight against this disease. Over the years, several methods have been developed to identify the bacillus, but bacterial culture remains one of the most affordable methods for most countries. For rapid and accurate identification, however, it is more feasible to implement molecular techniques, taking advantage of the availability of public databases containing protein sequences. Mass spectrometry (MS) has become an interesting technique for the identification of TB. Here, we review some of the most widely employed methods for identifying Mycobacterium tuberculosis and present an update on MS applied for the identification of mycobacterial species. © 2016 The Society for Applied Microbiology.

hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis in comparison to three other methods for identification of Mycobacterium species.

PubMed

Sajduda, Anna; Martin, Anandi; Portaels, Françoise; Palomino, Juan Carlos

2010-02-01

We developed a scheme for rapid identification of Mycobacterium species using an automated fluorescence capillary electrophoresis instrument. A 441-bp region of the hsp65 gene was examined using PCR-restriction analysis (PRA). The assay was initially evaluated on 38 reference strains. The observed sizes of restriction fragments were consistently smaller than the real sizes for each of the species as deduced from the sequence analysis (mean variance=7bp). Nevertheless, the obtained PRA patterns were highly reproducible and resulted in correct species identifications. A blind test was then successfully performed on 64 test isolates previously characterized by conventional biochemical methods, a commercial INNO-LiPA Mycobacteria assay and/or sequence determination of the 5' end of 16S rRNA gene. A total of 14 of 64 isolates were erroneously identified by conventional methods (78% accuracy). In contrast, PRA performed very well in comparison with the LiPA (89% concordance) and especially with DNA sequencing (93.3% of concordant results). Also, PRA identified seven isolates representing five previously unreported hsp65 alleles. We conclude that hsp65 PRA based on automated capillary electrophoresis is a rapid, simple and reliable method for identification of mycobacteria. Copyright 2010 Elsevier B.V. All rights reserved.

Rapid Identification of Pathogens from Positive Blood Cultures by Multiplex PCR using the FilmArray System

PubMed Central

Blaschke, Anne J.; Heyrend, Caroline; Byington, Carrie L.; Fisher, Mark A.; Barker, Elizabeth; Garrone, Nicholas F.; Thatcher, Stephanie A.; Pavia, Andrew T.; Barney, Trenda; Alger, Garrison D.; Daly, Judy A.; Ririe, Kirk M.; Ota, Irene; Poritz, Mark A.

2012-01-01

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Inc., Salt Lake City, UT) Blood Culture (BC) panel can identify > 25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 hour. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 of 92 pathogens (91%) covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven MRSA and VRE. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture. PMID:22999332

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

Microfluidic-Based Bacteria Isolation from Whole Blood for Diagnostics of Blood Stream Infection.

PubMed

Zelenin, Sergey; Ramachandraiah, Harisha; Faridi, Asim; Russom, Aman

2017-01-01

Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24-72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods. Rapid detection of microbial pathogens in blood related to bloodstream infections will allow the clinician to decide on or adjust the antimicrobial therapy potentially reducing the morbidity, mortality, and economic burden associated with BSI. For molecular-based methods, there is a lot to gain from an improved and straightforward method for isolation of bacteria from whole blood for downstream processing.We describe a microfluidic-based sample-preparation approach that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.

Rapid identification of group JK and other corynebacteria with the Minitek system.

PubMed Central

Slifkin, M; Gil, G M; Engwall, C

1986-01-01

Forty primary clinical isolates and 50 stock cultures of corynebacteria and coryneform bacteria were tested with the Minitek system (BBL Microbiology Systems, Cockeysville, Md.). The Minitek correctly identified all of these organisms, including JK group isolates, within 12 to 18 h of incubation. The method does not require serum supplements for testing carbohydrate utilization by the bacteria. The Minitek system is an extremely simple and rapid way to identify the JK group, as well as many other corynebacteria, by established identification schemata for these bacteria. PMID:3091632

Use of immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex from liquid culture

PubMed Central

Považan, Anika; Vukelić, Anka; Savković, Tijana; Kurucin, Tatjana

2012-01-01

A new, simple immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in liquid cultures has been developed. The principle of the assay is binding of the Mycobacterium tuberculosis complex specific antigen to the monoclonal antibody conjugated on the test strip. The aim of this study is evaluation of the performance of immunochromatographic assay in identification of Mycobacterium tuberculosis complex in primary positive liquid cultures of BacT/Alert automated system. A total of 159 primary positive liquid cultures were tested using the immunochromatographic assay (BD MGIT TBc ID) and the conventional subculture, followed by identification using biochemical tests. Of 159 positive liquid cultures, using the conventional method, Mycobacterium tuberculos is was identified in 119 (74.8%), nontuberculous mycobacteria were found in 4 (2.5%), 14 (8.8%) cultures were contaminated and 22 (13.8%) cultures were found to be negative. Using the immunochromatographic assay, Mycobacterium tuberculosis complex was detected in 118 (74.2%) liquid cultures, and 41 (25.8%) tests were negative. Sensitivity, specificity, positive and negative predictive values of the test were 98.3%; 97.5%; 99.15%; 95.12%, respectively. The value of kappa test was 0.950, and McNemar test was 1.00. The immunochromatographic assay is a simple and rapid test which represents a suitable alternative to the conventional subculture method for the primary identification of Mycobacterium tuberculosis complex in liquid cultures of BacT/Alert automated system. PMID:22364301

Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

PubMed Central

2010-01-01

Background Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. Results When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. Conclusion These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates. PMID:21073689

Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

PubMed

Ayyadurai, Saravanan; Flaudrops, Christophe; Raoult, Didier; Drancourt, Michel

2010-11-12

Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates.

[Studies on macroscopic and microscopic identification of Cordyceps sinensis and its counterfeits].

PubMed

Chan, Siutsau; Liu, Baoling; Zhao, Zhongzhen; Lam, Markin; Law, Kwokwai; Chen, Hubiao

2011-05-01

To provide a rapid, simple, accurate and reproducible identification method from which Cordyceps sinensis can be distinguished from other species. To observe the larva and stroma of Cordyceps family with macroscopic identification method, and with powder microscopic identification method. For macroscopic, only stroma of C. sinensis is mostly non-inflated, and un-obtuse at the tip, the caterpillar annulations of C. sinensis and the C. gracilis is distinct, about 20-30, and feet of above two are 8 pairs, 4 of 8 pairs are relatively distinct. The above appearance shows its unique characteristic. For microscopic identification, only C. sinensis exists microtrichia, the tip is pointed. The arranging order of stubby setae is irregular, the tip is blunt while the basal is gradually broader; the top of some setae bends slightly like a hook.

Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method.

PubMed

Ferreira, L; Sánchez-Juanes, F; Muñoz-Bellido, J L; González-Buitrago, J M

2011-07-01

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

CoBOP: Electro-Optic Identification Laser Line Sean Sensors

DTIC Science & Technology

1998-01-01

Electro - Optic Identification Sensors Project[1] is to develop and demonstrate high resolution underwater electro - optic (EO) imaging sensors, and associated image processing/analysis methods, for rapid visual identification of mines and mine-like contacts (MLCs). Identification of MLCs is a pressing Fleet need. During MCM operations, sonar contacts are classified as mine-like if they are sufficiently similar to signatures of mines. Each contact classified as mine-like must be identified as a mine or not a mine. During MCM operations in littoral areas,

A rapid identification of four medicinal chrysanthemum varieties with near infrared spectroscopy.

PubMed

Han, Bangxing; Yan, Hui; Chen, Cunwu; Yao, Houjun; Dai, Jun; Chen, Naifu

2014-07-01

For genuine medicinal material in Chinese herbs; the efficient, rapid, and precise identification is the focus and difficulty in the filed studying Chinese herbal medicines. Chrysanthemum morifolium as herbs has a long planting history in China, culturing high quality ones and different varieties. Different chrysanthemum varieties differ in quality, chemical composition, functions, and application. Therefore, chrysanthemum varieties in the market demands precise identification to provide reference for reasonable and correct application as genuine medicinal material. A total of 244 batches of chrysanthemum samples were randomly divided into calibration set (160 batches) and prediction set (84 batches). The near infrared diffuses reflectance spectra of chrysanthemum varieties were preprocessed by first order derivative (D1) and autoscaling and was built model with partial least squares (PLS). In this study of four chrysanthemum varieties identification, the accuracy rates in calibration sets of Boju, Chuju, Hangju, and Gongju are respectively 100, 100, 98.65, and 96.67%; while the accuracy rates in prediction sets are 100% except for 99.1% of Hangju. The research results demonstrate that the qualitative analysis can be conducted by machine learning combined with near infrared spectroscopy (NIR), which provides a new method for rapid and noninvasive identification of chrysanthemum varieties.

A manual and an automatic TERS based virus discrimination

NASA Astrophysics Data System (ADS)

Olschewski, Konstanze; Kämmer, Evelyn; Stöckel, Stephan; Bocklitz, Thomas; Deckert-Gaudig, Tanja; Zell, Roland; Cialla-May, Dana; Weber, Karina; Deckert, Volker; Popp, Jürgen

2015-02-01

Rapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses was enabled. In a further step, these methods were utilised to perform an automatic quality rating of the measured spectra. Spectra that passed this test were eventually used to calculate a classification model, through which a successful discrimination of the two viral species based on TERS spectra of single virus particles was also realised with a classification accuracy of 91%.Rapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses was enabled. In a further step, these methods were utilised to perform an automatic quality rating of the measured spectra. Spectra that passed this test were eventually used to calculate a classification model, through which a successful discrimination of the two viral species based on TERS spectra of single virus particles was also realised with a classification accuracy of 91%. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07033j

Comparative evaluation of matrix-assisted laser desorption ionisation-time of flight mass spectrometry and conventional phenotypic-based methods for identification of clinically important yeasts in a UK-based medical microbiology laboratory.

PubMed

Fatania, Nita; Fraser, Mark; Savage, Mike; Hart, Jason; Abdolrasouli, Alireza

2015-12-01

Performance of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was compared in a side-by side-analysis with conventional phenotypic methods currently in use in our laboratory for identification of yeasts in a routine diagnostic setting. A diverse collection of 200 clinically important yeasts (19 species, five genera) were identified by both methods using standard protocols. Discordant or unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene. MALDI-TOF and conventional methods were in agreement for 182 isolates (91%) with correct identification to species level. Eighteen discordant results (9%) were due to rarely encountered species, hence the difficulty in their identification using traditional phenotypic methods. MALDI-TOF MS enabled rapid, reliable and accurate identification of clinically important yeasts in a routine diagnostic microbiology laboratory. Isolates with rare, unusual or low probability identifications should be confirmed using robust molecular methods. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Rapid method for sampling metals for materials identification

NASA Technical Reports Server (NTRS)

Higgins, L. E.

1971-01-01

Nondamaging process similar to electrochemical machining is useful in obtaining metal samples from places inaccessible to conventional sampling methods or where methods would be hazardous or contaminating to specimens. Process applies to industries where metals or metal alloys play a vital role.

Distribution of different yeasts isolates among trauma patients and comparison of accuracy in identification of yeasts by automated method versus conventional methods for better use in low resource countries.

PubMed

Rajkumari, N; Mathur, P; Xess, I; Misra, M C

2014-01-01

As most trauma patients require long-term hospital stay and long-term antibiotic therapy, the risk of fungal infections in such patients is steadily increasing. Early diagnosis and rapid treatment is life saving in such critically ill trauma patients. To see the distribution of various species of Candida among trauma patients and compare the accuracy, rapid identification and cost effectiveness between VITEK 2, CHROMagar and conventional methods. Retrospective laboratory-based surveillance study performed over a period of 52 months (January 2009 to April 2013) at a level I trauma centre in New Delhi, India. All microbiological samples positive for Candida were processed for microbial identification using standard methods. Identification of Candida was done using chromogenic medium and by automated VITEK 2 Compact system and later confirmed using the conventional method. Time to identification in both was noted and accuracy compared with conventional method. Performed using the SPSS software for Windows (SPSS Inc. Chicago, IL, version 15.0). P values calculated using χ2 test for categorical variables. A P<0.05 was considered significant. Out of 445 yeasts isolates, Candida tropicalis (217, 49%) was the species that was maximally isolated. VITEK 2 was able to correctly identify 354 (79.5%) isolates but could not identify 48 (10.7%) isolates and wrongly identified or showed low discrimination in 43 (9.6%) isolates but CHROM agar correctly identified 381 (85.6%) isolates with 64 (14.4%) misidentification. Highest rate of misidentification was seen in C. tropicalis and C. glabrata (13, 27.1% each) by VITEK 2 and among C. albicans (9, 14%) by CHROMagar. Though CHROMagar gives identification at a lower cost compared with VITEK 2 and are more accurate, which is useful in low resource countries, its main drawback is the long duration taken for complete identification.

Microlensing observations rapid search for exoplanets: MORSE code for GPUs

NASA Astrophysics Data System (ADS)

McDougall, Alistair; Albrow, Michael D.

2016-02-01

The rapid analysis of ongoing gravitational microlensing events has been integral to the successful detection and characterization of cool planets orbiting low-mass stars in the Galaxy. In this paper, we present an implementation of search and fit techniques on graphical processing unit (GPU) hardware. The method allows for the rapid identification of candidate planetary microlensing events and their subsequent follow-up for detailed characterization.

Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting.

PubMed

Christner, Martin; Rohde, Holger; Wolters, Manuel; Sobottka, Ingo; Wegscheider, Karl; Aepfelbacher, Martin

2010-05-01

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

Development of a novel hexa-plex PCR method for identification and serotyping of Salmonella species.

PubMed

Li, Ruichao; Wang, Yang; Shen, Jianzhong; Wu, Congming

2014-01-01

Salmonella is one of the most important foodborne pathogens, which causes a huge economic burden worldwide. To detect Salmonella rapidly is very meaningful in preventing salmonellosis and decreasing economic losses. Currently, isolation of Salmonella is confirmed by biochemical and serobased serotyping methods, which are time consuming, labor intensive, and complicated. To solve this problem, a hexa-plex polymerase chain reaction (PCR) method was developed using comparative genomics analysis and multiplex PCR technology to detect Salmonella and Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Agona, Salmonella Choleraesuis, and Salmonella Pullorum simultaneously. The accuracy of this method was tested by a collection of 142 Salmonella. Furthermore, the strategy described in this article to mine serovar-specific fragments for Salmonella could be used to find specific fragments for other Salmonella serotypes and bacteria. The combination of this strategy and multiplex PCR is promising in the rapid identification of foodborne pathogens.

A simple, rapid and inexpensive screening method for the identification of Pythium insidiosum.

PubMed

Tondolo, Juliana Simoni Moraes; Loreto, Erico Silva; Denardi, Laura Bedin; Mario, Débora Alves Nunes; Alves, Sydney Hartz; Santurio, Janio Morais

2013-04-01

Growth of Pythium insidiosum mycelia around minocycline disks (30μg) did not occur within 7days of incubation at 35°C when the isolates were grown on Sabouraud, corn meal, Muller-Hinton or RPMI agar. This technique offers a simple and rapid method for the differentiation of P. insidiosum from true filamentous fungi. Copyright © 2013 Elsevier B.V. All rights reserved.

Same-Day Identification and Antimicrobial Susceptibility Testing of Bacteria in Positive Blood Culture Broths Using Short-Term Incubation on Solid Medium with the MicroFlex LT, Vitek-MS, and Vitek2 Systems

PubMed Central

Ha, Jihye; Han, Geum Hee; Kim, Myungsook; Lee, Kyungwon

2018-01-01

Background Early and appropriate antibiotic treatment improves the clinical outcome of patients with septicemia; therefore, reducing the turn-around time for identification (ID) and antimicrobial susceptibility test (AST) results is essential. We established a method for rapid ID and AST using short-term incubation of positive blood culture broth samples on solid media, and evaluated its performance relative to that of the conventional method using two rapid ID systems and a rapid AST method. Methods A total of 254 mono-microbial samples were included. Positive blood culture samples were incubated on blood agar plates for six hours and identified by the MicroFlex LT (Bruker Daltonics) and Vitek-MS (bioMeriéux) systems, followed by AST using the Vitek2 System (bioMeriéux). Results The correct species-level ID rates were 82.3% (209/254) and 78.3% (199/254) for the MicroFlex LT and Vitek-MS platforms, respectively. For the 1,174 microorganism/antimicrobial agent combinations tested, the rapid AST method showed total concordance of 97.8% (1,148/1,174) with the conventional method, with a very major error rate of 0.5%, major error rate of 0.7%, and minor error rate of 1.0%. Conclusions Routine implementation of this short-term incubation method could provide ID results on the day of blood culture-positivity detection and one day earlier than the conventional AST method. This simple method will be very useful for rapid ID and AST of bacteria from positive blood culture bottles in routine clinical practice. PMID:29401558

Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

DTIC Science & Technology

1982-06-18

areas !i). Presently, the only certain method of identification is through the use of rigidly standardized reference antiserum in a virus plaque...low passaged or unpassaged dengue virus from humans or insects using an indirect immunofluorescence 71 test. MATERIALS AND METHODS :, j Cell cultures...streptomycin. Maintanance medium for infected cell cultures consisted of the appropriate growth medium containing 0.4% bovine plasma albumin instead of FBS

Rapid Detection & Identification of Bacillus Species using MALDI-TOF/TOF and Biomarker Database

DTIC Science & Technology

2006-06-01

rRNA sequence analysis. Multilocus enzyme electrophoresis ( MEE ) and comparative DNA sequence analysis suggest that they may represent a single species...adaptation of the MEE method [63] but with greater discrimination [64]. All of these new PCR-based subtyping methods are certainly superior and more...Demirev, P.A., Lin, J.S., Pineda , F.J., and Fenselau, C. (2001). Bioinformatics and mass spectrometry for microorganism identification: proteome-wide

Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology.

PubMed

Clark, Andrew E; Kaleta, Erin J; Arora, Amit; Wolk, Donna M

2013-07-01

Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the "nuts and bolts" of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

PubMed Central

Clark, Andrew E.; Kaleta, Erin J.; Arora, Amit

2013-01-01

SUMMARY Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the “nuts and bolts” of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care. PMID:23824373

[Rapid identification of 22 abused drugs and organophosphorus pesticides in blood by LC-MS/MS].

PubMed

Liu, Hong-tao; Ma, An-de

2009-08-01

To develop a method for rapid identification of 22 abused drugs and organophosphorus pesticides in the blood. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple-reaction monitoring mode (MRM) was employed for detecting the drugs and pesticides in the blood. The MRM database and criteria for identification were established, and ethyl acetate was used for extraction of the drugs. After 3 rounds of extractions of the blood sample (1 mL) using 2 mL ethyl acetate, the extract was vortexed for 3 min and centrifuged at 5000 r/min. Each organic phase was combined and evaporated by gentle N2 gas. The residue was re-dissolved in 100 L mobile phase, from which 5 L was taken for LC-MS/MS detection. The detection of the 22 target compounds could be completed within 10 min. The limit of detection of the target compound ranged from 0.03 to 6.00 ng/ml. Satisfactory results were obtained in proficiency testing program organized by China National Accreditation Service for Conformity Assessment. The method we established is rapid, selective and sensitive for detecting the 22 abused drugs and organophosphorus pesticides.

Surface-enhanced Raman scattering spectroscopy characterization and identification of foodborne bacteria

NASA Astrophysics Data System (ADS)

Liu, Yongliang; Chen, Yud-Ren; Nou, Xiangwu; Chao, Kaunglin

2007-09-01

Rapid and routine identification of foodborne bacteria are considerably important, because of bio- / agro- terrorism threats, public health concerns, and economic loss. Conventional, PCR, and immunoassay methods for the detection of bacteria are generally time-consuming, chemical reagent necessary and multi-step procedures. Fast microbial detection requires minimal sample preparation, permits the routine analysis of large numbers of samples with negligible reagent costs, and is easy to operate. Therefore, we have developed silver colloidal nanoparticle based surface-enhanced Raman scattering (SERS) spectroscopy as a potential tool for the rapid and routine detection of E. coli and L. monocytogenes. This study presents the further results of our examination on S. typhimonium, one of the most commonly outbreak bacteria, for the characteristic bands and subsequent identification.

Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

PubMed

Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

2015-04-01

Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Cost Savings Realized by Implementation of Routine Microbiological Identification by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Alby, Kevin; Kerr, Alan; Jones, Melissa; Gilligan, Peter H.

2015-01-01

Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is an emerging technology for rapid identification of bacterial and fungal isolates. In comparison to conventional methods, this technology is much less labor intensive and can provide accurate and reliable results in minutes from a single isolated colony. We compared the cost of performing the bioMérieux Vitek MALDI-TOF MS with conventional microbiological methods to determine the amount saved by the laboratory by converting to the new technology. Identification costs for 21,930 isolates collected between April 1, 2013, and March 31, 2014, were directly compared for MALDI-TOF MS and conventional methodologies. These isolates were composed of commonly isolated organisms, including commonly encountered aerobic and facultative bacteria and yeast but excluding anaerobes and filamentous fungi. Mycobacterium tuberculosis complex and rapidly growing mycobacteria were also evaluated for a 5-month period during the study. Reagent costs and a total cost analysis that included technologist time in addition to reagent expenses and maintenance service agreement costs were analyzed as part of this study. The use of MALDI-TOF MS equated to a net savings of $69,108.61, or 87.8%, in reagent costs annually compared to traditional methods. When total costs are calculated to include technologist time and maintenance costs, traditional identification would have cost $142,532.69, versus $68,886.51 with the MALDI-TOF MS method, resulting in a laboratory savings of $73,646.18, or 51.7%, annually by adopting the new technology. The initial cost of the instrument at our usage level would be offset in about 3 years. MALDI-TOF MS not only represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates, it also provides a significant cost savings for the laboratory. PMID:25994167

Cost Savings Realized by Implementation of Routine Microbiological Identification by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

PubMed

Tran, Anthony; Alby, Kevin; Kerr, Alan; Jones, Melissa; Gilligan, Peter H

2015-08-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is an emerging technology for rapid identification of bacterial and fungal isolates. In comparison to conventional methods, this technology is much less labor intensive and can provide accurate and reliable results in minutes from a single isolated colony. We compared the cost of performing the bioMérieux Vitek MALDI-TOF MS with conventional microbiological methods to determine the amount saved by the laboratory by converting to the new technology. Identification costs for 21,930 isolates collected between April 1, 2013, and March 31, 2014, were directly compared for MALDI-TOF MS and conventional methodologies. These isolates were composed of commonly isolated organisms, including commonly encountered aerobic and facultative bacteria and yeast but excluding anaerobes and filamentous fungi. Mycobacterium tuberculosis complex and rapidly growing mycobacteria were also evaluated for a 5-month period during the study. Reagent costs and a total cost analysis that included technologist time in addition to reagent expenses and maintenance service agreement costs were analyzed as part of this study. The use of MALDI-TOF MS equated to a net savings of $69,108.61, or 87.8%, in reagent costs annually compared to traditional methods. When total costs are calculated to include technologist time and maintenance costs, traditional identification would have cost $142,532.69, versus $68,886.51 with the MALDI-TOF MS method, resulting in a laboratory savings of $73,646.18, or 51.7%, annually by adopting the new technology. The initial cost of the instrument at our usage level would be offset in about 3 years. MALDI-TOF MS not only represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates, it also provides a significant cost savings for the laboratory. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Direct identification of bacteria causing urinary tract infections by combining matrix-assisted laser desorption ionization-time of flight mass spectrometry with UF-1000i urine flow cytometry.

PubMed

Wang, X-H; Zhang, G; Fan, Y-Y; Yang, X; Sui, W-J; Lu, X-X

2013-03-01

Rapid identification of bacterial pathogens from clinical specimens is essential to establish an adequate empirical antibiotic therapy to treat urinary tract infections (UTIs). We used matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) combined with UF-1000i urine flow cytometry of urine specimens to quickly and accurately identify bacteria causing UTIs. We divided each urine sample into three aliquots for conventional identification, UF-1000i, and MALDI-TOF MS, respectively. We compared the results of the conventional method with those of MALDI-TOF MS combined with UF-1000i, and discrepancies were resolved by 16S rRNA gene sequencing. We analyzed 1456 urine samples from patients with UTI symptoms, and 932 (64.0%) were negative using each of the three testing methods. The combined method used UF-1000i to eliminate negative specimens and then MALDI-TOF MS to identify the remaining positive samples. The combined method was consistent with the conventional method in 1373 of 1456 cases (94.3%), and gave the correct result in 1381 of 1456 cases (94.8%). Therefore, the combined method described here can directly provide a rapid, accurate, definitive bacterial identification for the vast majority of urine samples, though the MALDI-TOF MS software analysis capabilities should be improved, with regard to mixed bacterial infection. Copyright © 2012 Elsevier B.V. All rights reserved.

On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system.

PubMed

Furutani, Shunsuke; Hagihara, Yoshihisa; Nagai, Hidenori

2017-09-01

Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

DEVELOPMENT OF CODOMINANT MARKERS FOR IDENTIFYING SPECIES HYBRIDS

EPA Science Inventory

Herein we describe a simple method for developing species-diagnostic markers that would permit the rapid identification of hybrid individuals. Our method relies on amplified length polymorphism (AFLP) and single strand conformation polymorphism (SSCP) technologies, both of which...

Same-Day Identification and Antimicrobial Susceptibility Testing of Bacteria in Positive Blood Culture Broths Using Short-Term Incubation on Solid Medium with the MicroFlex LT, Vitek-MS, and Vitek2 Systems.

PubMed

Ha, Jihye; Hong, Sung Kuk; Han, Geum Hee; Kim, Myungsook; Yong, Dongeun; Lee, Kyungwon

2018-05-01

Early and appropriate antibiotic treatment improves the clinical outcome of patients with septicemia; therefore, reducing the turn-around time for identification (ID) and antimicrobial susceptibility test (AST) results is essential. We established a method for rapid ID and AST using short-term incubation of positive blood culture broth samples on solid media, and evaluated its performance relative to that of the conventional method using two rapid ID systems and a rapid AST method. A total of 254 mono-microbial samples were included. Positive blood culture samples were incubated on blood agar plates for six hours and identified by the MicroFlex LT (Bruker Daltonics) and Vitek-MS (bioMeriéux) systems, followed by AST using the Vitek2 System (bioMeriéux). The correct species-level ID rates were 82.3% (209/254) and 78.3% (199/254) for the MicroFlex LT and Vitek-MS platforms, respectively. For the 1,174 microorganism/antimicrobial agent combinations tested, the rapid AST method showed total concordance of 97.8% (1,148/1,174) with the conventional method, with a very major error rate of 0.5%, major error rate of 0.7%, and minor error rate of 1.0%. Routine implementation of this short-term incubation method could provide ID results on the day of blood culture-positivity detection and one day earlier than the conventional AST method. This simple method will be very useful for rapid ID and AST of bacteria from positive blood culture bottles in routine clinical practice. © The Korean Society for Laboratory Medicine

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

PubMed Central

Li, Yiyan; Yang, Xing; Zhao, Weian

2018-01-01

Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies. PMID:28850804

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing.

PubMed

Li, Yiyan; Yang, Xing; Zhao, Weian

2017-12-01

Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies.

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

PubMed Central

Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

2002-01-01

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

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.

Screening and rapid identification of Campylobacter spp. DNA by FlaA PCR based method on chicken and human fecal samples in Egypt

USDA-ARS?s Scientific Manuscript database

Campylobacter is a foodborne pathogen which has a potential public health concern worldwide. Due to discriminatory problems encountered by conventional isolation of Campylobacter spp. and its genetic similarities, rapid molecular techniques for its genetic characterization are useful. In this study,...

Development of primer sets for loop-mediated isothermal amplification that enables rapid and specific detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

USDA-ARS?s Scientific Manuscript database

Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three ...

The use of Matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis.

PubMed

Karatuna, Onur; Celebi, Bekir; Can, Simge; Akyar, Isin; Kilic, Selcuk

2016-01-15

Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institute of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica due to RD1 subspecies-specific PCR result. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories.

The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis

PubMed Central

Karatuna, Onur; Çelebi, Bekir; Can, Simge; Akyar, Işın; Kiliç, Selçuk

2016-01-01

Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institution of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica according to region of difference 1 (RD1) subspecies-specific PCR results. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories. PMID:26773181

Fast methods of fungal and bacterial identification. MALDI-TOF mass spectrometry, chromogenic media.

PubMed

Siller-Ruiz, María; Hernández-Egido, Sara; Sánchez-Juanes, Fernando; González-Buitrago, José Manuel; Muñoz-Bellido, Juan Luis

2017-05-01

MALDI-TOF mass spectrometry is now a routine resource in Clinical Microbiology, because of its speed and reliability in the identification of microorganisms. Its performance in the identification of bacteria and yeasts is perfectly contrasted. The identification of mycobacteria and moulds is more complex, due to the heterogeneity of spectra within each species. The methodology is somewhat more complex, and expanding the size of species libraries, and the number of spectra of each species, will be crucial to achieve greater efficiency. Direct identification from blood cultures has been implemented, since its contribution to the management of severe patients is evident, but its application to other samples is more complex. Chromogenic media have also contributed to the rapid diagnosis in both bacteria and yeast, since they accelerate the diagnosis, facilitate the detection of mixed cultures and allow rapid diagnosis of resistant species. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Hyperspectral microscope imaging methods to classify gram-positive and gram-negative foodborne pathogenic bacteria

USDA-ARS?s Scientific Manuscript database

An acousto-optic tunable filter-based hyperspectral microscope imaging method has potential for identification of foodborne pathogenic bacteria from microcolony rapidly with a single cell level. We have successfully developed the method to acquire quality hyperspectral microscopic images from variou...

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine

PubMed Central

Huang, K. S.; Lee, S. E.; Yeh, Y.; Shen, G. S.; Mei, E.; Chang, C. M.

2010-01-01

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future. PMID:20129946

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine.

PubMed

Huang, K S; Lee, S E; Yeh, Y; Shen, G S; Mei, E; Chang, C M

2010-08-23

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future.

Development of Conductive Polymer Analysis for the Rapid Detection and Identification of Phytopathogenic Microbes

Treesearch

A. Dan Wilson; D.G. Lester; C.S. Oberle

2004-01-01

Conductive polymer analysis, a type of electronic aroma detection technology, was evaluated for its efficacy in the detection, identification, and discrimination of plant-pathogenic microorganisms on standardized media and in diseased plant tissues. The method is based on the acquisition of a diagnostic electronic fingerprint derived from multisensor responses to...

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

Micro-Raman spectroscopy for identification and classification of UTI bacteria

NASA Astrophysics Data System (ADS)

Yogesha, M.; Chawla, Kiran; Acharya, Mahendra; Chidangil, Santhosh; Bankapur, Aseefhali

2017-07-01

Urinary tract infection (UTI) is one of the major clinical problems known to mankind, especially among adult women. Conventional methods for identification of UTI causing bacteria are time consuming and expensive. Therefore, a rapid and cost-effective method is desired. In the present study, five bacteria (one Gram-positive and four Gram-negative), most commonly known to cause UTI, have been identified and classified using micro-Raman spectroscopy combined with principal component analysis (PCA).

Rapid Screening and Characterization of Acetylcholinesterase Inhibitors from Yinhuang Oral Liquid Using Ultrafiltration-liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry

PubMed Central

Zhang, Haomin; Guo, Yinan; Meng, Lingwen; Sun, Hui; Yang, Yinping; Gao, Ying; Sun, Jiaming

2018-01-01

Background: At present, approximately 17–25 million people in the world suffer from Alzheimer's disease (AD). The most efficacious and acceptable therapeutic drug clinically are the acetylcholinesterase inhibitors (AChEIs). Yinhuang oral liquid is a Chinese medicine preparation which contains AChEIs according to the literatures. However, no strategy has been presented for rapid screening and identification of AChEIs from Yinhuang oral liquid. Objective: To develop a method for rapid screening and identification of AChEIs from Yinhuang oral liquid using ultrafiltration–liquid chromatography–electrospray ionization tandem mass spectrometry (UF-LC-ESI-MS/MS). Materials and Methods: In this study, UF incubation conditions such as enzyme concentration, incubation time, and incubation temperature were optimized so as to get better screening results. The AChEIs from Yinhuang oral liquid were identified by high-performance liquid chromatography-ESI-MS and the improved Ellman method was used for the AChE inhibitory activity test in vitro. Results: The results showed that Yinhuang oral liquid can inhibit the activity of AChE. We screened and identified seven compounds with potential AChE inhibitory activity from Yinhuang oral liquid, which provided experimental basis for the treatment and prevention of AD. Conclusion: The current technique was used to directly screen the active ingredients with acetylcholinesterase inhibition from complex traditional Chinese medicine, which was simple, rapid, accurate, and suitable for high-throughput screening of AChEI from complex systems. SUMMARY A UF-LC-ESI-MS/MS method for rapid screening and identification of AChEIs from Yinhuang oral liquid was developedSeven compounds were screened and identified with potential AChE inhibitory activity from Yinhuang oral liquidIt provided experimental basis of Yinhuang oral liquid for the treating and preventing AD. Abbreviations used: (AD): Alzheimer's disease; (UF-LC-ESI-MS/MS): ultrafiltration–liquid chromatography–electrospray ionization tandem mass spectrometry; (AChEIs): acetylcholinesterase inhibitors. PMID:29720840

Molecular Identification of an Invasive Wood-Boring Insect Lyctus brunneus (Coleoptera: Bostrichidae: Lyctinae) Using Frass by Loop-Mediated Isothermal Amplification and Nested PCR Assays.

PubMed

Ide, Tatsuya; Kanzaki, Natsumi; Ohmura, Wakako; Okabe, Kimiko

2016-03-27

Lyctus brunneus(Stephens) is one of the most destructive and worldwide invasive pests of seasoned woods for wooden products. This and other pestLyctusspecies have had their distribution expanded by international and domestic human transportation of infested wood and wood products. Rapid detection and accurate identification ofLyctusspecies are effective tools for helping to eradicate them in new introduction sites. The accurate species-level identification of adults requires expert knowledge about their morphology. However, it takes much time and effort to recover suitable adult specimens because they are borers inside wood. Frass ofLyctusspecies can easily be detected and recovered in and around infested wood. Thus, frass was tested to see if it was a suitable sample to allow development of a rapid and technically easy molecular detection and identification method forL.brunneus.Species-specific primers were designed from the cytochrome c oxidase subunit I region ofL.brunneusand used in development and testing of methods for successfully identifying them from their frass using the loop-mediated isothermal amplification (LAMP) or species-specific nested polymerase chain reaction (PCR) assays. The LAMP assay was faster and more sensitive for detecting the presence of DNA derived fromL.brunneusin their frass than the nested PCR assay. These methodologies will be applicable for the rapid detection and identification of other wood-boring invasive pests in regulatory applications. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

PubMed Central

Zhang, Lin; Vranckx, Katleen; Janssens, Koen; Sandrin, Todd R.

2015-01-01

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level. PMID:25590854

Use of MALDI-TOF mass spectrometry and a custom database to characterize bacteria indigenous to a unique cave environment (Kartchner Caverns, AZ, USA).

PubMed

Zhang, Lin; Vranckx, Katleen; Janssens, Koen; Sandrin, Todd R

2015-01-02

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation.

PubMed

Schmitt, Bryan H; Cunningham, Scott A; Dailey, Aaron L; Gustafson, Daniel R; Patel, Robin

2013-03-01

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria.

GC/IR computer-aided identification of anaerobic bacteria

NASA Astrophysics Data System (ADS)

Ye, Hunian; Zhang, Feng S.; Yang, Hua; Li, Zhu; Ye, Song

1993-09-01

A new method was developed to identify anaerobic bacteria by using pattern recognition. The method is depended on GC / JR data. The system is intended for use as a precise rapid and reproduceable aid in the identification of unknown isolates. Key Words: Anaerobic bacteria Pattern recognition Computeraided identification GC / JR 1 . TNTRODUCTTON A major problem in the field of anaerobic bacteriology is the difficulty in accurately precisely and rapidly identifying unknown isolates. Tn the proceedings of the Third International Symposium on Rapid Methods and Automation in Microbiology C. M. Moss said: " Chromatographic analysis is a new future for clinical microbiology" . 12 years past and so far it seems that this is an idea whose time has not get come but it close. Now two major advances that have brought the technology forword in terms ofmaking it appropriate for use in the clinical laboratory can aldo be cited. One is the development and implementation of fused silica capillary columns. In contrast to packed columns and those of'' greater width these columns allow reproducible recovery of hydroxey fatty acids with the same carbon chain length. The second advance is the efficient data processing afforded by modern microcomputer systems. On the other hand the practical steps for sample preparation also are an advance in the clinical laboratory. Chromatographic Analysis means mainly of analysis of fatty acids. The most common

A simple and rapid technique for the authentication of the ginseng cultivar, Yunpoong, using an SNP marker in a large sample of ginseng leaves.

PubMed

Sun, Hua; Wang, Hong-Tao; Kwon, Woo-Saeng; Kim, Yeon-Ju; In, Jun-Gyo; Yang, Deok-Chun

2011-11-01

Yunpoong is an important Korean ginseng (Panax ginseng C. A. Meyer) cultivar, but no molecular marker has been available to identify Yunpoong from other cultivars. In this study, we developed a single nucleotide polymorphism (SNP) marker for Yunpoong based on analysis of expressed sequence tags (ESTs) in an exon region of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. This SNP marker had high specificity to authenticate Yunpoong in twelve different main ginseng cultivars. For application of the molecular marker, a rapid identification method was established based on the NaOH-Tris method and real-time polymerase chain reaction (PCR) in order to ensure more efficiency in the cultivar selection. The biggest feature of the NaOH-Tris method was that it made the extraction of DNA very simple and rapid in young leaf tissues. We only spent 1 min to extract DNA and directly used it to do PCR. In this report, the conventional DNA extraction method was used to develop molecular marker process, and the NaOH-Tris method was applied in screening large numbers of cultivars. Moreover, the greatest advantage of the real-time PCR compared with traditional PCR, is time saving and high efficiency. Thus, this strategy provides a rapid and reliable method for the specific identification of Yunpoong in a large number of samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Thin-layer chromatographic identification of Chinese propolis using chemometric fingerprinting.

PubMed

Tang, Tie-xin; Guo, Wei-yan; Xu, Ye; Zhang, Si-ming; Xu, Xin-jun; Wang, Dong-mei; Zhao, Zhi-min; Zhu, Long-ping; Yang, De-po

2014-01-01

Poplar tree gum has a similar chemical composition and appearance to Chinese propolis (bee glue) and has been widely used as a counterfeit propolis because Chinese propolis is typically the poplar-type propolis, the chemical composition of which is determined mainly by the resin of poplar trees. The discrimination of Chinese propolis from poplar tree gum is a challenging task. To develop a rapid thin-layer chromatographic (TLC) identification method using chemometric fingerprinting to discriminate Chinese propolis from poplar tree gum. A new TLC method using a combination of ammonia and hydrogen peroxide vapours as the visualisation reagent was developed to characterise the chemical profile of Chinese propolis. Three separate people performed TLC on eight Chinese propolis samples and three poplar tree gum samples of varying origins. Five chemometric methods, including similarity analysis, hierarchical clustering, k-means clustering, neural network and support vector machine, were compared for use in classifying the samples based on their densitograms obtained from the TLC chromatograms via image analysis. Hierarchical clustering, neural network and support vector machine analyses achieved a correct classification rate of 100% in classifying the samples. A strategy for TLC identification of Chinese propolis using chemometric fingerprinting was proposed and it provided accurate sample classification. The study has shown that the TLC identification method using chemometric fingerprinting is a rapid, low-cost method for the discrimination of Chinese propolis from poplar tree gum and may be used for the quality control of Chinese propolis. Copyright © 2014 John Wiley & Sons, Ltd.

Effects of solid-medium type on routine identification of bacterial isolates by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Anderson, Neil W; Buchan, Blake W; Riebe, Katherine M; Parsons, Lauren N; Gnacinski, Stacy; Ledeboer, Nathan A

2012-03-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.

Application of Pyrosequencing® in Food Biodefense.

PubMed

Amoako, Kingsley Kwaku

2015-01-01

The perpetration of a bioterrorism attack poses a significant risk for public health with potential socioeconomic consequences. It is imperative that we possess reliable assays for the rapid and accurate identification of biothreat agents to make rapid risk-informed decisions on emergency response. The development of advanced methodologies for the detection of biothreat agents has been evolving rapidly since the release of the anthrax spores in the mail in 2001, and recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence-based approaches such as Pyrosequencing(®), which has the capability to determine short DNA stretches in real time using biotinylated PCR amplicons, have potential biodefense applications. Using markers from the virulence plasmids and chromosomal regions, my laboratory has demonstrated the power of this technology in the rapid, specific, and sensitive detection of B. anthracis spores and Yersinia pestis in food. These are the first applications for the detection of the two organisms in food. Furthermore, my lab has developed a rapid assay to characterize the antimicrobial resistance (AMR) gene profiles for Y. pestis using Pyrosequencing. Pyrosequencing is completed in about 60 min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence, thus enabling rapid risk-informed decisions to be made. A typical run yields 40-84 bp reads with 94-100 % identity to the expected sequence. It also provides a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The method described is proposed as a novel detection system for potential application in food biodefense.

Rapid Diagnosis of Bloodstream Infections with PCR Followed by Mass Spectrometry

PubMed Central

Jordana-Lluch, Elena; Carolan, Heather E.; Giménez, Montserrat; Sampath, Rangarajan; Ecker, David J.; Quesada, M. Dolores; Mòdol, Josep M.; Arméstar, Fernando; Blyn, Lawrence B.; Cummins, Lendell L.; Ausina, Vicente; Martró, Elisa

2013-01-01

Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods. PMID:23626775

Identification of Low-Level Vancomycin Resistance in Staphylococcus aureus in the Era of Informatics.

PubMed

Ford, Bradley A

2016-04-01

Vancomycin-intermediateStaphylococcus aureus(VISA) and heteroresistant VISA (hVISA) are pathogens for which accurate antimicrobial susceptibility testing (AST) would rule out standard treatment with vancomycin. Unfortunately, AST for vancomycin is relatively slow and standard methods are unable to reliably detect VISA and hVISA. An article in this issue (C. A. Mather, B. J. Werth, S. Sivagnanam, D. J. SenGupta, and S. M. Butler-Wu, J Clin Microbiol 54:883-890, 2016, doi:http://dx.doi.org/10.1128/JCM.02428-15) describes a rapid whole-cell matrix-assisted laser desorption ionization-time of flight proxy susceptibility method that highlights current innovations and challenges with rapid AST, VISA/hVISA identification, and clinical bioinformatics. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Direct Application of the INNO-LiPA Rif.TB Line-Probe Assay for Rapid Identification of Mycobacterium tuberculosis Complex Strains and Detection of Rifampin Resistance in 360 Smear-Positive Respiratory Specimens from an Area of High Incidence of Multidrug-Resistant Tuberculosis

PubMed Central

Viveiros, Miguel; Leandro, Clara; Rodrigues, Liliana; Almeida, Josefina; Bettencourt, Rosário; Couto, Isabel; Carrilho, Lurdes; Diogo, José; Fonseca, Ana; Lito, Luís; Lopes, João; Pacheco, Teresa; Pessanha, Mariana; Quirim, Judite; Sancho, Luísa; Salfinger, Max; Amaral, Leonard

2005-01-01

The INNO-LiPA Rif.TB assay for the identification of Mycobacterium tuberculosis complex strains and the detection of rifampin (RIF) resistance has been evaluated with 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis (MDR-TB). The sensitivity when compared to conventional identification/culture methods was 82.2%, and the specificity was 66.7%; the sensitivity and specificity were 100.0% and 96.9%, respectively, for the detection of RIF resistance. This assay has the potential to provide rapid information that is essential for the effective management of MDR-TB. PMID:16145166

Rapid and accurate identification of in vivo-induced haploid seeds based on oil content in maize

PubMed Central

Melchinger, Albrecht E.; Schipprack, Wolfgang; Würschum, Tobias; Chen, Shaojiang; Technow, Frank

2013-01-01

The needs of a growing human population require rapid and efficient development of improved cultivars by plant breeders. The doubled haploid (DH) technology enables generating completely homozygous lines in a single step and, thus, is central to modern genetics and breeding approaches. Rapid and reliable identification of seeds with a haploid embryo after in vivo haploid induction is elementary in the method utilized in maize but current systems have severe shortcomings preventing their use in many germplasm types. Here, we describe an alternative method for discrimination of haploid from diploid seeds based on differences in their oil content stemming from pollination with high oil inducers. After presenting some fundamental theory, we provide a proof-of-concept with experimental results, demonstrating acceptable error rates across different germplasm. Our approach represents a breakthrough in DH technology in maize, because it is amenable to automated high-throughput screening and applicable to any maize germplasm worldwide. PMID:23820577

UPLC/Q-TOFMS/MS as a powerful technique for rapid identification of polymethoxylated flavones in Fructus aurantii.

PubMed

Zhou, Da-Yong; Zhang, Xiu-Li; Xu, Qing; Xue, Xing-Ya; Zhang, Fei-Fang; Liang, Xin-Miao

2009-08-15

Polymethoxylated flavones (PMFs), as potential cancer chemopreventive agents, are widely distributed in Citrus genus. In this study, a selected ion monitoring-tandem mass (SIM-MS/MS) method for the rapid identification of PMFs in Fructus aurantii (F. aurantii) with ultra-performance liquid chromatography (UPLC) coupled to quadrupole, hybrid orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOFMS/MS) was proposed. The MS data for candidates, containing accurate mass and isotopic patterns for both precursors and their fragment ions, were acquired selectively. Based on the MS data, the mass spectrometric fingerprint (MSFP) for candidates, consisting of chemical formula and dissociation pattern, was determined. Comparing the MSFPs of the observed compounds with the diagnostic MSFP of the species, 44 PMFs were tentatively identified. The method was validated by tangeretin and sinensetin, two representative compounds of PMFs, and was considered to be suitable for the rapid screening of PMFs in crude and partially purified samples.

Use of photopolymerization for the rapid and cost-effective identification of Shiga toxin-producing Escherichia coli on DNA microarrays

USDA-ARS?s Scientific Manuscript database

Shiga toxin-producing Escherichia coli O157:H7 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 virulent strains, the present study explored the use of photopolymerization, a colorimetric and photoinduced signal amplification d...

Expediting the Analysis of Qualitative Data in Evaluation: A Procedure for the Rapid Identification of Themes from Audio Recordings (RITA)

ERIC Educational Resources Information Center

Neal, Jennifer Watling; Neal, Zachary P.; VanDyke, Erika; Kornbluh, Mariah

2015-01-01

Qualitative data offer advantages to evaluators, including rich information about stakeholders' perspectives and experiences. However, qualitative data analysis is labor-intensive and slow, conflicting with evaluators' needs to provide punctual feedback to their clients. In this method note, we contribute to the literature on rapid evaluation and…

Rapid condition assessment of structural condition after a blast using state-space identification

NASA Astrophysics Data System (ADS)

Eskew, Edward; Jang, Shinae

2015-04-01

After a blast event, it is important to quickly quantify the structural damage for emergency operations. In order improve the speed, accuracy, and efficiency of condition assessments after a blast, the authors have previously performed work to develop a methodology for rapid assessment of the structural condition of a building after a blast. The method involved determining a post-event equivalent stiffness matrix using vibration measurements and a finite element (FE) model. A structural model was built for the damaged structure based on the equivalent stiffness, and inter-story drifts from the blast are determined using numerical simulations, with forces determined from the blast parameters. The inter-story drifts are then compared to blast design conditions to assess the structures damage. This method still involved engineering judgment in terms of determining significant frequencies, which can lead to error, especially with noisy measurements. In an effort to improve accuracy and automate the process, this paper will look into a similar method of rapid condition assessment using subspace state-space identification. The accuracy of the method will be tested using a benchmark structural model, as well as experimental testing. The blast damage assessments will be validated using pressure-impulse (P-I) diagrams, which present the condition limits across blast parameters. Comparisons between P-I diagrams generated using the true system parameters and equivalent parameters will show the accuracy of the rapid condition based blast assessments.

Evaluation of a simple phenotypic method for the detection of carbapenemase-producing Enterobacteriaceae.

PubMed

Saito, Ryoichi; Koyano, Saho; Dorin, Misato; Higurashi, Yoshimi; Misawa, Yoshiki; Nagano, Noriyuki; Kaneko, Takamasa; Moriya, Kyoji

2015-01-01

We investigated the performance of a phenotypic test, the Carbapenemase Detection Set (MAST-CDS), for the identification of carbapenemase-producing Enterobacteriaceae. Our results indicated that MAST-CDS is rapid, easily performed, simple to interpret, and highly sensitive for the identification of carbapenemase producers, particularly imipenemase producers. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid identification of Clostridium species by high-pressure liquid chromatography.

PubMed Central

Harpold, D J; Wasilauskas, B L; O'Connor, M L

1985-01-01

High-pressure liquid chromatography was evaluated as a rapid means of identifying various species of clostridia. Isolates were inoculated into a defined medium and incubated aerobically for 1 h at 35 degrees C. The organisms were removed, and the supernatants were derivatized for 1 min at room temperature by the addition of o-phthalaldehyde. The total time required to run each chromatogram was approximately 50 min. Standardized peak heights for each medium component and any new peaks formed were calculated for each isolate and compared with those for uninoculated control medium. Multiple isolates of various Clostridium species gave consistent patterns of medium utilization that could be used for identification. This rapid method can easily be adapted for laboratory use and has the potential for automation. PMID:3905852

Synthesis markers in illegally manufactured 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine.

PubMed

Bohn, M; Bohn, G; Blaschke, G

1993-01-01

In this paper the isolation and identification of 12 compounds as impurities in illicit 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) is reported. Isolation of these substances is performed by preparative TLC, while identification is performed by using mass spectrometry and 1H-NMR spectroscopy. A simple and rapid method for detection of these impurities in seized MDA and MDMA samples is described. The identification of the impurities can provide numerous points on which to base comparative analysis of different exhibits.

Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

PubMed

Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

2012-11-01

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

Discrimination of Chinese Sauce liquor using FT-IR and two-dimensional correlation IR spectroscopy

NASA Astrophysics Data System (ADS)

Sun, Su-Qin; Li, Chang-Wen; Wei, Ji-Ping; Zhou, Qun; Noda, Isao

2006-11-01

We applied the three-step IR macro-fingerprint identification method to obtain the IR characteristic fingerprints of so-called Chinese Sauce liquor (Moutai liquor and Kinsly liquor) and a counterfeit Moutai. These fingerprints can be used for the identification and discrimination of similar liquor products. The comparison of their conventional IR spectra, as the first step of identification, shows that the primary difference in Sauce liquor is the intensity of characteristic peaks at 1592 and 1225 cm -1. The comparison of the second derivative IR spectra, as the second step of identification, shows that the characteristic absorption in 1400-1800 cm -1 is substantially different. The comparison of 2D-IR correlation spectra, as the third and final step of identification, can discriminate the liquors from another direction. Furthermore, the method was successfully applied to the discrimination of a counterfeit Moutai from the genuine Sauce liquor. The success of the three-step IR macro-fingerprint identification to provide a rapid and effective method for the identification of Chinese liquor suggests the potential extension of this technique to the identification and discrimination of other wine and spirits, as well.

Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions.

PubMed

Pryce, Todd M; Palladino, Silvano; Price, Diane M; Gardam, Dianne J; Campbell, Peter B; Christiansen, Keryn J; Murray, Ronan J

2006-04-01

We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.

Direct maldi-tof mass spectrometry assay of blood culture broths for rapid identification of Candida species causing bloodstream infections: an observational study in two large microbiology laboratories.

PubMed

Spanu, Teresa; Posteraro, Brunella; Fiori, Barbara; D'Inzeo, Tiziana; Campoli, Serena; Ruggeri, Alberto; Tumbarello, Mario; Canu, Giulia; Trecarichi, Enrico Maria; Parisi, Gabriella; Tronci, Mirella; Sanguinetti, Maurizio; Fadda, Giovanni

2012-01-01

We evaluated the reliability of the Bruker Daltonik's MALDI Biotyper system in species-level identification of yeasts directly from blood culture bottles. Identification results were concordant with those of the conventional culture-based method for 95.9% of Candida albicans (187/195) and 86.5% of non-albicans Candida species (128/148). Results were available in 30 min (median), suggesting that this approach is a reliable, time-saving tool for routine identification of Candida species causing bloodstream infection.

Identification of bacteria isolated from veterinary clinical specimens using MALDI-TOF MS.

PubMed

Pavlovic, Melanie; Wudy, Corinna; Zeller-Peronnet, Veronique; Maggipinto, Marzena; Zimmermann, Pia; Straubinger, Alix; Iwobi, Azuka; Märtlbauer, Erwin; Busch, Ulrich; Huber, Ingrid

2015-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged as a rapid and accurate identification method for bacterial species. Although it has been successfully applied for the identification of human pathogens, it has so far not been well evaluated for routine identification of veterinary bacterial isolates. This study was performed to compare and evaluate the performance of MALDI-TOF MS based identification of veterinary bacterial isolates with commercially available conventional test systems. Discrepancies of both methods were resolved by sequencing 16S rDNA and, if necessary, the infB gene for Actinobacillus isolates. A total of 375 consecutively isolated veterinary samples were collected. Among the 357 isolates (95.2%) correctly identified at the genus level by MALDI-TOF MS, 338 of them (90.1% of the total isolates) were also correctly identified at the species level. Conventional methods offered correct species identification for 319 isolates (85.1%). MALDI-TOF identification therefore offered more accurate identification of veterinary bacterial isolates. An update of the in-house mass spectra database with additional reference spectra clearly improved the identification results. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for classification and identification of veterinary bacterial isolates.

A RAPID DNA EXTRACTION METHOD FOR PCR IDENTIFICATION OF FUNGAL INDOOR AIR CONTAMINANTS

EPA Science Inventory

Following air sampling, fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and polymerase chain reaction (PCR) appli...

Fluorescence excitation-emission matrix (EEM) spectroscopy for rapid identification and quality evaluation of cell culture media components.

PubMed

Li, Boyan; Ryan, Paul W; Shanahan, Michael; Leister, Kirk J; Ryder, Alan G

2011-11-01

The application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative analysis of complex, aqueous solutions of cell culture media components was investigated. These components, yeastolate, phytone, recombinant human insulin, eRDF basal medium, and four different chemically defined (CD) media, are used for the formulation of basal and feed media employed in the production of recombinant proteins using a Chinese Hamster Ovary (CHO) cell based process. The comprehensive analysis (either identification or quality assessment) of these materials using chromatographic methods is time consuming and expensive and is not suitable for high-throughput quality control. The use of EEM in conjunction with multiway chemometric methods provided a rapid, nondestructive analytical method suitable for the screening of large numbers of samples. Here we used multiway robust principal component analysis (MROBPCA) in conjunction with n-way partial least squares discriminant analysis (NPLS-DA) to develop a robust routine for both the identification and quality evaluation of these important cell culture materials. These methods are applicable to a wide range of complex mixtures because they do not rely on any predetermined compositional or property information, thus making them potentially very useful for sample handling, tracking, and quality assessment in biopharmaceutical industries.

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

Acidic Ribosomal Proteins from the Extreme ’Halobacterium cutirubrum’,

DTIC Science & Technology

the extreme halophilic bacterium, Halobacterium cutirubrum. The identification of the protein moieties involved in these and other interactions in...the halophile ribosome requires a rapid and reproducible screening method for the separation, enumeration and identification of these acidic...polypeptides in the complex ribosomal protein mixtures. In this paper the authors present the results of analyses of the halophile ribosomal proteins using a

Phenotypic Identification of Actinomyces and Related Species Isolated from Human Sources

PubMed Central

Sarkonen, Nanna; Könönen, Eija; Summanen, Paula; Könönen, Mauno; Jousimies-Somer, Hannele

2001-01-01

Recent advancements in chemotaxonomic and molecular biology-based identification methods have clarified the taxonomy of the genus Actinomyces and have led to the recognition of several new Actinomyces and related species. Actinomyces-like gram-positive rods have increasingly been isolated from various clinical specimens. Thus, an easily accessible scheme for reliable differentiation at the species level is needed in clinical and oral microbiology laboratories, where bacterial identification is mainly based on conventional biochemical methods. In the present study we designed a two-step protocol that consists of a flowchart that describes rapid, cost-efficient tests for preliminary identification of Actinomyces and closely related species and an updated more comprehensive scheme that also uses fermentation reactions for accurate differentiation of Actinomyces and closely related species. PMID:11682514

The significance of gtf genes in caries expression: a rapid identification of Streptococcus mutans from dental plaque of child patients.

PubMed

Mishra, Apurva; Pandey, Ramesh K; Manickam, Natesan

2015-01-01

Rapid phylogenetic and functional gene (gtfB) identification of S. mutans from the dental plaque derived from children. Dental plaque collected from fifteen patients of age group 7-12 underwent centrifugation followed by genomic DNA extraction for S. mutans. Genomic DNA was processed with S. mutans specific primers in suitable PCR condtions for phylogenetic and functional gene (gtfB) identification. The yield and results were confirmed by agarose gel electrophoresis. 1% agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1 kbp indicating the presence of S. mutans in the test sample. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bp when compared to 100 bp standards. With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques.

[Study of cuttings identification using laser-induced breakdown spectroscopy].

PubMed

Tian, Ye; Wang, Zhen-nan; Hou, Hua-ming; Zhai, Xiao-wei; Ci, Xing-hua; Zheng, Rong-er

2012-08-01

Cutting identification is one of the most important links in the course of cutting logging which is very significant in the process of oil drilling. In the present paper, LIBS was used for identification of four kinds of cutting samples coming from logging field, and then multivariate analysis was used in data processing. The whole spectra model and the feature model were built for cuttings identification using PLS-DA method. The accuracy of the whole spectra model was 88.3%, a little more than the feature model with an accuracy of 86.7%. While in the aspect of data size, the variables were decreased from 24,041 to 27 by feature extraction, which increased the efficiency of data processing observably. The obtained results demonstrate that LIBS combined with chemometrics method could be developed as a rapid and valid approach to cutting identification and has great potential to be used in logging field.

The rapid identification of human influenza neuraminidase N1 and N2 subtypes by ELISA.

PubMed

Barr, I G; McCaig, M; Durrant, C; Shaw, R

2006-11-10

An ELISA assay was developed to allow the rapid and accurate identification of human influenza A N1 and N2 neuraminidases. Initial testing using a fetuin pre-coating of wells correctly identified 81.7% of the neuraminidase type from a series of human A(H1N1), A(H1N2) and A(H3N2) viruses. This result could be improved to detect the neuraminidase subtype of almost all human influenza A viruses from a large panel of viruses isolated from 2000 to 2005, if the fetuin pre-coating was removed and the viruses were coated directly onto wells. This method is simple, rapid and can be used to screen large numbers of currently circulating human influenza A viruses for their neurraminidase subtype and is a good alternative to RT-PCR.

Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for the identification of clinical filamentous fungi.

PubMed

Huang, Yanfei; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Wang, Jinglin; Lu, Xinxin

2017-07-01

Infections caused by filamentous fungi have become a health concern, and require rapid and accurate identification in order for effective treatment of the pathogens. To compare the performance of two MALDI-TOF MS systems (Bruker Microflex LT and Xiamen Microtyper) in the identification of filamentous fungal species. A total of 374 clinical filamentous fungal isolates sequentially collected in the Clinical Laboratory at the Beijing Tongren Hospital between January 2014 and December 2015 were identified by traditional phenotypic methods, Bruker Microflex LT and Xiamen Microtyper MALDI-TOF MS, respectively. The discrepancy between these methods was resolved by sequencing for definitive identification. Bruker Microflex LT and Xiamen Microtyper had similar correct species ID (98.9 vs. 99.2%), genus ID (99.7 vs. 100%), mis-ID (0.3 vs. 0%) and no ID (0 vs. 0). The rate of correct species identification by both MALDI-TOF MS (98.9 and 99.2%, respectively) was much higher compared with phenotypic approach (91.9%). Both MALDI-TOF MS systems provide accurate identification of clinical filamentous fungi compared with conventional phenotypic method, and have the potential to replace identification for routine identification of these fungi in clinical mycology laboratories. Both systems have similar performance in the identification of clinical filamentous fungi.

Multiplex-PCR As a Rapid and Sensitive Method for Identification of Meat Species in Halal-Meat Products.

PubMed

Alikord, Mahsa; Keramat, Javad; Kadivar, Mahdi; Momtaz, Hassan; Eshtiaghi, Mohammad N; Homayouni-Rad, Aziz

2017-01-01

Species identification and authentication in meat products are important subjects for ensuring the health of consumers. The multiplex-PCR amplification and species- specific primer set were used for the identification of horse, donkey, pig and other ruminants in raw and processed meat products. Oligonucleotid primers were designed and patented for amplification of species-specific mitochondrial DNA sequences of each species and samples were prepared from binary meat mixtures. The results showed that meat species were accurately determined in all combinations by multiplex-PCR, and the sensitivity of this method was 0.001 ng, rendering this technique open to and suitable for use in industrial meat products. It is concluded that more fraud is seen in lower percentage industrial meat products than in higher percentage ones. There was also more fraud found in processed products than in raw ones. This rapid and useful test is recommended for quality control firms for applying more rigorous controls over industrial meat products, for the benefit of target consumers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Rapid analysis of aminoglycoside antibiotics in bovine tissues using disposable pipette extraction and ultrahigh performance liquid chromatography - tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

A high-throughput qualitative screening and identification method for 9 aminoglycosides of regulatory interest has been developed, validated, and implemented for bovine kidney, liver, and muscle tissues. The method involves extraction at previously validated conditions, cleanup using disposable pip...

Evaluation of Pyrrolidonyl Arylamidase Activity in Staphylococcus delphini.

PubMed

Compton, Samantha T; Kania, Stephen A; Robertson, Amy E; Lawhon, Sara D; Jenkins, Stephen G; Westblade, Lars F; Bemis, David A

2017-03-01

Clinical reference textbooks lack data for pyrrolidonyl arylamidase (PYR) activity in Staphylococcus delphini This study evaluated PYR activities of 21 S. delphini strains by reference broth, rapid disc, and rapid slide methods. Species and subgroup identifications were confirmed by nucleic acid-based methods and included nine group A and 12 group B strains. Testing by rapid PYR methods with products from four manufacturers was performed at two testing locations, and, with the exception of one strain tested at one location using reagents from one manufacturer, each S. delphini strain tested positive for PYR activity. Therefore, PYR may be a useful single-test adjunct for distinguishing Staphylococcus aureus from S. delphini and other members of the Staphylococcus intermedius group. Copyright © 2017 American Society for Microbiology.

MALDI-TOF MS for the identification of veterinary non-C. neoformans-C. gattii Cryptococcus spp. isolates from Italy.

PubMed

Danesi, Patrizia; Drigo, Ilenia; Iatta, Roberta; Firacative, Carolina; Capelli, Gioia; Cafarchia, Claudia; Meyer, Wieland

2014-08-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an effective alternative to phenotypic and molecular methods for the rapid identification of microorganisms. Our aim in this study was to create an in-house library for a set of strains of nine uncommonly reported human and animal cryptococcal species, including Cryptococcus adeliensis, C. albidosimilis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus, C. victoriae and C. uniguttulatus, and to use this library to make timely and correct identifications using MALDI-TOF MS for use in routine laboratory diagnostics. Protein extracts obtained via the formic acid extraction method of 62 veterinary non-C. neoformans-C. gattii cryptococcal isolates were studied. The obtained mass spectra correctly grouped all 62 studied isolates according to species identification previously obtained by internal transcribe spacer sequence analysis. The in-house database was than exported and successfully uploaded to the Microflex LT (Maldi Biotyper; Bruker Daltonics) instrument at a different diagnostic laboratory in Italy. Scores >2.7 obtained from isolates reanalyzed in the latter laboratory supported the high reproducibility of the method. The possibility of creating and transferring an in-house library adds to the usefulness MALDI-TOF MS an important tool for the rapid and inexpensive identification of pathogenic and saprophytic fungi as required for differential diagnosis of human and animal mycoses. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[Rapid identification of microorganisms by mass spectrometry in a blood culture system. Comparison of two procedures].

PubMed

Cattani, María E; Posse, Tamara; Hermes, Ricardo L; Kaufman, Sara C

2015-01-01

Rapid identification of microorganisms is critical in hospitalized infected patients. Blood culture is currently the gold standard for detecting and identifying microorganisms causing bacteremia or sepsis. The introduction of mass spectrometry by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) in microbiology laboratories, especially in microorganisms growing in blood culture bottles, provides rapid identification. This study evaluates the performance of the Maldi Sepsityper Biotyper procedure (hereinafter, MS) compared to that of an in-home method (hereinafter, HF). Eight hundred and forty (840) positive blood culture bottles were processed using the HF procedure, 542 of which were also processed using MS. The organisms were identified in 670 (79.76%) and 391 (72.14%) bottles respectively (p = 0,0013). This study demonstrates the effectiveness of both procedures for identifying microorganisms directly from positive blood culture bottles. However, the HF procedure proved to be more effective than MS, especially in the presence of Gram positive organisms. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Rapid Scanning Structure-Activity Relationships in Combinatorial Data Sets: Identification of Activity Switches

PubMed Central

Medina-Franco, José L.; Edwards, Bruce S.; Pinilla, Clemencia; Appel, Jon R.; Giulianotti, Marc A.; Santos, Radleigh G.; Yongye, Austin B.; Sklar, Larry A.; Houghten, Richard A.

2013-01-01

We present a general approach to describe the structure-activity relationships (SAR) of combinatorial data sets with activity for two biological endpoints with emphasis on the rapid identification of substitutions that have a large impact on activity and selectivity. The approach uses Dual-Activity Difference (DAD) maps that represent a visual and quantitative analysis of all pairwise comparisons of one, two, or more substitutions around a molecular template. Scanning the SAR of data sets using DAD maps allows the visual and quantitative identification of activity switches defined as specific substitutions that have an opposite effect on the activity of the compounds against two targets. The approach also rapidly identifies single- and double-target R-cliffs, i.e., compounds where a single or double substitution around the central scaffold dramatically modifies the activity for one or two targets, respectively. The approach introduced in this report can be applied to any analogue series with two biological activity endpoints. To illustrate the approach, we discuss the SAR of 106 pyrrolidine bis-diketopiperazines tested against two formylpeptide receptors obtained from positional scanning deconvolution methods of mixture-based libraries. PMID:23705689

Development of Amplified 16S Ribosomal DNA Restriction Analysis for Identification of Actinomyces Species and Comparison with Pyrolysis-Mass Spectrometry and Conventional Biochemical Tests

PubMed Central

Hall, Val; O’Neill, G. L.; Magee, J. T.; Duerden, B. I.

1999-01-01

Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinical isolates (n = 36) of Actinomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination of HaeIII and HpaII endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification of Actinomyces spp. of clinical origin. PMID:10364594

Review of methods used for identification of biothreat agents in environmental protection and human health aspects.

PubMed

Mirski, Tomasz; Bartoszcze, Michał; Bielawska-Drózd, Agata; Cieślik, Piotr; Michalski, Aleksander J; Niemcewicz, Marcin; Kocik, Janusz; Chomiczewski, Krzysztof

2014-01-01

Modern threats of bioterrorism force the need to develop methods for rapid and accurate identification of dangerous biological agents. Currently, there are many types of methods used in this field of studies that are based on immunological or genetic techniques, or constitute a combination of both methods (immuno-genetic). There are also methods that have been developed on the basis of physical and chemical properties of the analytes. Each group of these analytical assays can be further divided into conventional methods (e.g. simple antigen-antibody reactions, classical PCR, real-time PCR), and modern technologies (e.g. microarray technology, aptamers, phosphors, etc.). Nanodiagnostics constitute another group of methods that utilize the objects at a nanoscale (below 100 nm). There are also integrated and automated diagnostic systems, which combine different methods and allow simultaneous sampling, extraction of genetic material and detection and identification of the analyte using genetic, as well as immunological techniques.

Automatic and rapid identification of glycopeptides by nano-UPLC-LTQ-FT-MS and proteomic search engine.

PubMed

Giménez, Estela; Gay, Marina; Vilaseca, Marta

2017-01-30

Here we demonstrate the potential of nano-UPLC-LTQ-FT-MS and the Byonic™ proteomic search engine for the separation, detection, and identification of N- and O-glycopeptide glycoforms in standard glycoproteins. The use of a BEH C18 nanoACQUITY column allowed the separation of the glycopeptides present in the glycoprotein digest and a baseline-resolution of the glycoforms of the same glycopeptide on the basis of the number of sialic acids. Moreover, we evaluated several acquisition strategies in order to improve the detection and characterization of glycopeptide glycoforms with the maximum number of identification percentages. The proposed strategy is simple to set up with the technology platforms commonly used in proteomic labs. The method allows the straightforward and rapid obtention of a general glycosylated map of a given protein, including glycosites and their corresponding glycosylated structures. The MS strategy selected in this work, based on a gas phase fractionation approach, led to 136 unique peptides from four standard proteins, which represented 78% of the total number of peptides identified. Moreover, the method does not require an extra glycopeptide enrichment step, thus preventing the bias that this step could cause towards certain glycopeptide species. Data are available via ProteomeXchange with identifier PXD003578. We propose a simple and high-throughput glycoproteomics-based methodology that allows the separation of glycopeptide glycoforms on the basis of the number of sialic acids, and their automatic and rapid identification without prior knowledge of protein glycosites or type and structure of the glycans. Copyright © 2016 Elsevier B.V. All rights reserved.

Yeast species associated with orange juice: evaluation of different identification methods.

PubMed

Arias, Covadonga R; Burns, Jacqueline K; Friedrich, Lorrie M; Goodrich, Renee M; Parish, Mickey E

2002-04-01

Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.

Evaluation of a rapid diagnostic field test kit for identification of Phytophthora ramorum, P. kernoviae and other Phytophthora species at the point of inspection

Treesearch

C.R. Lane; E. Hobden; L. Laurenson; V.C. Barton; K.J.D. Hughes; H. Swan; N. Boonham; A.J. Inman

2008-01-01

Plant health regulations to prevent the introduction and spread of Phytophthora ramorum and P. kernoviae require rapid, cost effective diagnostic methods for screening large numbers of plant samples at the time of inspection. Current on-site techniques require expensive equipment, considerable expertise and are not suited for plant...

[Evaluation of mass spectrometry: MALDI-TOF MS for fast and reliable yeast identification].

PubMed

Relloso, María S; Nievas, Jimena; Fares Taie, Santiago; Farquharson, Victoria; Mujica, María T; Romano, Vanesa; Zarate, Mariela S; Smayevsky, Jorgelina

2015-01-01

The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. 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.

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.

Rapid identification of Enterobacter hormaechei and Enterobacter cloacae genetic cluster III.

PubMed

Ohad, S; Block, C; Kravitz, V; Farber, A; Pilo, S; Breuer, R; Rorman, E

2014-05-01

Enterobacter cloacae complex bacteria are of both clinical and environmental importance. Phenotypic methods are unable to distinguish between some of the species in this complex, which often renders their identification incomplete. The goal of this study was to develop molecular assays to identify Enterobacter hormaechei and Ent. cloacae genetic cluster III which are relatively frequently encountered in clinical material. The molecular assays developed in this study are qPCR technology based and served to identify both Ent. hormaechei and Ent. cloacae genetic cluster III. qPCR results were compared to hsp60 sequence analysis. Most clinical isolates were assigned to Ent. hormaechei subsp. steigerwaltii and Ent. cloacae genetic cluster III. The latter was proportionately more frequently isolated from bloodstream infections than from other material (P < 0·05). The qPCR assays detecting Ent. hormaechei and Ent. cloacae genetic cluster III demonstrated high sensitivity and specificity. The presented qPCR assays allow accurate and rapid identification of clinical isolates of the Ent. cloacae complex. The improved identifications obtained can specifically assist analysis of Ent. hormaechei and Ent. cloacae genetic cluster III in nosocomial outbreaks and can promote rapid environmental monitoring. An association was observed between Ent. cloacae cluster III and systemic infection that deserves further attention. © 2014 The Society for Applied Microbiology.

DNA barcoding and morphological analysis for rapid identification of most economically important crop-infesting Sunn pests belonging to Eurygaster Laporte, 1833 (Hemiptera, Scutelleridae).

PubMed

Syromyatnikov, Mikhail Y; Golub, Victor B; Kokina, Anastasia V; Victoria A Soboleva; Popov, Vasily N

2017-01-01

The genus Eurygaster Laporte, 1833 includes ten species five of which inhabit the European part of Russia. The harmful species of the genus is E. integriceps . Eurygaster species identification based on the morphological traits is very difficult, while that of the species at the egg or larval stages is extremely difficult or impossible. Eurygaster integriceps , E. maura , and E. testudinaria differ only slightly between each other morphologically, E. maura and E. testudinaria being almost indiscernible. DNA barcoding based on COI sequences have shown that E. integriceps differs significantly from these closely related species, which enables its rapid and accurate identification. Based on COI nucleotide sequences, three species of Sunn pests, E. maura , E. testudinarius , E. dilaticollis , could not be differentiated from each other through DNA barcoding. The difference in the DNA sequences between the COI gene of E. integriceps and COI genes of E. maura and E. testudinarius was more than 4%. In the present study DNA barcoding of two Eurygaster species was performed for the first time on E. integriceps , the most dangerous pest in the genus, and E. dilaticollis that only inhabits natural ecosystems. The PCR-RFLP method was developed in this work for the rapid identification of E. integriceps .

DNA barcoding and morphological analysis for rapid identification of most economically important crop-infesting Sunn pests belonging to Eurygaster Laporte, 1833 (Hemiptera, Scutelleridae)

PubMed Central

Syromyatnikov, Mikhail Y.; Golub, Victor B.; Kokina, Anastasia V.; Victoria A. Soboleva; Popov, Vasily N.

2017-01-01

Abstract The genus Eurygaster Laporte, 1833 includes ten species five of which inhabit the European part of Russia. The harmful species of the genus is E. integriceps. Eurygaster species identification based on the morphological traits is very difficult, while that of the species at the egg or larval stages is extremely difficult or impossible. Eurygaster integriceps, E. maura, and E. testudinaria differ only slightly between each other morphologically, E. maura and E. testudinaria being almost indiscernible. DNA barcoding based on COI sequences have shown that E. integriceps differs significantly from these closely related species, which enables its rapid and accurate identification. Based on COI nucleotide sequences, three species of Sunn pests, E. maura, E. testudinarius, E. dilaticollis, could not be differentiated from each other through DNA barcoding. The difference in the DNA sequences between the COI gene of E. integriceps and COI genes of E. maura and E. testudinarius was more than 4%. In the present study DNA barcoding of two Eurygaster species was performed for the first time on E. integriceps, the most dangerous pest in the genus, and E. dilaticollis that only inhabits natural ecosystems. The PCR-RFLP method was developed in this work for the rapid identification of E. integriceps. PMID:29118620

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

PubMed

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

2017-12-18

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

A commentary on the role of molecular technology and automation in clinical diagnostics

PubMed Central

O’Connor, Ciara; Fitzgibbon, Marie; Powell, James; Barron, Denis; O’Mahony, Jim; Power, Lorraine; O’Connell, Nuala H; Dunne, Colum

2014-01-01

Historically, the identification of bacterial or yeast isolates has been based on phenotypic characteristics such as growth on defined media, colony morphology, Gram stain, and various biochemical reactions, with significant delay in diagnosis. Clinical microbiology as a medical specialty has embraced advances in molecular technology for rapid species identification with broad-range 16S rDNA polymerase chain reaction (PCR) and matrix-assisted laser desorption and/or ionization time of flight (MALDI-TOF) mass spectrometry demonstrated as accurate, rapid, and cost-effective methods for the identification of most, but not all, bacteria and yeasts. Protracted conventional incubation times previously necessary to identify certain species have been mitigated, affording patients quicker diagnosis with associated reduction in exposure to empiric broad-spectrum antimicrobial therapy and shortened hospital stay. This short commentary details such molecular advances and their implications in the clinical microbiology setting. PMID:24658184

Real-Time Continuous Identification of Greenhouse Plant Pathogens Based on Recyclable Microfluidic Bioassay System.

PubMed

Qu, Xiangmeng; Li, Min; Zhang, Hongbo; Lin, Chenglie; Wang, Fei; Xiao, Mingshu; Zhou, Yi; Shi, Jiye; Aldalbahi, Ali; Pei, Hao; Chen, Hong; Li, Li

2017-09-20

The development of a real-time continuous analytical platform for the pathogen detection is of great scientific importance for achieving better disease control and prevention. In this work, we report a rapid and recyclable microfluidic bioassay system constructed from oligonucleotide arrays for selective and sensitive continuous identification of DNA targets of fungal pathogens. We employ the thermal denaturation method to effectively regenerate the oligonucleotide arrays for multiple sample detection, which could considerably reduce the screening effort and costs. The combination of thermal denaturation and laser-induced fluorescence detection technique enables real-time continuous identification of multiple samples (<10 min per sample). As a proof of concept, we have demonstrated that two DNA targets of fungal pathogens (Botrytis cinerea and Didymella bryoniae) can be sequentially analyzed using our rapid microfluidic bioassay system, which provides a new paradigm in the design of microfluidic bioassay system and will be valuable for chemical and biomedical analysis.

Comparing two methods to promote generalization of receptive identification in children with autism spectrum disorders.

PubMed

Dufour, Marie-Michèle; Lanovaz, Marc J

2017-11-01

The purpose of our study was to compare the effects of serial and concurrent training on the generalization of receptive identification in children with autism spectrum disorders (ASD). We taught one to three pairs of stimulus sets to nine children with ASD between the ages of three and six. One stimulus set within each pair was taught using concurrent training and the other using serial training. We alternated the training sessions within a multielement design and staggered the introduction of subsequent pairs for each participant as in a multiple baseline design. Overall, six participants generalized at least one stimulus set more rapidly with concurrent training whereas two participants showed generalization more rapidly with serial training. Our results differ from other comparison studies on the topic and indicate that practitioners should consider assessing the effects of both procedures prior to teaching receptive identification to children with ASD.

Rapid and Accurate Identification of Animal Species in Natural Leather Goods by Liquid Chromatography/Mass Spectrometry

PubMed Central

Izuchi, Yukari; Takashima, Tsuneo; Hatano, Naoya

2016-01-01

The demand for leather goods has grown globally in recent years. Industry revenue is forecast to reach $91.2 billion by 2018. There is an ongoing labelling problem in the leather items market, in that it is currently impossible to identify the species that a given piece of leather is derived from. To address this issue, we developed a rapid and simple method for the specific identification of leather derived from cattle, horses, pigs, sheep, goats, and deer by analysing peptides produced by the trypsin-digestion of proteins contained in leather goods using liquid chromatography/mass spectrometry. We determined species-specific amino acid sequences by liquid chromatography/tandem mass spectrometry analysis using the Mascot software program and demonstrated that collagen α-1(I), collagen α-2(I), and collagen α-1(III) from the dermal layer of the skin are particularly useful in species identification. PMID:27313979

A commentary on the role of molecular technology and automation in clinical diagnostics.

PubMed

O'Connor, Ciara; Fitzgibbon, Marie; Powell, James; Barron, Denis; O'Mahony, Jim; Power, Lorraine; O'Connell, Nuala H; Dunne, Colum

2014-01-01

Historically, the identification of bacterial or yeast isolates has been based on phenotypic characteristics such as growth on defined media, colony morphology, Gram stain, and various biochemical reactions, with significant delay in diagnosis. Clinical microbiology as a medical specialty has embraced advances in molecular technology for rapid species identification with broad-range 16S rDNA polymerase chain reaction (PCR) and matrix-assisted laser desorption and/or ionization time of flight (MALDI-TOF) mass spectrometry demonstrated as accurate, rapid, and cost-effective methods for the identification of most, but not all, bacteria and yeasts. Protracted conventional incubation times previously necessary to identify certain species have been mitigated, affording patients quicker diagnosis with associated reduction in exposure to empiric broad-spectrum antimicrobial therapy and shortened hospital stay. This short commentary details such molecular advances and their implications in the clinical microbiology setting.

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

The use of immunochromatographic rapid test for soft tissue remains identification in order to distinguish between human and non-human origin.

PubMed

Gascho, Dominic; Morf, Nadja V; Thali, Michael J; Schaerli, Sarah

2017-05-01

Clear identification of soft tissue remains as being of non-human origin may be visually difficult in some cases e.g. due to decomposition. Thus, an additional examination is required. The use of an immunochromatographic rapid tests (IRT) device can be an easy solution with the additional advantage to be used directly at the site of discovery. The use of these test devices for detecting human blood at crime scenes is a common method. However, the IRT is specific not only for blood but also for differentiation between human and non-human soft tissue remains. In the following this method is discussed and validated by means of two forensic cases and several samples of various animals. Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.

Screening and identification of radical scavengers from Neo-Taraxacum siphonanthum by online rapid screening method and nuclear magnetic resonance experiments.

PubMed

Shi, Shu Yun; Zhang, Yu Ping; Zhou, Hong Hao; Huang, Ke Long; Jiang, Xin Yu

2010-01-01

An online rapid screening method, the high-performance liquid chromatography (HPLC)-diode array detector (DAD)-radical scavenging detection (RSD)-electrospray ionization (ESI)-mass spectroscopy (MS)/MS system, was developed for the screening and identification of radical scavengers from Neo-Taraxacum siphonanthum, a new species found in China in 1989. For further characterization, the target compounds were isolated by silica column chromatography, preparative high-performance liquid chromatography (HPLC), HSCCC, and Sephadex LH-20 column chromatography and elucidated on the basis of ultraviolet (UV), ESI-MS/MS, and nuclear magnetic resonance (NMR) spectroscopy, as well as the chemical analysis. Eighteen antioxidative polyphenols (5 caffeic acid derivatives and 13 flavonoid derivatives) were characterized from Neo-T. siphonanthum. The distribution of all compounds was discussed in a chemosystematic context, which suggested that the genera Neo-Taraxacum and Taraxacum might relate chemosystematically.

Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

PubMed

Wang, Deguo; Liu, Yanhong

2015-05-26

Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies.

Rapid authentication and identification of different types of A. roxburghii by Tri-step FT-IR spectroscopy

NASA Astrophysics Data System (ADS)

Chen, Ying; Huang, Jinfang; Yeap, Zhao Qin; Zhang, Xue; Wu, Shuisheng; Ng, Chiew Hoong; Yam, Mun Fei

2018-06-01

Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) is a precious traditional Chinese medicinal herb and has been perennially used to treat various illness. However, there were unethical sellers who adulterated wild A. roxburghii with tissue cultured and cultivated ones. Therefore, there is an urgent need for an effective authentication method to differentiate between these different types of A. roxburghii. In this research, the infrared spectroscopic tri-step identification approach including Fourier transform infrared spectroscopy (FT-IR), Second derivative infrared spectra (SD-IR) and two-dimensional correlation infrared spectra (2D-IR) was used to develop a simple and rapid method to discriminate between wild, cultivated and tissue cultivated A. roxburghii plant. Through this study, all three types of A. roxburghii plant were successfully identified and discriminated through the infrared spectroscopic tri-step identification method. Besides that, all the samples of wild, cultivated and tissue cultivated A. roxburghii plant were analysed with the Soft Independent Modelling of Class Analogy (SIMCA) pattern recognition technique to test and verify the experimental results. The results showed that the three types of A. roxburghii can be discriminated clearly as the recognition rate was 100% for all three types and the rejection rate was more than 60%. 70% of the validated samples were also identified correctly by the SIMCA model. The SIMCA model was also validated by comparing 70 standard herbs to the model. As a result, it was demonstrated that the macroscopic IR fingerprint method and the classification analysis could discriminate not only between the A. roxburghi samples and the standard herbs, it could also distinguish between the three different types of A. roxburghi plant in a direct, rapid and holistic manner.

Rapid authentication and identification of different types of A. roxburghii by Tri-step FT-IR spectroscopy.

PubMed

Chen, Ying; Huang, Jinfang; Yeap, Zhao Qin; Zhang, Xue; Wu, Shuisheng; Ng, Chiew Hoong; Yam, Mun Fei

2018-06-15

Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) is a precious traditional Chinese medicinal herb and has been perennially used to treat various illness. However, there were unethical sellers who adulterated wild A. roxburghii with tissue cultured and cultivated ones. Therefore, there is an urgent need for an effective authentication method to differentiate between these different types of A. roxburghii. In this research, the infrared spectroscopic tri-step identification approach including Fourier transform infrared spectroscopy (FT-IR), Second derivative infrared spectra (SD-IR) and two-dimensional correlation infrared spectra (2D-IR) was used to develop a simple and rapid method to discriminate between wild, cultivated and tissue cultivated A. roxburghii plant. Through this study, all three types of A. roxburghii plant were successfully identified and discriminated through the infrared spectroscopic tri-step identification method. Besides that, all the samples of wild, cultivated and tissue cultivated A. roxburghii plant were analysed with the Soft Independent Modelling of Class Analogy (SIMCA) pattern recognition technique to test and verify the experimental results. The results showed that the three types of A. roxburghii can be discriminated clearly as the recognition rate was 100% for all three types and the rejection rate was more than 60%. 70% of the validated samples were also identified correctly by the SIMCA model. The SIMCA model was also validated by comparing 70 standard herbs to the model. As a result, it was demonstrated that the macroscopic IR fingerprint method and the classification analysis could discriminate not only between the A. roxburghi samples and the standard herbs, it could also distinguish between the three different types of A. roxburghi plant in a direct, rapid and holistic manner. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid screening of guar gum using portable Raman spectral identification methods.

PubMed

Srivastava, Hirsch K; Wolfgang, Steven; Rodriguez, Jason D

2016-01-25

Guar gum is a well-known inactive ingredient (excipient) used in a variety of oral pharmaceutical dosage forms as a thickener and stabilizer of suspensions and as a binder of powders. It is also widely used as a food ingredient in which case alternatives with similar properties, including chemically similar gums, are readily available. Recent supply shortages and price fluctuations have caused guar gum to come under increasing scrutiny for possible adulteration by substitution of cheaper alternatives. One way that the U.S. FDA is attempting to screen pharmaceutical ingredients at risk for adulteration or substitution is through field-deployable spectroscopic screening. Here we report a comprehensive approach to evaluate two field-deployable Raman methods--spectral correlation and principal component analysis--to differentiate guar gum from other gums. We report a comparison of the sensitivity of the spectroscopic screening methods with current compendial identification tests. The ability of the spectroscopic methods to perform unambiguous identification of guar gum compared to other gums makes them an enhanced surveillance alternative to the current compendial identification tests, which are largely subjective in nature. Our findings indicate that Raman spectral identification methods perform better than compendial identification methods and are able to distinguish guar gum from other gums with 100% accuracy for samples tested by spectral correlation and principal component analysis. Published by Elsevier B.V.

Simultaneous identification of natural dyes in the collection of drawings and maps from The Royal Chancellery Archives in Granada (Spain) by CE.

PubMed

López-Montes, Ana; Blanc García, Rosario; Espejo, Teresa; Huertas-Perez, José F; Navalón, Alberto; Vílchez, José Luis

2007-04-01

A simple and rapid capillary electrophoretic method with UV detection (CE-UV) has been developed for the identification of five natural dyes namely, carmine, indigo, saffron, gamboge and Rubia tinctoria root. The separation was performed in a fused-silica capillary of 64.5 cm length and 50 microm id. The running buffer was 40 mM sodium tetraborate buffer solution (pH 9.25). The applied potential was 30 kV, the temperature was 25 degrees C and detections were performed at 196, 232, 252, 300 and 356 nm. The injections were under pressure of 50 mbar during 13 s. The method was applied to the identification of carminic acid, gambogic acid, crocetin, indigotin, alizarin and purpurin in the collection of drawings and maps at the Royal Chancellery Archives in Granada (Spain). The method was validated by using HPLC as a reference method.

Rapid identification of Bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

PubMed

Dybwad, Marius; van der Laaken, Anton L; Blatny, Janet Martha; Paauw, Armand

2013-09-01

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.

Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

PubMed Central

van der Laaken, Anton L.; Blatny, Janet Martha; Paauw, Armand

2013-01-01

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 106 spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min. PMID:23811517

The status of Phytophthora ramorum in Ireland

Treesearch

Carmel O?Connor; Elizabeth Gosling

2008-01-01

This paper reports on the first 2 years of data collected to study the ecology of Phytophthora ramorum in Ireland. Since spring 2005, sampling has been carried out for the presence of the pathogen in soil and watercourses from 11 susceptible forest sites in Ireland, using a rapid DNA method in conjunction with morphological identification methods....

Einstein Slew Survey: Data analysis innovations

NASA Technical Reports Server (NTRS)

Elvis, Martin S.; Plummer, David; Schachter, Jonathan F.; Fabbiano, G.

1992-01-01

Several new methods were needed in order to make the Einstein Slew X-ray Sky Survey. The innovations which enabled the Slew Survey to be done are summarized. These methods included experimental approach to large projects, parallel processing on a LAN, percolation source detection, minimum action identifications, and rapid dissemination of the whole data base.

Selective One-Dimensional Total Correlation Spectroscopy Nuclear Magnetic Resonance Experiments for a Rapid Identification of Minor Components in the Lipid Fraction of Milk and Dairy Products: Toward Spin Chromatography?

PubMed

Papaemmanouil, Christina; Tsiafoulis, Constantinos G; Alivertis, Dimitrios; Tzamaloukas, Ouranios; Miltiadou, Despoina; Tzakos, Andreas G; Gerothanassis, Ioannis P

2015-06-10

We report a rapid, direct, and unequivocal spin-chromatographic separation and identification of minor components in the lipid fraction of milk and common dairy products with the use of selective one-dimensional (1D) total correlation spectroscopy (TOCSY) nuclear magnetic resonance (NMR) experiments. The method allows for the complete backbone spin-coupling network to be elucidated even in strongly overlapped regions and in the presence of major components from 4 × 10(2) to 3 × 10(3) stronger NMR signal intensities. The proposed spin-chromatography method does not require any derivatization steps for the lipid fraction, is selective with excellent resolution, is sensitive with quantitation capability, and compares favorably to two-dimensional (2D) TOCSY and gas chromatography-mass spectrometry (GC-MS) methods of analysis. The results of the present study demonstrated that the 1D TOCSY NMR spin-chromatography method can become a procedure of primary interest in food analysis and generally in complex mixture analysis.

Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview.

PubMed

Deshmukh, Rehan A; Joshi, Kopal; Bhand, Sunil; Roy, Utpal

2016-12-01

Waterborne diseases have emerged as global health problems and their rapid and sensitive detection in environmental water samples is of great importance. Bacterial identification and enumeration in water samples is significant as it helps to maintain safe drinking water for public consumption. Culture-based methods are laborious, time-consuming, and yield false-positive results, whereas viable but nonculturable (VBNCs) microorganisms cannot be recovered. Hence, numerous methods have been developed for rapid detection and quantification of waterborne pathogenic bacteria in water. These rapid methods can be classified into nucleic acid-based, immunology-based, and biosensor-based detection methods. This review summarizes the principle and current state of rapid methods for the monitoring and detection of waterborne bacterial pathogens. Rapid methods outlined are polymerase chain reaction (PCR), digital droplet PCR, real-time PCR, multiplex PCR, DNA microarray, Next-generation sequencing (pyrosequencing, Illumina technology and genomics), and fluorescence in situ hybridization that are categorized as nucleic acid-based methods. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are classified into immunology-based methods. Optical, electrochemical, and mass-based biosensors are grouped into biosensor-based methods. Overall, these methods are sensitive, specific, time-effective, and important in prevention and diagnosis of waterborne bacterial diseases. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

NMR experiments for the rapid identification of P=O···H-X type hydrogen bonds in nucleic acids.

PubMed

Duchardt-Ferner, Elke; Wöhnert, Jens

2017-10-01

Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927-7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1 H, 31 P-correlation experiments as well as a BEST-HNP experiment exploiting 3h J N,P rather than 2h J H,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~ 100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2'-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.

Rapid Characterization and Identification of Flavonoids in Radix Astragali by Ultra-High-Pressure Liquid Chromatography Coupled with Linear Ion Trap-Orbitrap Mass Spectrometry.

PubMed

Zhang, Jing; Xu, Xiao-Jie; Xu, Wen; Huang, Juan; Zhu, Da-yuan; Qiu, Xiao-Hui

2015-07-01

A simple and effective method was established for separation and characterization of flavonoid constituents in Radix Astragali (RA) by combination of ultra-high-pressure liquid chromatography with LTQ-Orbitrap tandem mass spectrometry (u-HPLC-LTQ-Orbitrap-MS(n)). For three major structural types of flavonoids, the proposed fragmentation pathways and major diagnostic fragment ions of isoflavones, pterocarpans and isoflavans were investigated to trace isoflavonoid derivatives in crude plant extracts. Based on the systematic identification strategy, 48 constituents were rapidly detected and characterized or tentatively identified, many of which were first reported in RA. The u-PHLC-LTQ-Orbitrap MS(n) platform was proved as an effective tool for rapid qualitative analysis of secondary metabolite productions from natural resources. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Neutral Fragment Filtering for Rapid Identification of New Diester-Diterpenoid Alkaloids in Roots of Aconitum carmichaeli by Ultra-High-Pressure Liquid Chromatography Coupled with Linear Ion Trap-Orbitrap Mass Spectrometry

PubMed Central

Qiu, Xiao Hui; Yang, Yi Ming; Zhu, Da Yuan; Xu, Wen

2012-01-01

A rapid and effective method was developed for separation and identification of diester-diterpenoid alkaloids (DDA) in the roots of Aconitum carmichaeli by ultra-high-pressure liquid chromatography coupled with high resolution LTQ-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MSn). According to accurate mass measurement and the characteristic neutral loss filtering strategy, a total of 42 diester-diterpenoid alkaloids (DDA) were rapidly detected and characterized or tentatively identified. Meanwhile, the proposed fragmentation pathways and the major diagnostic fragment ions of aconitine, mesaconitine and hypaconitine were investigated to trace DDA derivatives in crude plant extracts. 23 potential new compounds were successfully screened and characterized in Aconitum carmichaeli, including 16 short chain fatty acyls DDA, 4 N-dealkyl DDA and several isomers of aconitine, mesaconitine and hypaconitine. PMID:23285005

Identification of the Species of Origin for Meat Products by Rapid Evaporative Ionization Mass Spectrometry.

PubMed

Balog, Julia; Perenyi, Dora; Guallar-Hoyas, Cristina; Egri, Attila; Pringle, Steven D; Stead, Sara; Chevallier, Olivier P; Elliott, Chris T; Takats, Zoltan

2016-06-15

Increasingly abundant food fraud cases have brought food authenticity and safety into major focus. This study presents a fast and effective way to identify meat products using rapid evaporative ionization mass spectrometry (REIMS). The experimental setup was demonstrated to be able to record a mass spectrometric profile of meat specimens in a time frame of <5 s. A multivariate statistical algorithm was developed and successfully tested for the identification of animal tissue with different anatomical origin, breed, and species with 100% accuracy at species and 97% accuracy at breed level. Detection of the presence of meat originating from a different species (horse, cattle, and venison) has also been demonstrated with high accuracy using mixed patties with a 5% detection limit. REIMS technology was found to be a promising tool in food safety applications providing a reliable and simple method for the rapid characterization of food products.

Rapid diagnostic methods for influenza virus in clinical specimens - A comparative study

NASA Technical Reports Server (NTRS)

Evans, A. S.; Olson, B.

1982-01-01

A comparison of five rapid viral diagnostic techniques for identifying influenza virus in nasopharyngeal aspirates has been made on patients with influenza-like illnesses. Initial results with immune electron microscopy were positive in only one of 11 specimens from which virus was isolated and further work abandoned. Four other rapid tests were carried out on 39 specimens from which influenza virus had been isolated in tissue culture in 28. Of these 28 specimens yielding virus, 24 (85.7 percent) were positive by an indirect fluorescent antibody test (IFAT) on nasopharyngeal cells, 18 (64.3 percent) by enzyme-linked immunosorbent assay (ELISA), 19 (67.8 percent) by enzyme-linked fluorescent assay (ELFA), and 26 (92.8 percent) by a rapid tissue culture amplification method (TCA) in a continuous Rhesus monkey kidney line (LLC-MK2) with identification of virus by fluorescent antibody. In terms of sensitivity, simplicity, and rapidity, a combination of the IFAT and TCA methods seems to be very useful.

Automatic identification of the reference system based on the fourth ventricular landmarks in T1-weighted MR images.

PubMed

Fu, Yili; Gao, Wenpeng; Chen, Xiaoguang; Zhu, Minwei; Shen, Weigao; Wang, Shuguo

2010-01-01

The reference system based on the fourth ventricular landmarks (including the fastigial point and ventricular floor plane) is used in medical image analysis of the brain stem. The objective of this study was to develop a rapid, robust, and accurate method for the automatic identification of this reference system on T1-weighted magnetic resonance images. The fully automated method developed in this study consisted of four stages: preprocessing of the data set, expectation-maximization algorithm-based extraction of the fourth ventricle in the region of interest, a coarse-to-fine strategy for identifying the fastigial point, and localization of the base point. The method was evaluated on 27 Brain Web data sets qualitatively and 18 Internet Brain Segmentation Repository data sets and 30 clinical scans quantitatively. The results of qualitative evaluation indicated that the method was robust to rotation, landmark variation, noise, and inhomogeneity. The results of quantitative evaluation indicated that the method was able to identify the reference system with an accuracy of 0.7 +/- 0.2 mm for the fastigial point and 1.1 +/- 0.3 mm for the base point. It took <6 seconds for the method to identify the related landmarks on a personal computer with an Intel Core 2 6300 processor and 2 GB of random-access memory. The proposed method for the automatic identification of the reference system based on the fourth ventricular landmarks was shown to be rapid, robust, and accurate. The method has potentially utility in image registration and computer-aided surgery.

Real-time PCR detection of Listeria monocytogenes in infant formula and lettuce following macrophage-based isolation and enrichment.

PubMed

Day, J B; Basavanna, U

2015-01-01

To develop a rapid detection procedure for Listeria monocytogenes in infant formula and lettuce using a macrophage-based enrichment protocol and real-time PCR. A macrophage cell culture system was employed for the isolation and enrichment of L. monocytogenes from infant formula and lettuce for subsequent identification using real-time PCR. Macrophage monolayers were exposed to infant formula and lettuce contaminated with a serial dilution series of L. monocytogenes. As few as approx. 10 CFU ml(-1) or g(-1) of L. monocytogenes were detected in infant formula and lettuce after 16 h postinfection by real-time PCR. Internal positive PCR controls were utilized to eliminate the possibility of false-negative results. Co-inoculation with Listeria innocua did not reduce the L. monocytogenes detection sensitivity. Intracellular L. monocytogenes could also be isolated on Listeria selective media from infected macrophage lysates for subsequent confirmation. The detection method is highly sensitive and specific for L. monocytogenes in infant formula and lettuce and establishes a rapid identification time of 20 and 48 h for presumptive and confirmatory identification, respectively. The method is a promising alternative to many currently used q-PCR detection methods which employ traditional selective media for enrichment of contaminated food samples. Macrophage enrichment of L. monocytogenes eliminates PCR inhibitory food elements and contaminating food microflora which produce cleaner samples that increase the rapidity and sensitivity of detection. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Nonenzymatic microorganism identification based on ribosomal RNA

NASA Astrophysics Data System (ADS)

Ives, Jeffrey T.; Pierini, Alicia M.; Stokes, Jeffrey A.; Wahlund, Thomas M.; Read, Betsy; Bechtel, James H.; Bronk, Burt V.

1999-11-01

Effective defense against biological warfare (BW) agents requires rapid, fieldable and accurate systems. For micro- organisms like bacteria and viruses, ribosomal RNA (rRNA) provides a valuable target with multiple advantages of species specificity and intrinsic target amplification. Vegetative and spore forms of bacteria contain approximately 104 copies of rRNA. Direct detection of rRNA copies can eliminate some of the interference and preparation difficulties involved in enzymatic amplification methods. In order to apply the advantages of rRNA to BW defense, we are developing a fieldable system based on 16S rRNA, physical disruption of the micro-organism, solid phase hybridization, and fluorescence detection. Our goals include species-specific identification, complete operation from raw sample to identification in 15 minutes or less, and compact, fieldable instrumentation. Initial work on this project has investigated the lysis and hybridization steps, the species-specificity of oligonucleotides probes, and the development of a novel electromagnetic method to physically disrupt the micro- organisms. Target bacteria have been Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Continuing work includes further development of methods to rapidly disrupt the micro-organisms and release the rRNA, improved integration and processing, and extension to bacterial and mammalian viruses like MS2 and vesicular stomatitis virus.

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.

Recommendations for Improving Identification and Quantification in Non-Targeted, GC-MS-Based Metabolomic Profiling of Human Plasma

PubMed Central

Wang, Hanghang; Muehlbauer, Michael J.; O’Neal, Sara K.; Newgard, Christopher B.; Hauser, Elizabeth R.; Shah, Svati H.

2017-01-01

The field of metabolomics as applied to human disease and health is rapidly expanding. In recent efforts of metabolomics research, greater emphasis has been placed on quality control and method validation. In this study, we report an experience with quality control and a practical application of method validation. Specifically, we sought to identify and modify steps in gas chromatography-mass spectrometry (GC-MS)-based, non-targeted metabolomic profiling of human plasma that could influence metabolite identification and quantification. Our experimental design included two studies: (1) a limiting-dilution study, which investigated the effects of dilution on analyte identification and quantification; and (2) a concentration-specific study, which compared the optimal plasma extract volume established in the first study with the volume used in the current institutional protocol. We confirmed that contaminants, concentration, repeatability and intermediate precision are major factors influencing metabolite identification and quantification. In addition, we established methods for improved metabolite identification and quantification, which were summarized to provide recommendations for experimental design of GC-MS-based non-targeted profiling of human plasma. PMID:28841195

Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

PubMed

Alibi, S; Ferjani, A; Gaillot, O; Marzouk, M; Courcol, R; Boukadida, J

2015-09-01

We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods. Copyright © 2015. Published by Elsevier SAS.

Identification of clinical yeasts by Vitek MS system compared with API ID 32 C.

PubMed

Durán-Valle, M Teresa; Sanz-Rodríguez, Nuria; Muñoz-Paraíso, Carmen; Almagro-Moltó, María; Gómez-Garcés, José Luis

2014-05-01

We performed a clinical evaluation of the Vitek MS matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) system with the commercial database version 2.0 for rapid identification of medically important yeasts as compared with the conventional phenotypic method API ID 32 C. We tested 161 clinical isolates, nine isolates from culture collections and five reference strains. In case of discrepant results or no identification with one or both methods, molecular identification techniques were employed. Concordance between both methods was observed with 160/175 isolates (91.42%) and misidentifications by both systems occurred only when taxa were not included in the respective databases, i.e., one isolate of Candida etchellsii was identified as C. globosa by Vitek MS and two isolates of C. orthopsilosis were identified as C. parapsilosis by API ID 32 C. Vitek MS could not identify nine strains (5.14%) and API ID 32 C did not identify 13 (7.42%). Vitek MS was more reliable than API ID 32 C and reduced the time required for the identification of clinical isolates to only a few minutes.

Protein Chips for Detection of Salmonella spp. from Enrichment Culture

PubMed Central

Poltronieri, Palmiro; Cimaglia, Fabio; De Lorenzis, Enrico; Chiesa, Maurizio; Mezzolla, Valeria; Reca, Ida Barbara

2016-01-01

Food pathogens are the cause of foodborne epidemics, therefore there is a need to detect the pathogens in food productions rapidly. A pre-enrichment culture followed by selective agar plating are standard detection methods. Molecular methods such as qPCR have provided a first rapid protocol for detection of pathogens within 24 h of enrichment culture. Biosensors also may provide a rapid tool to individuate a source of Salmonella contamination at early times of pre-enrichment culture. Forty mL of Salmonella spp. enrichment culture were processed by immunoseparation using the Pathatrix, as in AFNOR validated qPCR protocols. The Salmonella biosensor combined with immunoseparation showed a limit of detection of 100 bacteria/40 mL, with a 400 fold increase to previous results. qPCR analysis requires processing of bead-bound bacteria with lysis buffer and DNA clean up, with a limit of detection of 2 cfu/50 μL. Finally, a protein chip was developed and tested in screening and identification of 5 common pathogen species, Salmonella spp., E. coli, S. aureus, Campylobacter spp. and Listeria spp. The protein chip, with high specificity in species identification, is proposed to be integrated into a Lab-on-Chip system, for rapid and reproducible screening of Salmonella spp. and other pathogen species contaminating food productions. PMID:27110786

A simple and rapid electrophoretic method to characterize simple phenols, lignans, complex phenols, phenolic acids, and flavonoids in extra-virgin olive oil.

PubMed

Carrasco-Pancorbo, Alegria; Gómez-Caravaca, Ana Maria; Cerretani, Lorenzo; Bendini, Alessandra; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

2006-09-01

We have devised a simple and rapid capillary electrophoretic method which provides the analyst with a useful tool for the characterization of the polyphenolic fraction of extra-virgin olive oil. This method that uses a capillary with 50 microm id and a total length of 47 cm (40 cm to the detector) with a detection window of 100 x 200 microm, and a buffer solution containing 45 mM of sodium tetraborate pH 9.3 offers valuable information about all the families of compounds present in the polar fraction of the olive oil. The detection was carried out by UV absorption at 200, 240, 280, and 330 nm in order to facilitate the identification of the compounds. Concretely, the method permits the identification of simple phenols, lignans, complex phenols (isomeric forms of secoiridoids), phenolic acids, and flavonoids in the SPE-Diol extracts from extra-virgin olive oil in a short time (less than 10 min) and provides a satisfactory resolution. Peak identification was done by comparing both migration time and spectral data obtained from olive oil samples and standards (commercial or isolated (by HPLC-MS) standards), with spiked methanol-water extracts of olive oil with HPLC-collected compounds and commercially available standards at several concentration levels, studying the information of the electropherograms obtained at several wavelengths and also using the information previously reported.

Extraction and identification of cyclobutanones from irradiated cheese employing a rapid direct solvent extraction method.

PubMed

Tewfik, Ihab

2008-01-01

2-Alkylcyclobutanones (cyclobutanones) are accepted as chemical markers for irradiated foods containing lipid. However, current extraction procedures (Soxhlet-florisil chromatography) for the isolation of these markers involve a long and tedious clean-up regime prior to gas chromatography-mass spectrophotometry identification. This paper outlines an alternative isolation and clean-up method for the extraction of cyclobutanones in irradiated Camembert cheese. The newly developed direct solvent extraction method enables the efficient screening of large numbers of food samples and is not as resource intensive as the BS EN 1785:1997 method. Direct solvent extraction appears to be a simple, robust method and has the added advantage of a considerably shorter extraction time for the analysis of foods containing lipid.

Molecular Identification and Databases in Fusarium

USDA-ARS?s Scientific Manuscript database

DNA sequence-based methods for identifying pathogenic and mycotoxigenic Fusarium isolates have become the gold standard worldwide. Moreover, fusarial DNA sequence data are increasing rapidly in several web-accessible databases for comparative purposes. Unfortunately, the use of Basic Alignment Sea...

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

PubMed

Chen, Shi-Lin; Yao, Hui; Han, Jian-Ping; Xin, Tian-Yi; Pang, Xiao-Hui; Shi, Lin-Chun; Luo, Kun; Song, Jing-Yuan; Hou, Dian-Yun; Shi, Shang-Mei; Qian, Zhong-Zhi

2013-01-01

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

Identification of Pseudallescheria and Scedosporium species by three molecular methods.

PubMed

Lu, Qiaoyun; Gerrits van den Ende, A H G; Bakkers, J M J E; Sun, Jiufeng; Lackner, M; Najafzadeh, M J; Melchers, W J G; Li, Ruoyu; de Hoog, G S

2011-03-01

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction.

PubMed

Meex, Cécile; Neuville, Florence; Descy, Julie; Huynen, Pascale; Hayette, Marie-Pierre; De Mol, Patrick; Melin, Pierrette

2012-11-01

In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66 % of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52 % of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90 % of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method.

The use of real-time polymerase chain reaction for rapid diagnosis of skeletal tuberculosis.

PubMed

Kobayashi, Naomi; Fraser, Thomas G; Bauer, Thomas W; Joyce, Michael J; Hall, Gerri S; Tuohy, Marion J; Procop, Gary W

2006-07-01

We identified Mycobacterium tuberculosis DNA using real-time polymerase chain reaction on a specimen from an osteolytic lesion of a femoral condyle, in which the frozen section demonstrated granulomas. The process was much more rapid than is possible with culture. The rapid detection of M tuberculosis and the concomitant exclusion of granulomatous disease caused by nontuberculous mycobacteria or systemic fungi are necessary to appropriately treat skeletal tuberculosis. The detection and identification of M tuberculosis by culture may require several weeks using traditional methods. The real-time polymerase chain reaction method used has been shown to be rapid and reliable, and is able to detect and differentiate both tuberculous and nontuberculous mycobacteria. Real-time polymerase chain reaction may become a diagnostic standard for the evaluation of clinical specimens for the presence of mycobacteria; this case demonstrates the potential utility of this assay for the rapid diagnosis of skeletal tuberculosis.

The Changing Role of the Clinical Microbiology Laboratory in Defining Resistance in Gram-negatives.

PubMed

Endimiani, Andrea; Jacobs, Michael R

2016-06-01

The evolution of resistance in Gram-negatives has challenged the clinical microbiology laboratory to implement new methods for their detection. Multidrug-resistant strains present major challenges to conventional and new detection methods. More rapid pathogen identification and antimicrobial susceptibility testing have been developed for use directly on specimens, including fluorescence in situ hybridization tests, automated polymerase chain reaction systems, microarrays, mass spectroscopy, next-generation sequencing, and microfluidics. Review of these methods shows the advances that have been made in rapid detection of resistance in cultures, but limited progress in direct detection from specimens. Copyright © 2016 Elsevier Inc. All rights reserved.

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

PubMed

Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

2014-03-07

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Rapid Detection of the Chlamydiaceae and Other Families in the Order Chlamydiales: Three PCR Tests

PubMed Central

Everett, Karin D. E.; Hornung, Linda J.; Andersen, Arthur A.

1999-01-01

Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation. PMID:9986815

Frequency regularities of acoustic modes and multi-colour mode identification in rapidly rotating stars

NASA Astrophysics Data System (ADS)

Reese, D. R.; Lignières, F.; Ballot, J.; Dupret, M.-A.; Barban, C.; van't Veer-Menneret, C.; MacGregor, K. B.

2017-05-01

Context. Mode identification has remained a major obstacle in the interpretation of pulsation spectra in rapidly rotating stars. This has motivated recent work on calculating realistic multi-colour mode visibilities in this type of star. Aims: We would like to test mode identification methods and seismic diagnostics in rapidly rotating stars, using oscillation spectra that are based on these new theoretical predictions. Methods: We investigate the auto-correlation function and Fourier transform of theoretically calculated frequency spectra, in which modes are selected according to their visibilities. Given that intrinsic mode amplitudes are determined by non-linear saturation and cannot currently be theoretically predicted, we experimented with various ad-hoc prescriptions for setting the mode amplitudes, including using random values. Furthermore, we analyse the ratios between mode amplitudes observed in different photometric bands to see up to what extent they can identify modes. Results: When non-random intrinsic mode amplitudes are used, our results show that it is possible to extract a mean value for the large frequency separation or half its value and, sometimes, twice the rotation rate, from the auto-correlation of the frequency spectra. Furthermore, the Fourier transforms are mostly sensitive to the large frequency separation or half its value. The combination of the two methods may therefore measure and distinguish the two types of separations. When the intrinsic mode amplitudes include random factors, which seems more representative of real stars, the results are far less favourable. It is only when the large separation or half its value coincides with twice the rotation rate, that it might be possible to detect the signature of a frequency regularity. We also find that amplitude ratios are a good way of grouping together modes with similar characteristics. By analysing the frequencies of these groups, it is possible to constrain mode identification, as well as determine the large frequency separation and the rotation rate.

Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory.

PubMed

Rodríguez-Sánchez, Belén; Marín, Mercedes; Sánchez-Carrillo, Carlos; Cercenado, Emilia; Ruiz, Adrián; Rodríguez-Créixems, Marta; Bouza, Emilio

2014-05-01

This study evaluates matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following 2 methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%), we obtained a reliable identification by MALDI-TOF: in 103 isolates, the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result), and in 18 isolates, the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods. Copyright © 2014 Elsevier Inc. All rights reserved.

Prospective evaluation of a high multiplexing real-time polymerase chain reaction array for the rapid identification and characterization of bacteria causative of nosocomial pneumonia from clinical specimens: a proof-of-concept study.

PubMed

Roisin, S; Huang, T-D; de Mendonça, R; Nonhoff, C; Bogaerts, P; Hites, M; Delaere, B; Hamels, S; de Longueville, F; Glupczynski, Y; Denis, O

2018-01-01

The purpose of this study was evaluation of the VAPChip assay based on the "Rapid-Array-PCR-technology" which targets 13 respiratory pathogens and 24 β-lactam resistance genes directly on respiratory clinical specimens. The first step included analysis of 45 respiratory specimens in order to calibrate and determine the threshold for target genes. The second prospective step involved 85 respiratory samples from patients suspected of nosocomial pneumonia collected in two academic hospitals over an 8-month period. Results of the VAPChip assay were compared to routine methods. The first step showed a large proportion of positive signals for H. influenzae and/or S. pneumoniae. For identification, discrepancies were observed in seven samples. Thresholds were adapted and two probes were re-designed to create a new version of the cartridge. In the second phase, sensitivity and specificity of the VAPchip for bacterial identification were 72.9% and 99.1%, respectively. Seventy (82%) pathogens were correctly identified by both methods. Nine pathogens detected by the VAPChip were culture negative and 26 pathogens identified by culture were VAPChip negative. For resistance mechanisms, 11 probes were positive without identification of pathogens with an antimicrobial-susceptibility testing compatible by culture. However, the patient's recent microbiological history was able to explain most of these positive signals. The VAPChip assay simultaneously detects different pathogens and resistance mechanisms directly from clinical samples. This system seems very promising but the extraction process needs to be automated for routine implementation. This kind of rapid point-of-care automated platform permitting a syndromic approach will be the future challenge in the management of infectious diseases.

An unusual method of forensic human identification: use of selfie photographs.

PubMed

Miranda, Geraldo Elias; Freitas, Sílvia Guzella de; Maia, Luiza Valéria de Abreu; Melani, Rodolfo Francisco Haltenhoff

2016-06-01

As with other methods of identification, in forensic odontology, antemortem data are compared with postmortem findings. In the absence of dental documentation, photographs of the smile play an important role in this comparison. As yet, there are no reports of the use of the selfie photograph for identification purposes. Owing to advancements in technology, electronic devices, and social networks, this type of photograph has become increasingly common. This paper describes a case in which selfie photographs were used to identify a carbonized body, by using the smile line and image superimposition. This low-cost, rapid, and easy to analyze technique provides highly reliable results. Nevertheless, there are disadvantages, such as the limited number of teeth that are visible in a photograph, low image quality, possibility of morphological changes in the teeth after the antemortem image was taken, and difficulty of making comparisons depending on the orientation of the photo. In forensic odontology, new methods of identification must be sought to accompany technological evolution, particularly when no traditional methods of comparison, such as clinical record charts or radiographs, are available. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Rapid Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) by the Vitek MS Saramis system.

PubMed

Shan, Weiguang; Li, Jiaping; Fang, Ying; Wang, Xuan; Gu, Danxia; Zhang, Rong

2016-01-01

A rapid, sensitive, and accurate Vitek MS assay was developed to distinguish clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) from clinical isolates of methicillin-sensitive Staphylococcus aureus (MSSA) by developing an in-house knowledgebase of SuperSpectra. Three unique peaks, including peaks at 2305.6 and 3007.3 Da specific to MRSA, and 6816.7 Da specific to MSSA, were selected for differentiating MRSA and MSSA. This assay accurately identified 84 and 91% of clinical MRSA and MSSA strains out of the total 142 clinically acquired S. aureus strains that were tested. This method will greatly improve the efficiency of single clinical sample identification of MRSA, thereby facilitating a reduction in the transmission of MRSA in clinical settings.

Noninvasive identification of fluids by swept-frequency acoustic interferometry

DOEpatents

Sinha, Dipen N.

1998-01-01

A method for rapid, noninvasive identification and monitoring of chemicals in sealed containers or containers where direct access to the chemical is not possible is described. Multiple ultrasonic acoustic properties (up to four) of a fluid are simultaneously determined. The present invention can be used for chemical identification and for determining changes in known chemicals from a variety of sources. It is not possible to identify all known chemicals based on the measured parameters, but known classes of chemicals in suspected containers, such as in chemical munitions, can be characterized. In addition, a large number of industrial chemicals can be identified.

Direct Gram staining and its various benefits in the diagnosis of bacterial infections.

PubMed

Boyanova, Lyudmila

2018-01-01

In the era of rapid development of molecular and other diagnostic methods, direct Gram staining (DGS) tends to remain in the background, although it can provide both microbiologists and clinicians numerous benefits. The aim of this review was to emphasize the importance of DGS for the diagnosis of many clinically important infections. A PubMed search was carried out using relevant keywords for articles published primarily since 2010. The DGS can provide early information for a timely diagnosis of infections, can reveal the causative agents of the infections even under suboptimal conditions of specimen collection, transport or identification methods, can detect the presence of rare/unusual pathogens, moreover, the method shows the specimen quality, by distinguishing between contamination and true infection, it can direct or change initial antibiotic treatment before the availability of culture results, can indicate the need of other methods for pathogen identification and, in some cases, can show the need for emergency attention such as urgent antibiotic therapy and surgical measures. Briefly, the DGS remains an easy, rapid, inexpensive and important method, which use should be encouraged in conditions of a standardized and controlled performance to avoid technical or interpretation errors.

Rapid on-site TLC-SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements.

PubMed

Zhu, Qingxia; Cao, Yongbing; Cao, Yingying; Chai, Yifeng; Lu, Feng

2014-03-01

A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC-SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS). The study showed that TLC-SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants.

Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants.

PubMed

Zhang, Xiao; Liu, Zhijie; Yang, Jifei; Chen, Ze; Guan, Guiquan; Ren, Qiaoyun; Liu, Aihong; Luo, Jianxun; Yin, Hong; Li, Youquan

2014-02-01

Infections with Theileria sp. may cause significant economic losses to the sheep industry. Species identification based on microscopic examination is difficult, and more suitable methods are required for the rapid detection and identification of Theileria sp, in clinical specimens. In this study, a multiplex polymerase chain reaction (mPCR) assay was developed to simultaneously identify three individual Theileria species in small ruminants. Three pairs of specific, sensitive primers were designed on the basis of the 5.8S ribosomal RNA gene (Theileria luwenshuni and Theileria ovis) and the 18S ribosomal RNA gene (Theileria uilenbergi) to generate target products of 303, 884, and 530 bp, respectively. Standard DNA for each of the three species was extracted from blood recovered from infected sheep, and a preliminary study was conducted on 56 sheep to verify the reliability of the system. Optimal PCR conditions, including primer concentration, annealing time, and the number of amplification cycles, were established. The assay sensitivity under these conditions was 10(-3) % parasitemia, and its specificity was 100 %. The results of the study suggest that mPCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of Theileria species in small ruminants.

Identification of blood culture isolates directly from positive blood cultures by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry and a commercial extraction system: analysis of performance, cost, and turnaround time.

PubMed

Lagacé-Wiens, Philippe R S; Adam, Heather J; Karlowsky, James A; Nichol, Kimberly A; Pang, Paulette F; Guenther, Jodi; Webb, Amanda A; Miller, Crystal; Alfa, Michelle J

2012-10-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry represents a revolution in the rapid identification of bacterial and fungal pathogens in the clinical microbiology laboratory. Recently, MALDI-TOF has been applied directly to positive blood culture bottles for the rapid identification of pathogens, leading to reductions in turnaround time and potentially beneficial patient impacts. The development of a commercially available extraction kit (Bruker Sepsityper) for use with the Bruker MALDI BioTyper has facilitated the processing required for identification of pathogens directly from positive from blood cultures. We report the results of an evaluation of the accuracy, cost, and turnaround time of this method for 61 positive monomicrobial and 2 polymicrobial cultures representing 26 species. The Bruker MALDI BioTyper with the Sepsityper gave a valid (score, >1.7) identification for 85.2% of positive blood cultures with no misidentifications. The mean reduction in turnaround time to identification was 34.3 h (P < 0.0001) in the ideal situation where MALDI-TOF was used for all blood cultures and 26.5 h in a more practical setting where conventional identification or identification from subcultures was required for isolates that could not be directly identified by MALDI-TOF. Implementation of a MALDI-TOF-based identification system for direct identification of pathogens from blood cultures is expected to be associated with a marginal increase in operating costs for most laboratories. However, the use of MALDI-TOF for direct identification is accurate and should result in reduced turnaround time to identification.

Quantitation by Portable Gas Chromatography: Mass Spectrometry of VOCs Associated with Vapor Intrusion

PubMed Central

Fair, Justin D.; Bailey, William F.; Felty, Robert A.; Gifford, Amy E.; Shultes, Benjamin; Volles, Leslie H.

2010-01-01

Development of a robust reliable technique that permits for the rapid quantitation of volatile organic chemicals is an important first step to remediation associated with vapor intrusion. This paper describes the development of an analytical method that allows for the rapid and precise identification and quantitation of halogenated and nonhalogenated contaminants commonly found within the ppbv level at sites where vapor intrusion is a concern. PMID:20885969

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.

PubMed

Song, Y; Kato, N; Liu, C; Matsumiya, Y; Kato, H; Watanabe, K

2000-06-15

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

[Evaluation of mass spectrometry for the identification of clinically interesting yeasts].

PubMed

Galán, Fátima; García-Agudo, Lidia; Guerrero, Inmaculada; Marín, Pilar; García-Tapia, Ana; García-Martos, Pedro; Rodríguez-Iglesias, Manuel

2015-01-01

Identification of yeasts is based on morphological, biochemical and nutritional characteristics, and using molecular methods. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, a new method for the identification of microorganisms, has demonstrated to be very useful. The aim of this study is to evaluate this new method in the identification of yeasts. A total of 600 strains of yeasts isolated from clinical specimens belonging to 9 genera and 43 species were tested. Identification was made by sequencing of the ITS regions of ribosomal DNA, assimilation of carbon compounds (ID 32C), and mass spectrometry on a Microflex spectrometer (Bruker Daltonics GmbH, Germany). A total of 569 strains (94.8%) were identified to species level by ID 32C, and 580 (96.7%) by MALDI-TOF. Concordance between both methods was observed for 553 strains (92.2%), with 100% in clinically relevant species: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and almost 100% in C. krusei. MALDI-TOF identified species requiring molecular methods: Candida dubliniensis, C. nivariensis, C. metapsilosis and C. orthopsilosis. Some irregularities were observed in the identification of arthroconidia yeast and basidiomycetes. MALDI-TOF is a rapid, effective and economic method, which enables the identification of most clinically important yeasts and the differentiation of closely related species. It would be desirable to include more species in its database to expand its performance. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Knowns and unknowns in metabolomics identified by multidimensional NMR and hybrid MS/NMR methods

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

Bingol, Kerem; Brüschweiler, Rafael

Metabolomics continues to make rapid progress through the development of new and better methods and their applications to gain insight into the metabolism of a wide range of different biological systems from a systems biology perspective. Customization of NMR databases and search tools allows the faster and more accurate identification of known metabolites, whereas the identification of unknowns, without a need for extensive purification, requires new strategies to integrate NMR with mass spectrometry, cheminformatics, and computational methods. For some applications, the use of covalent and non-covalent attachments in the form of labeled tags or nanoparticles can significantly reduce the complexitymore » of these tasks.« less

Determination of alpha-hydroxy acids in cosmetic products by high-performance liquid chromatography with a narrow-bore column.

PubMed

Nicoletti, I; Corradini, C; Cogliandro, E; Cavazza, A

1999-08-01

This paper reports the results of a study carried out to develop a simple, rapid and sensitive method for the separation, identification and quantitative measurement of alpha-hydroxy acids in commercial cosmetics using high-performance liquid chromatography (HPLC). This method is successfully applied to the simultaneous identification and quantitative determination of glycolic, lactic, malic, tartaric and citric acids employing a reversed phase narrow-bore column under isocratic condition and UV detection. The method is validated by determining the precision of replicate analyses and accuracy by analyzing samples with and without adding know amount of the alpha-hydroxy acids. The procedure is suitable for routine analyses of commercial cosmetics.

Molecular identification of Malassezia species isolated from dermatitis affections.

PubMed

Affes, M; Ben Salah, S; Makni, F; Sellami, H; Ayadi, A

2009-05-01

The lipophilic yeast of the genus Malassezia are opportunistic microorganisms of the skin microflora but they can be agents of various dermatomycoses. The aim of this study was to perform molecular identification of the commonly isolated Malassezia species from various dermatomycoses in our region. Thirty strains of Malassezia were isolated from different dermatologic affections: pityriasis versicolor (17), dandruff (5), seborrheic dermatitis (4), onyxis (2), folliculitis (1) and blepharitis (1). These species were identified by their morphological features and biochemical characterisation. The molecular identification was achieved by amplification of the internal transcribed spacer region by simple PCR. PCR technique was used for molecular characterisation of four Malassezia species: Malassezia globosa (270 bp), Malassezia furfur (230 bp), Malassezia sympodialis (190 bp) and Malassezia restricta (320 bp). We have detected the association between M. furfur and M. sympodialis in 16% and confirmed presumptive identification in 70% of the cases. The phenotypic identification based on microscopic and physiological method is difficult and time consuming. The application of a simple PCR method provides a sensitive and rapid identification system for Malassezia species, which may be applied in epidemiological surveys and routine practice.

Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

PubMed

Ricchi, M; Mazzarelli, A; Piscini, A; Di Caro, A; Cannas, A; Leo, S; Russo, S; Arrigoni, N

2017-03-01

The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex. © 2016 The Society for Applied Microbiology.

Use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analyser in a diagnostic microbiology laboratory in a developing country.

PubMed

Bulane, Atang; Hoosen, Anwar

2017-01-01

Rapid and accurate identification of pathogens is of utmost importance for management of patients. Current identification relies on conventional phenotypic methods which are time consuming. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) is based on proteomic profiling and allows for rapid identification of pathogens. We compared MALDI-TOF MS against two commercial systems, MicroScan Walkaway and VITEK 2 MS. Over a three-month period from July 2013 to September 2013, a total of 227 bacteria and yeasts were collected from an academic microbiology laboratory ( N = 121; 87 Gram-negatives, seven Gram-positives, 27 yeasts) and other laboratories ( N = 106; 35 Gram-negatives, 34 Gram-positives, 37 yeasts). Sixty-five positive blood cultures were initially processed with Bruker Sepsityper kit for direct identification. From the 65 blood culture bottles, four grew more than one bacterial pathogen and MALDI-TOF MS identified only one isolate. The blood cultures yielded 21 Gram-negatives, 43 Gram-positives and one Candida . There were 21 Escherirchia coli isolates which were reported by the MALDI-TOF MS as E. coli / Shigella . Of the total 292 isolates, discrepant results were found for one bacterial and three yeast isolates. Discrepant results were resolved by testing with the API system with MALDI-TOF MS showing 100% correlation. The MALDI-TOF MS proved to be very useful for rapid and reliable identification of bacteria and yeasts directly from blood cultures and after culture of other specimens. The difference in time to identification was significant for all isolates. However, for positive blood cultures with minimal sample preparation time there was a massive difference in turn-around time with great appreciation by clinicians.

Standoff detection: distinction of bacteria by hyperspectral laser induced fluorescence

NASA Astrophysics Data System (ADS)

Walter, Arne; Duschek, Frank; Fellner, Lea; Grünewald, Karin M.; Hausmann, Anita; Julich, Sandra; Pargmann, Carsten; Tomaso, Herbert; Handke, Jürgen

2016-05-01

Sensitive detection and rapid identification of hazardous bioorganic material with high sensitivity and specificity are essential topics for defense and security. A single method can hardly cover these requirements. While point sensors allow a highly specific identification, they only provide localized information and are comparatively slow. Laser based standoff systems allow almost real-time detection and classification of potentially hazardous material in a wide area and can provide information on how the aerosol may spread. The coupling of both methods may be a promising solution to optimize the acquisition and identification of hazardous substances. The capability of the outdoor LIF system at DLR Lampoldshausen test facility as an online classification tool has already been demonstrated. Here, we present promising data for further differentiation among bacteria. Bacteria species can express unique fluorescence spectra after excitation at 280 nm and 355 nm. Upon deactivation, the spectral features change depending on the deactivation method.

[Research on Identification and Determination of Pesticides in Apples Using Raman Spectroscopy].

PubMed

Zhai, Chen; Peng, Yan-kun; Li, Yong-yu; Dhakal, Sagar; Xu, Tian-feng; Guo, Lang-hua

2015-08-01

Raman spectroscopy combined with chemometric methods has been thought to an efficient method for identification and determination of pesticide residues in fruits and vegetables. In the present research, a rapid and nondestructive method was proposed and testified based on self-developed Raman system for the identification and determination of deltamethrin and acetamiprid remaining in apple. The peaks of Raman spectra at 574 and 843 cm(-1) can be used to identify deltamethrin and acetamiprid, respectively, the characteristic peaks of deltamethrin and acetamiprid were still visible when the concentrations of the two pesticides were 0.78 and 0.15 mg · kg(-1) in apples samples, respectively. Calibration models of pesticide content were developed by partial least square (PLS) algorithm with different spectra pretreatment methods (Savitzky-Golay smoothing, first derivative transformation, second derivative transformation, baseline calibration, standard normal variable transformation). The baseline calibration methods by 8th order polynomial fitting gave the best results. For deltamethrin, the obtained prediction coefficient (Rp) value from PLS model for the results of prediction and gas chromatography measurement was 0.94; and the root mean square error of prediction (RMSEP) was 0.55 mg · kg(-1). The values of Rp and RMSEP were respective 0.85 and 0.12 mg · kg(-1) for acetamiprid. According to the detect performance, applying Raman technology in the nondestructive determination of pesticide residuals in apples is feasible. In consideration of that it needs no pretreatment before spectra collection and causes no damage to sample, this technology can be used in detection department, fruit and vegetable processing enterprises, supermarket, and vegetable market. The result of this research is promising for development of industrially feasible technology for rapid, nondestructive and real time detection of different types of pesticide with its concentration in apples. This supplies a rapid nondestructive and environmentally friendly way for the determination of fruit and vegetable quality and safety.

Establishment of a multiplex real-time RT-PCR assay for rapid identification of H6 subtype avian influenza viruses.

PubMed

Yang, Fan; Wu, Haibo; Liu, Fumin; Lu, Xiangyun; Peng, Xiuming; Wu, Nanping

2018-06-01

The H6 subtype avian influenza viruses (AIVs) possess the capacity for zoonotic transmission from avian species to humans. Establishment of a specific, rapid and sensitive method to screen H6 AIVs is necessary. Based on the conserved domain of the matrix and H6 AIV hemagglutinin genes, two TaqMan minor-groove-binder probes and multiplex real-time RT-PCR primers were designed in this study. The multiplex real-time RT-PCR assay developed in this study had high specificity and repeatability and a detection limit of 30 copies per reaction. This rapid diagnostic method will be useful for clinical detection and surveillance of H6 AIVs in China.

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.

Identification of specialists and abundance-occupancy relationships among intestinal bacteria of Aves, Mammalia, and Actinopterygii

EPA Science Inventory

The coalescence of next generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineating host-microbial associations has a...

DNA-BASED METHODS FOR MONITORING INVASIVE SPECIES: A REVIEW AND PROSPECTUS

EPA Science Inventory

The recent explosion of interest in DNA-based tools for species identification has prompted widespread speculation on the future availability of inexpensive, rapid and accurate means of identifying specimens and assessing biodiversity. One applied field that may benefit dramatic...

RNAi screen for rapid therapeutic target identification in leukemia patients

PubMed Central

Tyner, Jeffrey W.; Deininger, Michael W.; Loriaux, Marc M.; Chang, Bill H.; Gotlib, Jason R.; Willis, Stephanie G.; Erickson, Heidi; Kovacsovics, Tibor; O'Hare, Thomas; Heinrich, Michael C.; Druker, Brian J.

2009-01-01

Targeted therapy has vastly improved outcomes in certain types of cancer. Extension of this paradigm across a broad spectrum of malignancies will require an efficient method to determine the molecular vulnerabilities of cancerous cells. Improvements in sequencing technology will soon enable high-throughput sequencing of entire genomes of cancer patients; however, determining the relevance of identified sequence variants will require complementary functional analyses. Here, we report an RNAi-assisted protein target identification (RAPID) technology that individually assesses targeting of each member of the tyrosine kinase gene family. We demonstrate that RAPID screening of primary leukemia cells from 30 patients identifies targets that are critical to survival of the malignant cells from 10 of these individuals. We identify known, activating mutations in JAK2 and K-RAS, as well as patient-specific sensitivity to down-regulation of FLT1, CSF1R, PDGFR, ROR1, EPHA4/5, JAK1/3, LMTK3, LYN, FYN, PTK2B, and N-RAS. We also describe a previously undescribed, somatic, activating mutation in the thrombopoietin receptor that is sensitive to down-stream pharmacologic inhibition. Hence, the RAPID technique can quickly identify molecular vulnerabilities in malignant cells. Combination of this technique with whole-genome sequencing will represent an ideal tool for oncogenic target identification such that specific therapies can be matched with individual patients. PMID:19433805

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®

Evaluation of the Verigene Gram-Positive Blood Culture Nucleic Acid Test for Rapid Detection of Bacteria and Resistance Determinants

PubMed Central

Wojewoda, Christina M.; Sercia, Linda; Navas, Maria; Tuohy, Marion; Wilson, Deborah; Hall, Geraldine S.; Procop, Gary W.

2013-01-01

Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results. PMID:23596240

Evaluation of the Verigene Gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants.

PubMed

Wojewoda, Christina M; Sercia, Linda; Navas, Maria; Tuohy, Marion; Wilson, Deborah; Hall, Geraldine S; Procop, Gary W; Richter, Sandra S

2013-07-01

Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.

Molecular identification of Neofabraea species associated with bull's-eye rot on apple using rolling-circle amplification of partial EF-1α sequence.

PubMed

Lin, Huijiao; Jiang, Xiang; Yi, Jianping; Wang, Xinguo; Zuo, Ranling; Jiang, Zide; Wang, Weifang; Zhou, Erxun

2018-01-01

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull's-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull's-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull's-eye rot pathogens in port quarantine and orchard epidemiological studies.

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

PubMed

Chae, H S; Park, G N; Kim, S H; Jo, H J; Kim, J T; Jeoung, H Y; An, D J; Kim, N H; Shin, B W; Kang, Y I; Chang, K S

2012-08-01

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

Novel Antigen Identification Method for Discovery of Protective Malaria Antigens by Rapid Testing of DNA Vaccines Encoding Exons from the Parasite Genome

PubMed Central

Haddad, Diana; Bilcikova, Erika; Witney, Adam A.; Carlton, Jane M.; White, Charles E.; Blair, Peter L.; Chattopadhyay, Rana; Russell, Joshua; Abot, Esteban; Charoenvit, Yupin; Aguiar, Joao C.; Carucci, Daniel J.; Weiss, Walter R.

2004-01-01

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens. PMID:14977966

Direct-injection mass spectrometric method for the rapid identification of fentanyl and norfentanyl in postmortem urine of six drug-overdose cases.

PubMed

Peer, Cody J; Shakleya, Diaa M; Younis, Islam R; Kraner, James C; Callery, Patrick S

2007-10-01

A rapid mass spectrometric method was developed for the identification of fentanyl and its major hepatic metabolite norfentanyl in postmortem urine of six drug-overdose victims involving fentanyl use. To reduce matrix effects or ion suppression, sample preparation consisted of centrifugation and solid-phase extraction. Deuterium-labeled internal standards ((2)H(5)-fentanyl and (2)H(5)-norfentanyl) were used to compensate for instrument variation in signal, analyte recovery during sample preparation, and ion suppression. Structural information for fentanyl and norfentanyl were collected using mass spectrometry (MS) with electrospray ionization (ESI) operated in the positive ion mode. Fentanyl (m/z 337) was found in each of the six overdose cases by the appearance of the MS-MS daughter ion on both an ion trap and a triple-quadrupole MS resulting from the fragmentation pathway of fentanyl (m/z 337 --> 188). Norfentanyl was detected in all six cases by the appearance of the MH(+) ion, m/z 233, with a single-quadrupole MS and confirmed in an ion trap MS. Ion suppression, as determined by the comparison of ion intensities from spiked samples in water with postmortem urine from the cases, ranged from 18% to 98% in three ESI sources. The use of stable isotope-labeled internal standards obviates sample preparation because ratios of analyte/internal standard remain constant in the presence of extensive matrix effects. This MS method provided sufficient sensitivity and selectivity for the rapid identification of fentanyl and norfentanyl in urine at levels >/= 10 ng/mL without prior analyte resolution by chromatography and with a total analysis time of less than 1 h.

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America.

PubMed

Rasmussen Hellberg, Rosalee S; Morrissey, Michael T; Hanner, Robert H

2010-09-01

The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5' cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) "barcode" sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.

Evaluation of MALDI-TOF-MS for the Identification of Yeast Isolates Causing Bloodstream Infection.

PubMed

Turhan, Ozge; Ozhak-Baysan, Betil; Zaragoza, Oscar; Er, Halil; Sarıtas, Zubeyde Eres; Ongut, Gozde; Ogunc, Dilara; Colak, Dilek; Cuenca-Estrella, Manuel

2017-04-01

Infections due to Candida species are major causes of morbidity and mortality in humans, causing a diverse spectrum of clinical disease ranging from superficial and mucosal infections to invasive disease. Several authors have demonstrated that mortality is closely linked to both timing of therapy and/or source control. The rapid identification of pathogenic species is helpful to start timely and effective antifungal therapy. The aim of this study was to assess the performance of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system for the correct and rapid identification of yeast isolates causing bloodstream infection. Between January 2014 and January 2015, a total of 117 yeast like organisms isolated from blood culture samples of 117 episodes from 102 patients who had blood stream infections were included in the study. The isolates were identified by MALDI-TOF MS. The results were compared with those obtained by the standard mycological methods and/or sequence analysis. One hundred and seventeen yeast isolates including 115 Candida spp and two non-Candida yeasts were analysed. The Biotyper correctly identified 115 (98.3%) isolates to the genus level and 102 (87.2%) isolates to the species level using the manufacturer's recommended cutoff scores. The Bruker Biotyper is a rapid, easy, inexpensive, and highly reliable system for the identification of yeast isolates. Early identification with MALDI-TOF MS would save time for determination of antifungal susceptibility and proper treatment strategy. The expansion of the database of the library by addition of less common species will improve the performance of the system.

Highly efficient classification and identification of human pathogenic bacteria by MALDI-TOF MS.

PubMed

Hsieh, Sen-Yung; Tseng, Chiao-Li; Lee, Yun-Shien; Kuo, An-Jing; Sun, Chien-Feng; Lin, Yen-Hsiu; Chen, Jen-Kun

2008-02-01

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.

Rapid identification of probiotic Lactobacillus species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA.

PubMed

Kwon, Hyuk-Sang; Yang, Eun-Hee; Yeon, Seung-Woo; Kang, Byoung-Hwa; Kim, Tae-Yong

2004-10-15

This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of seven probiotic Lactobacillus species such as Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus rhamnosus. The primer set, comprising of seven specific and two conserved primers, was derived from the integrated sequences of 16S and 23S rRNA genes and their rRNA intergenic spacer region of each species. It was able to identify the seven target species with 93.6% accuracy, which exceeds that of the general biochemical methods. The phylogenetic analyses, using 16S rDNA sequences of the probiotic isolates, also provided further support that the results from the multiplex PCR assay were trustworthy. Taken together, we suggest that the multiplex primer set is an efficient tool for simple, rapid and reliable identification of seven Lactobacillus species.

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting.

PubMed

Andrés-Barrao, Cristina; Benagli, Cinzia; Chappuis, Malou; Ortega Pérez, Ruben; Tonolla, Mauro; Barja, François

2013-03-01

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species. Copyright © 2012 Elsevier GmbH. All rights reserved.

An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS.

PubMed

Loonen, A J M; Jansz, A R; Stalpers, J; Wolffs, P F G; van den Brule, A J C

2012-07-01

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker's MALDI Sepsityper kit, the commercially available Molzym's MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.

A novel monodisperse SiO2@C-dot for the rapid and facile identification of latent fingermarks using self-quenching resistant solid-state fluorescence.

PubMed

Peng, Di; Liu, Xiang; Huang, Mengjun; Wang, Dan; Liu, Renlong

2018-04-24

Solid powder fluorescence shows great potential for application in medicine, biology, and engineering, especially in the identification of latent fingermarks in forensic science. However, conventional developing methods suffer from some drawbacks, such as low contrast, low sensitivity, low selectivity, and high toxicity. To conquer these challenges, novel SiO2@C-dot microspheres were prepared via a facile one-pot hydrothermal method by using citric acid as a carbon source and aminosilane as a nitrogen source. Interestingly, the results showed that the resultant powders possess good monodispersity, high fluorescence emission, and resistance to self-quenching. Additionally, the mechanism for the solid-state fluorescence of SiO2@C-dot compounds was also investigated. More importantly, the fingermarks on various surfaces, including transparent glasses, ceramic tiles, transparent plastics, aluminum alloys, plastic cards, painted woods, artificial leathers, and Chinese paper money, developed by the powders have indicated well-defined papillary ridges under a 365 nm UV lamp. The novel strategy of using monodisperse SiO2@C-dot microspheres as a fluorescent label for developing latent fingermarks showed greater advantages compared to conventional methods, which was also demonstrated using the automatic fingerprint identification system. It is simple, rapid, low-cost, nontoxic, and effective, and is expected to be a promising alternative for the development of latent fingerprints in forensic science.

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

PubMed Central

Obručová, Hana; Tihelková, Radka; Kotásková, Iva; Růžička, Filip; Holá, Veronika; Němcová, Eva

2016-01-01

Early diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the “psilosis” complex. PMID:26935732

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections.

PubMed

Obručová, Hana; Tihelková, Radka; Kotásková, Iva; Růžička, Filip; Holá, Veronika; Němcová, Eva; Freiberger, Tomáš

2016-05-01

Early diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Comparison of the MUREX C. albicans, Albicans-Sure, and BactiCard Candida test kits with the germ tube test for presumptive identification of Candida albicans.

PubMed Central

Crist, A E; Dietz, T J; Kampschroer, K

1996-01-01

The MUREX C. albicans (MC)(Murex Diagnostics), Albicans-Sure (AS) (Clinical Standards Laboratories), and BactiCard Candida (BC) (Remel) test kits were compared with the germ tube (GT) test for the rapid, presumptive identification of Candida albicans. All three test kits detect the enzymes L-proline aminopeptidase and beta-galactosaminidase in yeast cells grown on culture media and are based on the principle that C. albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C system and conventional methods) and included 303 C. albicans isolates, 153 Candida glabrata isolates, 70 Candida tropicalis isolates, 36 Candida parapsilosis isolates, 13 isolates of other Candida spp., 5 Cryptococcus neoformans isolates, and 3 Saccharomyces cerevisiae isolates. The MC, AS, BC, and GT tests detected 299 (98.7%), 300 (99.0%), 301 (99.3%), and 287 (94.7%) C. albicans isolates, respectively. There was one false-positive result with both the MC and BC kits and two false-positive results with the GT test. The enzymatic methods evaluated in this study provide rapid and accurate alternatives to the GT test for the presumptive identification of C. albicans. PMID:8880535

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system.

PubMed

Garner, O; Mochon, A; Branda, J; Burnham, C-A; Bythrow, M; Ferraro, M; Ginocchio, C; Jennemann, R; Manji, R; Procop, G W; Richter, S; Rychert, J; Sercia, L; Westblade, L; Lewinski, M

2014-04-01

Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Rapid identification of staphylococci by Raman spectroscopy.

PubMed

Rebrošová, Katarína; Šiler, Martin; Samek, Ota; Růžička, Filip; Bernatová, Silvie; Holá, Veronika; Ježek, Jan; Zemánek, Pavel; Sokolová, Jana; Petráš, Petr

2017-11-01

Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.

High resolution melting analysis: rapid and precise characterisation of recombinant influenza A genomes

PubMed Central

2013-01-01

Background High resolution melting analysis (HRM) is a rapid and cost-effective technique for the characterisation of PCR amplicons. Because the reverse genetics of segmented influenza A viruses allows the generation of numerous influenza A virus reassortants within a short time, methods for the rapid selection of the correct recombinants are very useful. Methods PCR primer pairs covering the single nucleotide polymorphism (SNP) positions of two different influenza A H5N1 strains were designed. Reassortants of the two different H5N1 isolates were used as a model to prove the suitability of HRM for the selection of the correct recombinants. Furthermore, two different cycler instruments were compared. Results Both cycler instruments generated comparable average melting peaks, which allowed the easy identification and selection of the correct cloned segments or reassorted viruses. Conclusions HRM is a highly suitable method for the rapid and precise characterisation of cloned influenza A genomes. PMID:24028349

Neuronal models for evaluation of proliferation in vitro using high content screening

EPA Science Inventory

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental proc...

Rapid glucosinolate detection and identification using accurate mass MS-MS

USDA-ARS?s Scientific Manuscript database

Currently, there is a demand for accurate evaluation of brassica plat species for their glucosinolate content. An optimized method has been developed for detecting and identifying glucosinolates in plant extracts using MS-MS fragmentation with ion trap collision induced dissociation (CID) and higher...

Identification of Pseudallescheria and Scedosporium Species by Three Molecular Methods▿

PubMed Central

Lu, Qiaoyun; Gerrits van den Ende, A. H. G.; Bakkers, J. M. J. E.; Sun, Jiufeng; Lackner, M.; Najafzadeh, M. J.; Melchers, W. J. G.; Li, Ruoyu; de Hoog, G. S.

2011-01-01

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 103, and 5 × 102 cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species. PMID:21177887

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.

PubMed

Zhou, Yuexin; Zhu, Shiyou; Cai, Changzu; Yuan, Pengfei; Li, Chunmei; Huang, Yanyi; Wei, Wensheng

2014-05-22

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

PCR Methods for Rapid Identification and Characterization of Actinobacillus seminis Strains

PubMed Central

Appuhamy, S.; Coote, J. G.; Low, J. C.; Parton, R.

1998-01-01

Twenty-four isolates of Actinobacillus seminis were typed by PCR ribotyping, repetitive extragenic palindromic element (REP)-based PCR, and enterobacterial repetitive intergenic consensus (ERIC)-based PCR. Five types were distinguished by REP-PCR, and nine types were distinguished by ERIC-PCR. PCR ribotyping produced the simplest pattern and could be useful for identification of A. seminis and for its differentiation from related species. REP- and ERIC-PCR could be used for strain differentiation in epidemiological studies of A. seminis. PMID:9508320

Research of Face Recognition with Fisher Linear Discriminant

NASA Astrophysics Data System (ADS)

Rahim, R.; Afriliansyah, T.; Winata, H.; Nofriansyah, D.; Ratnadewi; Aryza, S.

2018-01-01

Face identification systems are developing rapidly, and these developments drive the advancement of biometric-based identification systems that have high accuracy. However, to develop a good face recognition system and to have high accuracy is something that’s hard to find. Human faces have diverse expressions and attribute changes such as eyeglasses, mustache, beard and others. Fisher Linear Discriminant (FLD) is a class-specific method that distinguishes facial image images into classes and also creates distance between classes and intra classes so as to produce better classification.

High-Throughput Block Optical DNA Sequence Identification.

PubMed

Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Flow cytomeric measurement of DNA and incorporated nucleoside analogs

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1989-01-01

A method is provided for simultaneously measuring total cellular DNA and incorporated nucleoside analog. The method entails altering the cellular DNA of cells grown in the presence of a nucleoside analog so that single stranded and double stranded portions are present. Separate stains are used against the two portions. An immunochemical stain is used against the single stranded portion to provide a measure of incorporated nucleoside analog, and a double strand DNA-specific stain is used against the double stranded portion to simultaneously provide a measure of total cellular DNA. The method permits rapid flow cytometric analysis of cell populations, rapid identification of cycling and noncycling subpopulations, and determination of the efficacy of S phase cytotoxic anticancer agents.

PCR/LDR/universal array platforms for the diagnosis of infectious disease.

PubMed

Pingle, Maneesh; Rundell, Mark; Das, Sanchita; Golightly, Linnie M; Barany, Francis

2010-01-01

Infectious diseases account for between 14 and 17 million deaths worldwide each year. Accurate and rapid diagnosis of bacterial, fungal, viral, and parasitic infections is therefore essential to reduce the morbidity and mortality associated with these diseases. Classical microbiological and serological methods have long served as the gold standard for diagnosis but are increasingly being replaced by molecular diagnostic methods that demonstrate increased sensitivity and specificity and provide an identification of the etiologic agent in a shorter period of time. PCR/LDR coupled with universal array detection provides a highly sensitive and specific platform for the detection and identification of bacterial and viral infections.

PCR/LDR/Universal Array Platforms for the Diagnosis of Infectious Disease

PubMed Central

Pingle, Maneesh; Rundell, Mark; Das, Sanchita; Golightly, Linnie M.; Barany, Francis

2015-01-01

Infectious diseases account for between 14 and 17 million deaths worldwide each year. Accurate and rapid diagnosis of bacterial, fungal, viral, and parasitic infections is therefore essential to reduce the morbidity and mortality associated with these diseases. Classical microbiological and serological methods have long served as the gold standard for diagnosis but are increasingly being replaced by molecular diagnostic methods that demonstrate increased sensitivity and specificity and provide an identification of the etiologic agent in a shorter period of time. PCR/LDR coupled with universal array detection provides a highly sensitive and specific platform for the detection and identification of bacterial and viral infections. PMID:20217576

Rapid identification of triterpenoid sulfates and hydroxy fatty acids including two new constituents from Tydemania expeditionis by LC-MS

PubMed Central

Zhang, Jian-Long; Kubanek, Julia; Hay, Mark E.; Aalbersberg, William; Ye, Wen-Cai; Jiang, Ren-Wang

2011-01-01

Tydemania expeditionis Weber-van Bosse (Udoteaceae) is a weakly calcified green alga. In the present paper, liquid chromatography coupled with photodiode array detection and electrospray mass spectrometry was developed to identify the fingerprint components. A total of four triterpenoid sulfates and three hydroxy fatty acids in the ethyl acetate fraction of the crude extract were structurally characterized on the basis of retention time, online UV spectrum and mass fragmentation pattern. Furthermore, detailed LC-MS analysis revealed two new hydroxy fatty acids, which were then prepared and characterized by extensive NMR analyses. The proposed method provides a scientific and technical platform for the rapid identification of triterpenoid sulfates and hydroxy fatty acids in similar marine algae and terrestrial plants. PMID:21915955

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.

Comparison of methods for the identification of microorganisms isolated from blood cultures.

PubMed

Monteiro, Aydir Cecília Marinho; Fortaleza, Carlos Magno Castelo Branco; Ferreira, Adriano Martison; Cavalcante, Ricardo de Souza; Mondelli, Alessandro Lia; Bagagli, Eduardo; da Cunha, Maria de Lourdes Ribeiro de Souza

2016-08-05

Bloodstream infections are responsible for thousands of deaths each year. The rapid identification of the microorganisms causing these infections permits correct therapeutic management that will improve the prognosis of the patient. In an attempt to reduce the time spent on this step, microorganism identification devices have been developed, including the VITEK(®) 2 system, which is currently used in routine clinical microbiology laboratories. This study evaluated the accuracy of the VITEK(®) 2 system in the identification of 400 microorganisms isolated from blood cultures and compared the results to those obtained with conventional phenotypic and genotypic methods. In parallel to the phenotypic identification methods, the DNA of these microorganisms was extracted directly from the blood culture bottles for genotypic identification by the polymerase chain reaction (PCR) and DNA sequencing. The automated VITEK(®) 2 system correctly identified 94.7 % (379/400) of the isolates. The YST and GN cards resulted in 100 % correct identifications of yeasts (15/15) and Gram-negative bacilli (165/165), respectively. The GP card correctly identified 92.6 % (199/215) of Gram-positive cocci, while the ANC card was unable to correctly identify any Gram-positive bacilli (0/5). The performance of the VITEK(®) 2 system was considered acceptable and statistical analysis showed that the system is a suitable option for routine clinical microbiology laboratories to identify different microorganisms.

Rapid identification and simultaneous analysis of multiple constituents from Rheum tanguticum Maxim. ex Balf. by UPLC/Q-TOF-MS.

PubMed

Gao, Liang-Liang; Guo, Tao; Xu, Xu-Dong; Yang, Jun-Shan

2017-07-01

Rhubarb contains biologically active compounds such as anthraquinones, anthrones, stilbenes and tannins. A rapid and efficient UPLC/Q-TOF-MS/MS method was developed and applied towards identifying the constituents of Rheum tanguticum Maxim. ex Balf. for the first time. Chemical constituents were separated and investigated by UPLC/Q-TOF-MS/MS in the negative ion mode. The ESI-MS 2 fragmentation pathways of four types of compounds were interpreted, providing a very useful guidance for the characterisation of different types of compounds. Based on the exact mass information, fragmentation characteristic and LC retention time of 7 reference standards, 30 constituents were tentatively identified from the methanol extract of R. tanguticum. Among them, seven compounds were described for the first time from R. tanguticum and two from the genus Rheum were described for the first time. The analytical tool used here is valuable for the rapid separation and identification of multiple and minor constituents in methanol extracts of R. tanguticum.

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.

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.

[Mass spectrometry in the clinical microbiology laboratory].

PubMed

Jordana-Lluch, Elena; Martró Català, Elisa; Ausina Ruiz, Vicente

2012-12-01

Infectious diseases are still a cause of high mortality and morbidity rates. Current microbiological diagnostic methods are based on culture and phenotypic identification of isolated microorganisms, which can be obtained in about 24-48 h. Given that the microbiological identification is of major importance for patient management, new diagnostic methods are needed in order to detect and identify microorganisms in a timely and accurate manner. Over the last few years, several molecular techniques based on the amplification of microbial nucleic acids have been developed with the aim of reducing the time needed for the identification of the microorganisms involved in different infectious processes. On the other hand, mass spectrometry has emerged as a rapid and consistent alternative to conventional methods for microorganism identification. This review describes the most widely used mass spectrometry technologies -matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization time-of-flight (ESI-TOF)-, both for protein and nucleic acid analysis, as well as the commercial platforms available. Related publications of most interest in clinical microbiology are also reviewed. Copyright © 2011 Elsevier España, S.L. All rights reserved.

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 Separation of Bacteria from Blood—Review and Outlook

PubMed Central

Alizadeh, Mahsa; Husseini, Ghaleb A.; McClellan, Daniel S.; Buchanan, Clara M.; Bledsoe, Colin G.; Robison, Richard A.; Blanco, Rae; Roeder, Beverly L.; Melville, Madison; Hunter, Alex K.

2017-01-01

The high morbidity and mortality rate of bloodstream infections involving antibiotic-resistant bacteria necessitate a rapid identification of the infectious organism and its resistance profile. Traditional methods based on culturing the blood typically require at least 24 h, and genetic amplification by PCR in the presence of blood components has been problematic. The rapid separation of bacteria from blood would facilitate their genetic identification by PCR or other methods so that the proper antibiotic regimen can quickly be selected for the septic patient. Microfluidic systems that separate bacteria from whole blood have been developed, but these are designed to process only microliter quantities of whole blood or only highly diluted blood. However, symptoms of clinical blood infections can be manifest with bacterial burdens perhaps as low as 10 CFU/mL, and thus milliliter quantities of blood must be processed to collect enough bacteria for reliable genetic analysis. This review considers the advantages and shortcomings of various methods to separate bacteria from blood, with emphasis on techniques that can be done in less than 10 min on milliliter-quantities of whole blood. These techniques include filtration, screening, centrifugation, sedimentation, hydrodynamic focusing, chemical capture on surfaces or beads, field-flow fractionation, and dielectrophoresis. Techniques with the most promise include screening, sedimentation, and magnetic bead capture, as they allow large quantities of blood to be processed quickly. Some microfluidic techniques can be scaled up. PMID:27160415

Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

PubMed

Saito, Takahiro; Kikuchi, Aoi; Kaneko, Akira; Isozumi, Rie; Teramoto, Isao; Kimura, Masatsugu; Hirasawa, Noriyasu; Hiratsuka, Masahiro

2018-06-01

Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid screening of abused drugs by direct analysis in real time (DART) coupled to time-of-flight mass spectrometry (TOF-MS) combined with ion mobility spectrometry (IMS).

PubMed

Lian, Ru; Wu, Zhongping; Lv, Xiaobao; Rao, Yulan; Li, Haiyang; Li, Jinghua; Wang, Rong; Ni, Chunfang; Zhang, Yurong

2017-10-01

Increasing in cases involving drugs of abuse leads to heavy burden for law enforcement agencies, exacerbating demand for rapid screening technique. In this study, atmospheric pressure ionization technologies including direct analysis in real time (DART) ion source coupled to a time-of-flight mass spectrometer (DART-TOF-MS)as well asdopant-assisted positive photoionization ion mobility spectrometry (DAPP-IMS) without radioactivity were utilized together as the powerful analytical tool for the rapid screening and identification of 53 abused drugs.The limits of detection (LOD) were 0.05-2μg/mL when using DART-TOF-MS and 0.02-2μg when using DAPP-IMS which could satisfy the actual requirement in forensic science laboratory. The advantages of this method included fast response, high-throughput potential, high specificity, and minimal sample preparation. A screening library of reduced mobility (K 0 ), accurate mass of informative precursor ion ([M+H] + ) and fragment ions was established respectively by employing a bench-top DAPP-IMS and TOF-MS in-source collision induced dissociation (CID) mode. Then the standardized screening procedure was developed with criteria for the confirmation of positive result. A total of 50 seized drug samples provided by local forensic laboratory we reanalyzed to testify the utility of the method. This study suggests that a method combing DART-TOF-MS and DAPP-IMS is promising for the rapid screening and identification of abused drugs with minimal sample preparation and absence of chromatography. Copyright © 2017 Elsevier B.V. All rights reserved.

Mastitis detection: current trends and future perspectives.

PubMed

Viguier, Caroline; Arora, Sushrut; Gilmartin, Niamh; Welbeck, Katherine; O'Kennedy, Richard

2009-08-01

Bovine mastitis, the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. Traditionally, methods of detection have included estimation of somatic cell counts, an indication of inflammation, measurement of biomarkers associated with the onset of the disease (e.g. the enzymes N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase) and identification of the causative microorganisms, which often involves culturing methods. These methods have their limitations and there is a need for new rapid, sensitive and reliable assays. Recently, significant advances in the identification of nucleic acid markers and other novel biomarkers and the development of sensor-based platforms have taken place. These novel strategies have shown promise, and their advantages over the conventional tests are discussed.

Classification of gram-positive and gram-negative foodborne pathogenic bacteria with hyperspectral microscope imaging

USDA-ARS?s Scientific Manuscript database

Optical method with hyperspectral microscope imaging (HMI) has potential for identification of foodborne pathogenic bacteria from microcolonies rapidly with a cell level. A HMI system that provides both spatial and spectral information could be an effective tool for analyzing spectral characteristic...

Sensitive and Rapid Identification of Biological Threat Agents

DTIC Science & Technology

1999-12-01

plague, and Bruceila abortis , the etiologic agent of brucellosis, indicate that the Qiagen procedures will also be effective in preparing these agents for...procedure for two bacteria, Yersinia pestis and Bruceila abortis , and found that DNA isolated with this method yielded PCR detection limits at least

Esculin hydrolysis by Gram positive bacteria. A rapid test and it's comparison with other methods.

PubMed

Qadri, S M; Smith, J C; Zubairi, S; DeSilva, M I

1981-01-01

A number of bacteria hydrolyze esculin enzymatically to esculetin. This characteristic is used by taxonomists and clinical microbiologists in the differentiation and identification of bacteria, especially to distinguish Lance-field group D streptococci from non-group D organisms and Listeria monocytogenes from morphologically similar Erysipelothrix rhusipoathiae and diphtheroids. Conventional methods used for esculin hydrolysis require 4--48 h for completion. We developed and evaluated a medium which gives positive results more rapidly. The 2,330 isolates used in this study consisted of 1,680 esculin positive and 650 esculin negative organisms. The sensitivity and specificity of this method were compared with the PathoTec esculin hydrolysis strip and the procedure of Vaughn and Levine (VL). Of the 1,680 esculin positive organisms, 97% gave positive reactions within 30 minutes with the rapid test whereas PathoTec required 3--4 h incubation for the same number of organisms to yield a positive reaction.

Rapid Authentication of Ginkgo biloba Herbal Products Using the Recombinase Polymerase Amplification Assay.

PubMed

Liu, Yang; Wang, Xiao-Yue; Wei, Xue-Min; Gao, Zi-Tong; Han, Jian-Ping

2018-05-22

Species adulteration in herbal products (HPs) exposes consumers to health risks. Chemical and morphological methods have their own deficiencies when dealing with the detection of species containing the same active compounds in HPs. In this study, we developed a rapid identification method using the recombinase polymerase amplification (RPA) assay to detect two species, Ginkgo biloba and Sophora japonica (as adulteration), in Ginkgo biloba HPs. Among 36 Ginkgo biloba HP samples, 34 were found to have Ginkgo biloba sequences, and 9 were found to have Sophora japonica sequences. During the authentication process, the RPA-LFS assay showed a higher specificity, sensitivity and efficiency than PCR-based methods. We initially applied the RPA-LSF technique to detect plant species in HPs, demonstrating that this assay can be developed into an efficient tool for the rapid on-site authentication of plant species in Ginkgo biloba HPs.

Simple Sample Preparation Method for Direct Microbial Identification and Susceptibility Testing From Positive Blood Cultures.

PubMed

Pan, Hong-Wei; Li, Wei; Li, Rong-Guo; Li, Yong; Zhang, Yi; Sun, En-Hua

2018-01-01

Rapid identification and determination of the antibiotic susceptibility profiles of the infectious agents in patients with bloodstream infections are critical steps in choosing an effective targeted antibiotic for treatment. However, there has been minimal effort focused on developing combined methods for the simultaneous direct identification and antibiotic susceptibility determination of bacteria in positive blood cultures. In this study, we constructed a lysis-centrifugation-wash procedure to prepare a bacterial pellet from positive blood cultures, which can be used directly for identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and antibiotic susceptibility testing by the Vitek 2 system. The method was evaluated using a total of 129 clinical bacteria-positive blood cultures. The whole sample preparation process could be completed in <15 min. The correct rate of direct MALDI-TOF MS identification was 96.49% for gram-negative bacteria and 97.22% for gram-positive bacteria. Vitek 2 antimicrobial susceptibility testing of gram-negative bacteria showed an agreement rate of antimicrobial categories of 96.89% with a minor error, major error, and very major error rate of 2.63, 0.24, and 0.24%, respectively. Category agreement of antimicrobials against gram-positive bacteria was 92.81%, with a minor error, major error, and very major error rate of 4.51, 1.22, and 1.46%, respectively. These results indicated that our direct antibiotic susceptibility analysis method worked well compared to the conventional culture-dependent laboratory method. Overall, this fast, easy, and accurate method can facilitate the direct identification and antibiotic susceptibility testing of bacteria in positive blood cultures.

MALDI-TOF MS is more accurate than VITEK II ANC card and API Rapid ID 32 A system for the identification of Clostridium species.

PubMed

Kim, Young Jin; Kim, Si Hyun; Park, Hyun-Jung; Park, Hae-Geun; Park, Dongchul; Song, Sae Am; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kook, Joong-Ki; Kim, Hye Ran; Shin, Jeong Hwan

2016-08-01

All 50 Clostridium difficile strains were definitely identified by Vitek2 system, Rapid ID 32A system, and MALDI-TOF. For 18 non-difficile Clostridium strains, the identification results were correct in 0, 2, and 17 strains by Vitek2, Rapid ID 32A, and MALDI-TOF, respectively. MALDI-TOF could be used as the primary tool for identification of Clostridium species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel Optical Methods for Identification, Imaging, and Preservation of the Cavernous Nerves Responsible for Penile Erections during Prostate Cancer Surgery

DTIC Science & Technology

2009-03-01

wavelength, pulse energy, and pulse rate) to produce strongest and most rapid erectile response as measured by intracavernosal pressure in the penis ...PC Fiber Rod Housing Optics 5-mm-ID Port Probe Handle Probe Stem Enlarged View of Probe Tip Oscilloscope FunctionGenerator Thulium Fiber Laser Shutter...rapid erectile response as measured by intracavernosal pressure (ICP) in the penis . ICP values were increased from an initial range of 30-40 mmHg

Rapid, Contactless and Non-Destructive Testing of Chemical Composition of Samples

NASA Astrophysics Data System (ADS)

Ivanov, O.; Vaseashta, A.; Stoychev, L.

Our results demonstrate that a new effect can be induced in each solid in a wide spectral range of electromagnetic irradiation. In the present manuscript we prove experimentally that one of the possible applications of this effect is for an express contactless control of the chemical composition of a series of samples, in this case, coins. The method has wide applicability ranging from defense and homeland security to several applications requiring rapid and nondestructive identification of chemical composition.

Identification of four squid species by quantitative real-time polymerase chain reaction.

PubMed

Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

2016-02-01

Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of select Dalbergia and trade timber using direct analysis in real time and time-of-flight mass spectrometry for CITES enforcement.

PubMed

Lancaster, Cady; Espinoza, Edgard

2012-05-15

International trade of several Dalbergia wood species is regulated by The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). In order to supplement morphological identification of these species, a rapid chemical method of analysis was developed. Using Direct Analysis in Real Time (DART) ionization coupled with Time-of-Flight (TOF) Mass Spectrometry (MS), selected Dalbergia and common trade species were analyzed. Each of the 13 wood species was classified using principal component analysis and linear discriminant analysis (LDA). These statistical data clusters served as reliable anchors for species identification of unknowns. Analysis of 20 or more samples from the 13 species studied in this research indicates that the DART-TOFMS results are reproducible. Statistical analysis of the most abundant ions gave good classifications that were useful for identifying unknown wood samples. DART-TOFMS and LDA analysis of 13 species of selected timber samples and the statistical classification allowed for the correct assignment of unknown wood samples. This method is rapid and can be useful when anatomical identification is difficult but needed in order to support CITES enforcement. Published 2012. This article is a US Government work and is in the public domain in the USA.

Noninvasive forward-scattering system for rapid detection, characterization, and identification of Listeria colonies: image processing and data analysis

NASA Astrophysics Data System (ADS)

Rajwa, Bartek; Bayraktar, Bulent; Banada, Padmapriya P.; Huff, Karleigh; Bae, Euiwon; Hirleman, E. Daniel; Bhunia, Arun K.; Robinson, J. Paul

2006-10-01

Bacterial contamination by Listeria monocytogenes puts the public at risk and is also costly for the food-processing industry. Traditional methods for pathogen identification require complicated sample preparation for reliable results. Previously, we have reported development of a noninvasive optical forward-scattering system for rapid identification of Listeria colonies grown on solid surfaces. The presented system included application of computer-vision and patternrecognition techniques to classify scatter pattern formed by bacterial colonies irradiated with laser light. This report shows an extension of the proposed method. A new scatterometer equipped with a high-resolution CCD chip and application of two additional sets of image features for classification allow for higher accuracy and lower error rates. Features based on Zernike moments are supplemented by Tchebichef moments, and Haralick texture descriptors in the new version of the algorithm. Fisher's criterion has been used for feature selection to decrease the training time of machine learning systems. An algorithm based on support vector machines was used for classification of patterns. Low error rates determined by cross-validation, reproducibility of the measurements, and robustness of the system prove that the proposed technology can be implemented in automated devices for detection and classification of pathogenic bacteria.

Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene.

PubMed

Virkler, Kelly; Lednev, Igor K

2009-07-01

Body fluid traces recovered at crime scenes are among the most important types of evidence to forensic investigators. They contain valuable DNA evidence which can identify a suspect or victim as well as exonerate an innocent individual. The first step of identifying a particular body fluid is highly important since the nature of the fluid is itself very informative to the investigation, and the destructive nature of a screening test must be considered when only a small amount of material is available. The ability to characterize an unknown stain at the scene of the crime without having to wait for results from a laboratory is another very critical step in the development of forensic body fluid analysis. Driven by the importance for forensic applications, body fluid identification methods have been extensively developed in recent years. The systematic analysis of these new developments is vital for forensic investigators to be continuously educated on possible superior techniques. Significant advances in laser technology and the development of novel light detectors have dramatically improved spectroscopic methods for molecular characterization over the last decade. The application of this novel biospectroscopy for forensic purposes opens new and exciting opportunities for the development of on-field, non-destructive, confirmatory methods for body fluid identification at a crime scene. In addition, the biospectroscopy methods are universally applicable to all body fluids unlike the majority of current techniques which are valid for individual fluids only. This article analyzes the current methods being used to identify body fluid stains including blood, semen, saliva, vaginal fluid, urine, and sweat, and also focuses on new techniques that have been developed in the last 5-6 years. In addition, the potential of new biospectroscopic techniques based on Raman and fluorescence spectroscopy is evaluated for rapid, confirmatory, non-destructive identification of a body fluid at a crime scene.

Rapid microscale in-gel processing and digestion of proteins using surface acoustic waves.

PubMed

Kulkarni, Ketav P; Ramarathinam, Sri H; Friend, James; Yeo, Leslie; Purcell, Anthony W; Perlmutter, Patrick

2010-06-21

A new method for in-gel sample processing and tryptic digestion of proteins is described. Sample preparation, rehydration, in situ digestion and peptide extraction from gel slices are dramatically accelerated by treating the gel slice with surface acoustic waves (SAWs). Only 30 minutes total workflow time is required for this new method to produce base peak chromatograms (BPCs) of similar coverage and intensity to those observed for traditional processing and overnight digestion. Simple set up, good reproducibility, excellent peptide recoveries, rapid turnover of samples and high confidence protein identifications put this technology at the fore-front of the next generation of proteomics sample processing tools.

Rapid Identification and Quantification of Natural Antioxidants in the Seeds of Rhubarb from Different Habitats in China Using Accelerated Solvent Extraction and HPLC-DAD-ESI-MSn-DPPH Assay.

PubMed

Tan, Liang; Geng, Dan-dan; Hu, Feng-zu; Dong, Qi

2016-01-01

In this study, the 10 accessions of rhubarb seeds from different habitats in China were investigated. Lipids were removed using petroleum ether, and the effective components were then separated using accelerated solvent extraction with 80% aqueous methanol. An off-line 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging method was used as the marker to evaluate the total antioxidant capability of extracts. On-line high-performance liquid chromatography-diode-array detectors-electrospray ionization-tandem mass spectrometry (HPLC-DAD-ESI-MS(n)) and HPLC-DAD-DPPH assays were developed for rapid identification and quantification of individual free-radical scavengers in extracts of rhubarb seeds. Ten free-radical scavengers from methanolic extracts of the rhubarb seeds were screened, five of which were identified and quantitatively analyzed: epicatechin, myricetin, hyperoside, quercitrin and quercetin. All were identified in rhubarb seeds for the first time and can be regarded as the major potent antioxidants in rhubarb seeds due to representing most of the total free-radical scavenging activity. Preliminary analysis of structures was performed for another five antioxidants. Based on our validation results, the developed method can be used for rapid separation, convenient identification and quantification of the multiple antioxidative constituents in rhubarb seeds, featuring good quantification parameters, accuracy and precision. The results are important to clarify the material basis and therapeutic mechanism of rhubarb seeds. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Laboratory Diagnosis and Susceptibility Testing for Mycobacterium tuberculosis.

PubMed

Procop, Gary W

2016-12-01

The laboratory, which utilizes some of the most sophisticated and rapidly changing technologies, plays a critical role in the diagnosis of tuberculosis. Some of these tools are being employed in resource-challenged countries for the rapid detection and characterization of Mycobacterium tuberculosis. Foremost, the laboratory defines appropriate specimen criteria for optimal test performance. The direct detection of mycobacteria in the clinical specimen, predominantly done by acid-fast staining, may eventually be replaced by rapid-cycle PCR. The widespread use of the Xpert MTB/RIF (Cepheid) assay, which detects both M. tuberculosis and key genetic determinants of rifampin resistance, is important for the early detection of multidrug-resistant strains. Culture, using both broth and solid media, remains the standard for establishing the laboratory-based diagnosis of tuberculosis. Cultured isolates are identified far less commonly by traditional biochemical profiling and more commonly by molecular methods, such as DNA probes and broad-range PCR with DNA sequencing. Non-nucleic acid-based methods of identification, such as high-performance liquid chromatography and, more recently, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, may also be used for identification. Cultured isolates of M. tuberculosis should be submitted for susceptibility testing according to standard guidelines. The use of broth-based susceptibility testing is recommended to significantly decrease the time to result. Cultured isolates may also be submitted for strain typing for epidemiologic purposes. The use of massive parallel sequencing, also known as next-generation sequencing, promises to continue to this molecular revolution in mycobacteriology, as whole-genome sequencing provides identification, susceptibility, and typing information simultaneously.

Rapid identification of pathogens directly from blood culture bottles by Bruker matrix-assisted laser desorption laser ionization-time of flight mass spectrometry versus routine methods.

PubMed

Jamal, Wafaa; Saleem, Rola; Rotimi, Vincent O

2013-08-01

The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of microorganisms directly from blood culture is an exciting dimension to the microbiologists. We evaluated the performance of Bruker SepsiTyper kit™ (STK) for direct identification of bacteria from positive blood culture. This was done in parallel with conventional methods. Nonrepetitive positive blood cultures from 160 consecutive patients were prospectively evaluated by both methods. Of 160 positive blood cultures, the STK identified 114 (75.6%) isolates and routine conventional method 150 (93%). Thirty-six isolates were misidentified or not identified by the kit. Of these, 5 had score of >2.000 and 31 had an unreliable low score of <1.7. Four of 8 yeasts were identified correctly. The average turnaround time using the STK was 35 min, including extraction steps and 30:12 to 36:12 h with routine method. The STK holds promise for timely management of bacteremic patients. Copyright © 2013 Elsevier Inc. All rights reserved.

Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

PubMed

Cao, Jijuan; Xu, Junyi; Zhao, Tongtong; Cao, Dongmei; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2014-01-01

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.

Raman spectroscopic studies on bacteria

NASA Astrophysics Data System (ADS)

Maquelin, Kees; Choo-Smith, Lin-P'ing; Endtz, Hubert P.; Bruining, Hajo A.; Puppels, Gerwin J.

2000-11-01

Routine clinical microbiological identification of pathogenic micro-organisms is largely based on nutritional and biochemical tests. Laboratory results can be presented to a clinician after 2 - 3 days for most clinically relevant micro- organisms. Most of this time is required to obtain pure cultures and enough biomass for the tests to be performed. In the case of severely ill patients, this unavoidable time delay associated with such identification procedures can be fatal. A novel identification method based on confocal Raman microspectroscopy will be presented. With this method it is possible to obtain Raman spectra directly from microbial microcolonies on the solid culture medium, which have developed after only 6 hours of culturing for most commonly encountered organisms. Not only does this technique enable rapid (same day) identifications, but also preserves the sample allowing it to be double-checked with traditional tests. This, combined with the speed and minimal sample handling indicate that confocal Raman microspectroscopy has much potential as a powerful new tool in clinical diagnostic microbiology.

Methods for identifying an essential gene in a prokaryotic microorganism

DOEpatents

Shizuya, Hiroaki

2006-01-31

Methods are provided for the rapid identification of essential or conditionally essential DNA segments in any species of haploid cell (one copy chromosome per cell) that is capable of being transformed by artificial means and is capable of undergoing DNA recombination. This system offers an enhanced means of identifying essential function genes in diploid pathogens, such as gram-negative and gram-positive bacteria.

Rapid and improved gas-liquid chromatography technique for detection of hippurate hydrolysis by Campylobacter jejuni and Campylobacter coli.

PubMed Central

Bär, W; Fricke, G

1987-01-01

A gas-liquid chromatographic method which requires no chloroform extraction of the split products has been investigated for the detection of hippurate hydrolysis by Campylobacter spp. This technique gave better reproducibility than other tests also used in this study and allows the routine use of the gas-liquid chromatographic method for identification of Campylobacter isolates. PMID:3654950

Rapid and efficient differentiation of Yersinia species using high-resolution melting analysis.

PubMed

Souza, Roberto A; Frazão, Miliane R; Almeida, Alzira M P; Falcão, Juliana P

2015-08-01

The primary goal of clinical microbiology is the accurate identification of the causative agent of the disease. Here, we describe a method for differentiation between Yersinia species using PCR-HRMA. The results revealed species-specific melting profiles. The herein developed assay can be used as an effective method to differentiate Yersinia species. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid identification of transience in streambed conductance by inversion of floodwave responses

NASA Astrophysics Data System (ADS)

Gianni, Guillaume; Richon, Julien; Perrochet, Pierre; Vogel, Alexandre; Brunner, Philip

2016-04-01

Streambed conductance controls the interaction between surface and groundwater. However, the streambed conductance is often subject to transience. Directly measuring hydraulic properties in a river yields only point values, is time-consuming and therefore not suited to detect transience of physical properties. Here, we present a method to continuously monitor transience in streambed conductance. Input data are time series of stream stage and near stream hydraulic head. The method is based on the inversion of floodwave responses. The analytical model consists of three parameters: x, the distance between streambank and an observation well, α, the aquifer diffusivity, and a the retardation coefficient that is inversely proportional to the streambed conductance. Estimation of a is carried out over successive time steps in order to identify transience in streambed conductance. The method is tested using synthetic data and is applied to field data from the Rhône River and its alluvial aquifer (Switzerland). The synthetic method demonstrated the robustness of the proposed methodology. Application of the method to the field data allowed identifying transience in streambed properties, following flood events in the Rhône. This method requires transience in the surface water, and the river should not change its width significantly with a rising water level. If these conditions are fulfilled, this method allows for a rapid and effective identification of transience in streambed conductance.

Laser-scanning techniques for rapid ballistics identification

NASA Technical Reports Server (NTRS)

Woodburgy, R. C.; Nakich, R. B.

1974-01-01

Two different laser-scanning methods may be utilized. In each case scanned cylindrical bullet surface is displayed ""unwrapped'' on oscilloscope screen. Bullets are compared by photographing each display and superimposing negatives of two images. With some modifications bullets can be scanned and compared by superimposing images on screen of dual-beam oscilloscope.

Inter-laboratory Comparison of Real-time PCR Methods for Quantification of General Fecal Indicator Bacteria

EPA Science Inventory

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized prot...

NANOSTRUCTURED PLANAR WAVEGUIDE DEVICE FOR MOLECULAR IDENTIFICATION OF HAZARDOUS COMPOUNDS IN WATER BY EVANESCENT SURFACE ENHANCED RAMAN SPECTROSCOPY - PHASE I

EPA Science Inventory

Senspex, Inc. proposes to investigate a novel diagnostic tool based upon evanescent field planar waveguide sensing and complementary nanostructured mediated molecular vibration spectroscopy methods for rapid detection and analysis of hazardous biological and chemical targets i...

Computational Issues in Damping Identification for Large Scale Problems

NASA Technical Reports Server (NTRS)

Pilkey, Deborah L.; Roe, Kevin P.; Inman, Daniel J.

1997-01-01

Two damping identification methods are tested for efficiency in large-scale applications. One is an iterative routine, and the other a least squares method. Numerical simulations have been performed on multiple degree-of-freedom models to test the effectiveness of the algorithm and the usefulness of parallel computation for the problems. High Performance Fortran is used to parallelize the algorithm. Tests were performed using the IBM-SP2 at NASA Ames Research Center. The least squares method tested incurs high communication costs, which reduces the benefit of high performance computing. This method's memory requirement grows at a very rapid rate meaning that larger problems can quickly exceed available computer memory. The iterative method's memory requirement grows at a much slower pace and is able to handle problems with 500+ degrees of freedom on a single processor. This method benefits from parallelization, and significant speedup can he seen for problems of 100+ degrees-of-freedom.

Development, standardization and validation of molecular techniques for malaria vector species identification, trophic preferences, and detection of Plasmodium falciparum.

PubMed

Rath, Animesha; Prusty, Manas R; Barik, Sushanta K; Das, Mumani; Tripathy, Hare K; Mahapatra, Namita; Hazra, Rupenangshu K

2017-01-01

Knowledge on prevalence of malaria vector species of a certain area provides important information for implementation of appropriate control strategies. The present study describes a rapid method for screening of major Anopheline vector species and at the same time detection of Plasmodium falciparum sporozoite infection and blood meal preferences/trophic preferences. The study was carried from February 2012 to March 2013 in three seasons, i.e. rainy, winter and summer in Jhumpura PHC of Keonjhar district, Odisha, India. Processing of mosquitoes was carried out in two different methods, viz. mosquito pool (P1) and mosquito DNA pool (P2). Pool size for both the methods was standardized for DNA isolation and multiplex PCR assay. This PCR based assay was employed to screen the major vector com- position in three different seasons of four different ecotypes of Keonjhar district. Pearson's correlation coefficient was determined for a comparative analysis of the morphological identification with the pool prevalence assay for each ecotype. A pool size of 10 was standardized for DNA isolation as well as PCR. PCR assay revealed that the average pool prevalence for all ecotypes was highest for An. annularis in winter and summer whereas for An. culicifacies it was rainy season. Foothill and plain ecotypes contributed to highest and lowest vectorial abundance respectively. The results of the prevalence of vector species in pool from PCR based assay were found to be highly correlated with that of the results of morphological identification. Screening by pool based PCR assay is relatively rapid as compared to conventional identification and can be employed as an important tool in malaria control programmes.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

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

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

[Rapid identification of potato cultivars using NIR-excited fluorescence and Raman spectroscopy].

PubMed

Dai, Fen; Bergholt, Mads Sylvest; Benjamin, Arnold Julian Vinoj; Hong, Tian-Sheng; Zhiwei, Huang

2014-03-01

Potato is one of the most important food in the world. Rapid and noninvasive identification of potato cultivars plays a important role in the better use of varieties. In this study, The identification ability of optical spectroscopy techniques, including near-infrared (NIR) Raman spectroscopy and NIR fluorescence spectroscopy, for invasive detection of potato cultivars was evaluated. A rapid NIR Raman spectroscopy system was applied to measure the composite Raman and NIR fluorescence spectroscopy of 3 different species of potatoes (98 samples in total) under 785 nm laser light excitation. Then pure Raman and NIR fluorescence spectroscopy were abstracted from the composite spectroscopy, respectively. At last, the partial least squares-discriminant analysis (PLS-DA) was utilized to analyze and classify Raman spectra of 3 different types of potatoes. All the samples were divided into two sets at random: the calibration set (74samples) and prediction set (24 samples), the model was validated using a leave-one-out, cross-validation method. The results showed that both the NIR-excited fluorescence spectra and pure Raman spectra could be used to identify three cultivars of potatoes. The fluorescence spectrum could distinguish the Favorita variety well (sensitivity: 1, specificity: 0.86 and accuracy: 0.92), but the result for Diamant (sensitivity: 0.75, specificity: 0.75 and accuracy: 0. 75) and Granola (sensitivity: 0.16, specificity: 0.89 and accuracy: 0.71) cultivars identification were a bit poorer. We demonstrated that Raman spectroscopy uncovered the main biochemical compositions contained in potato species, and provided a better classification sensitivity, specificity and accuracy (sensitivity: 1, specificity: 1 and accuracy: 1 for all 3 potato cultivars identification) among the three types of potatoes as compared to fluorescence spectroscopy.

Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI.

PubMed

Diba, K; Mirhendi, H; Kordbacheh, P; Rezaie, S

2014-01-01

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species.

Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI

PubMed Central

Diba, K.; Mirhendi, H.; Kordbacheh, P.; Rezaie, S.

2014-01-01

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species. PMID:25242934

Answering medical questions at the point of care: a cross-sectional study comparing rapid decisions based on PubMed and Epistemonikos searches with evidence-based recommendations developed with the GRADE approach.

PubMed

Izcovich, Ariel; Criniti, Juan Martín; Popoff, Federico; Ragusa, Martín Alberto; Gigler, Cristel; Gonzalez Malla, Carlos; Clavijo, Manuela; Manzotti, Matias; Diaz, Martín; Catalano, Hugo Norberto; Neumann, Ignacio; Guyatt, Gordon

2017-08-07

Using the best current evidence to inform clinical decisions remains a challenge for clinicians. Given the scarcity of trustworthy clinical practice guidelines providing recommendations to answer clinicians' daily questions, clinical decision support systems (ie, assistance in question identification and answering) emerge as an attractive alternative. The trustworthiness of the recommendations achieved by such systems is unknown. To evaluate the trustworthiness of a question identification and answering system that delivers timely recommendations. Cross-sectional study. We compared the responses to 100 clinical questions related to inpatient management provided by two rapid response methods with 'Gold Standard' recommendations. One of the rapid methods was based on PubMed and the other on Epistemonikos database. We defined our 'Gold Standard' as trustworthy published evidence-based recommendations or, when unavailable, recommendations developed locally by a panel of six clinicians following the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. Recommendations provided by the rapid strategies were classified as potentially misleading or reasonable. We also determined if the potentially misleading recommendations could have been avoided with the appropriate implementation of searching and evidence summary tools. We were able to answer all of the 100 questions with both rapid methods. Of the 200 recommendations obtained, 6.5% (95% CI 3% to 9.9%) were classified as potentially misleading and 93.5% (95% CI 90% to 96.9%) as reasonable. 6 of the 13 potentially misleading recommendations could have been avoided by the appropriate usage of the Epistemonikos matrix tool or by constructing summary of findings tables. No significant differences were observed between the evaluated rapid response methods. A question answering service based on the GRADE approach proved feasible to implement and provided appropriate guidance for most identified questions. Our approach could help stakeholders in charge of managing resources and defining policies for patient care to improve evidence-based decision-making in an efficient and feasible manner. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Rapid identification of pathogenic streptococci isolated from moribund red tilapia (Oreochromis spp.).

PubMed

Abdelsalam, Mohamed; Elgendy, Mamdouh Y; Shaalan, Mohamed; Moustafa, Mohamed; Fujino, Masayuki

2017-03-01

Accurate and rapid identification of bacterial pathogens of fish is essential for the effective treatment and speedy control of infections. Massive mortalities in market-sized red tilapia (Oreochromis spp.) were noticed in mariculture concrete ponds in northern Egypt. Histopathological examination revealed marked congestion in the central vein of the liver with the presence of bacterial aggregates inside the lumen and in the vicinity of the central vein. A total of 12 isolates of streptococci were obtained from the moribund fish. This study documented the ability of the MicroSeq 500 16S bacterial sequencing method to accurately identify Streptococcus agalactiae and S. dysgalactiae mixed infections from moribund red tilapia that were difficult to be recognised by the commercial biochemical systems. The continuously decreasing cost of the sequencing technique should encourage its application in routine diagnostic procedures.

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing.

PubMed

Karger, Axel; Stock, Rüdiger; Ziller, Mario; Elschner, Mandy C; Bettin, Barbara; Melzer, Falk; Maier, Thomas; Kostrzewa, Markus; Scholz, Holger C; Neubauer, Heinrich; Tomaso, Herbert

2012-10-10

Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

PubMed Central

2012-01-01

Background Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. Results A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. Conclusions Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed. PMID:23046611

Flight Test Results of a GPS-Based Pitot-Static Calibration Method Using Output-Error Optimization for a Light Twin-Engine Airplane

NASA Technical Reports Server (NTRS)

Martos, Borja; Kiszely, Paul; Foster, John V.

2011-01-01

As part of the NASA Aviation Safety Program (AvSP), a novel pitot-static calibration method was developed to allow rapid in-flight calibration for subscale aircraft while flying within confined test areas. This approach uses Global Positioning System (GPS) technology coupled with modern system identification methods that rapidly computes optimal pressure error models over a range of airspeed with defined confidence bounds. This method has been demonstrated in subscale flight tests and has shown small 2- error bounds with significant reduction in test time compared to other methods. The current research was motivated by the desire to further evaluate and develop this method for full-scale aircraft. A goal of this research was to develop an accurate calibration method that enables reductions in test equipment and flight time, thus reducing costs. The approach involved analysis of data acquisition requirements, development of efficient flight patterns, and analysis of pressure error models based on system identification methods. Flight tests were conducted at The University of Tennessee Space Institute (UTSI) utilizing an instrumented Piper Navajo research aircraft. In addition, the UTSI engineering flight simulator was used to investigate test maneuver requirements and handling qualities issues associated with this technique. This paper provides a summary of piloted simulation and flight test results that illustrates the performance and capabilities of the NASA calibration method. Discussion of maneuver requirements and data analysis methods is included as well as recommendations for piloting technique.

Rapid characterisation of Klebsiella oxytoca isolates from contaminated liquid hand soap using mass spectrometry, FTIR and Raman spectroscopy.

PubMed

Dieckmann, Ralf; Hammerl, Jens Andre; Hahmann, Hartmut; Wicke, Amal; Kleta, Sylvia; Dabrowski, Piotr Wojciech; Nitsche, Andreas; Stämmler, Maren; Al Dahouk, Sascha; Lasch, Peter

2016-06-23

Microbiological monitoring of consumer products and the efficiency of early warning systems and outbreak investigations depend on the rapid identification and strain characterisation of pathogens posing risks to the health and safety of consumers. This study evaluates the potential of three rapid analytical techniques for identification and subtyping of bacterial isolates obtained from a liquid hand soap product, which has been recalled and reported through the EU RAPEX system due to its severe bacterial contamination. Ten isolates recovered from two bottles of the product were identified as Klebsiella oxytoca and subtyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS), near-infrared Fourier transform (NIR FT) Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Comparison of the classification results obtained by these phenotype-based techniques with outcomes of the DNA-based methods pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis of whole-genome sequencing (WGS) data revealed a high level of concordance. In conclusion, a set of analytical techniques might be useful for rapid, reliable and cost-effective microbial typing to ensure safe consumer products and allow source tracking.

Same Day Identification and Full Panel Antimicrobial Susceptibility Testing of Bacteria from Positive Blood Culture Bottles Made Possible by a Combined Lysis-Filtration Method with MALDI-TOF VITEK Mass Spectrometry and the VITEK2 System

PubMed Central

Machen, Alexandra; Drake, Tim; Wang, Yun F. (Wayne)

2014-01-01

Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001). Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship. PMID:24551067

Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system.

PubMed

Machen, Alexandra; Drake, Tim; Wang, Yun F Wayne

2014-01-01

Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001). Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.

Establishment of a simple and rapid identification method for Listeria spp. by using high-resolution melting analysis, and its application in food industry.

PubMed

Ohshima, Chihiro; Takahashi, Hajime; Phraephaisarn, Chirapiphat; Vesaratchavest, Mongkol; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

2014-01-01

Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying Listeria spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying Listeria spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.

Establishment of a Simple and Rapid Identification Method for Listeria spp. by Using High-Resolution Melting Analysis, and Its Application in Food Industry

PubMed Central

Ohshima, Chihiro; Takahashi, Hajime; Phraephaisarn, Chirapiphat; Vesaratchavest, Mongkol; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

2014-01-01

Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying Listeria spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying Listeria spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment. PMID:24918440

[Rapid determination of volatile organic compounds in workplace air by protable gas chromatography-mass spectrometer].

PubMed

Zhu, H B; Su, C J; Tang, H F; Ruan, Z; Liu, D H; Wang, H; Qian, Y L

2017-10-20

Objective: To establish a method for rapid determination of 47 volatile organic compounds in the air of workplace using portable gas chromatography - mass spectrometer(GC - MS). Methods: The mixed standard gas with different concentration levels was made by using the static gas distribution method with the high purity nitrogen as dilution gas. The samples were injected into the GC - MS by a hand - held probe. Retention time and characteristic ion were used for qualitative analysis,and the internal standard method was usd for quantitation. Results: The 47 poisonous substances were separated and determined well. The linear range of this method was 0.2 - 16.0 mg/m(3),and the relative standard deviation of 45 volatile ovganic compounds was 3.8% - 15.8%. The average recovery was 79.3% - 119.0%. Conclusion: The method is simple,accurate,sensitive,has good separation effect,short analysis period, can be used for qualitative and quantitative analysis of volatile organic compounds in the workplace, and also supports the rapid identification and detection of occupational hazards.

MALDI-TOF mass spectrometry provides high accuracy in identification of Salmonella at species level but is limited to type or subtype Salmonella serovars.

PubMed

Kang, Lin; Li, Nan; Li, Ping; Zhou, Yang; Gao, Shan; Gao, Hongwei; Xin, Wenwen; Wang, Jinglin

2017-04-01

Salmonella can cause global foodborne illnesses in humans and many animals. The current diagnostic gold standard used for detecting Salmonella infection is microbiological culture followed by serological confirmation tests. However, these methods are complicated and time-consuming. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis offers some advantages in rapid identification, for example, simple and fast sample preparation, fast and automated measurement, and robust and reliable identification up to genus and species levels, possibly even to the strain level. In this study, we established a reference database for species identification using whole-cell MALDI-TOF MS; the database consisted of 12 obtained main spectra of the Salmonella culture collection strains belonged to seven serotypes. Eighty-two clinical isolates of Salmonella were identified using established database, and partial 16S rDNA gene sequencing and serological method were used as comparison. We found that MALDI-TOF mass spectrometry provided high accuracy in identification of Salmonella at species level but was limited to type or subtype Salmonella serovars. We also tried to find serovar-specific biomarkers and failed. Our study demonstrated that (a) MALDI-TOF MS was suitable for identification of Salmonella at species level with high accuracy and (b) that MALDI-TOF MS method presented in this study was not useful for serovar assignment of Salmonella currently, because of its low matching with serological method and (c) MALDI-TOF MS method presented in this study was not suitable to subtype S. typhimurium because of its low discriminatory ability.

Isolation and molecular characterization of multiresistant Staphylococcus sciuri and Staphylococcus haemolyticus associated with skin and soft-tissue infections.

PubMed

Shittu, Adebayo; Lin, Johnson; Morrison, Donald; Kolawole, Deboye

2004-01-01

The isolation, molecular identification and genotyping of multiresistant Staphylococcus sciuri and Staphylococcus haemolyticus from skin and soft-tissue infections are reported. Accurate and full identification of three coagulase-negative staphylococcal isolates was achieved using PCR, while the API STAPH method failed to identify an isolate of S. haemolyticus fully. The PCR assay, which detects polymorphism in the 16S-23S rRNA spacer region, is shown to be potentially useful for rapid and accurate identification of coagulase-negative staphylococci. Identical PFGE type and antibiotic-resistance profiles of two methicillin-resistant S. haemolyticus isolates in this study suggest the existence of a multiresistant community clone.

Using DNA barcoding to differentiate invasive Dreissena species (Mollusca, Bivalvia)

PubMed Central

Marescaux, Jonathan; Van Doninck, Karine

2013-01-01

Abstract The zebra mussel (Dreissena polymorpha) and the quagga mussel (Dreissena rostriformis bugensis) are considered as the most competitive invaders in freshwaters of Europe and North America. Although shell characteristics exist to differentiate both species, phenotypic plasticity in the genus Dreissena does not always allow a clear identification. Therefore, the need to find an accurate identification method is essential. DNA barcoding has been proven to be an adequate procedure to discriminate species. The cytochrome c oxidase subunit I mitochondrial gene (COI) is considered as the standard barcode for animals. We tested the use of this gene as an efficient DNA barcode and found that it allow rapid and accurate identification of adult Dreissena individuals. PMID:24453560

Dynamic biometric identification from multiple views using the GLBP-TOP method.

PubMed

Wang, Yu; Shen, Xuanjing; Chen, Haipeng; Zhai, Yujie

2014-01-01

To realize effective and rapid dynamic biometric identification with low computational complexity, a video-based facial texture program that extracts local binary patterns from three orthogonal planes in the frequency domain of the Gabor transform (GLBP-TOP) was proposed. Firstly, each normalized face was transformed by Gabor wavelet to get the enhanced Gabor magnitude map, and then the LBP-TOP operator was applied to the maps to extract video texture. Finally, weighted Chi square statistics based on the Fisher Criterion were used to realize the identification. The proposed algorithm was proved effective through the biometric experiments using the Honda/UCSD database, and was robust against changes of illumination and expressions.

Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Schmidt, V; Jarosch, A; März, P; Sander, C; Vacata, V; Kalka-Moll, W

2012-03-01

Blood culture is probably the most significant specimen used for the diagnosis of bacterial infections, especially for bloodstream infections. In the present study, we compared the resin-containing BD BACTEC™ Plus-Aerobic (Becton Dickinson), non-charcoal-containing BacT/Alert(®) SA (bioMérieux), and charcoal-containing BacT/Alert(®) FA (bioMérieux) blood culture bottles with direct identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 103 bacterial isolates, from clinical blood cultures, representing the most frequent 13 genera and 24 species were examined. Bacteria were extracted from positive blood culture broth by density centrifugation and then subjected to identification by MALDI-TOF MS using two different volumes and chemical treatments. Overall, correct identification by MALDI-TOF MS was obtained for the BD BACTEC™ Plus-Aerobic, BacT/Alert(®) SA, and BacT/Alert(®) FA blood culture bottles in 72%, 45.6%, and 23%, respectively, for gram-negative bacteria in 86.6%, 69.2%, and 47.1%, respectively, and for gram-positive bacteria in 60.0%, 28.8%, and 5.4%, respectively. The lack of identification was observed mainly with viridans streptococci. Depending on the blood culture bottles used in routine diagnostic procedures and the protocol for bacterial preparation, the applied MALDI-TOF MS represents an efficient and rapid method for direct bacterial identification.

Prospective Evaluation of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System in a Hospital Clinical Microbiology Laboratory for Identification of Bacteria and Yeasts: a Bench-by-Bench Study for Assessing the Impact on Time to Identification and Cost-Effectiveness

PubMed Central

Tan, K. E.; Ellis, B. C.; Lee, R.; Stamper, P. D.; Zhang, S. X.

2012-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories. PMID:22855510

Prospective evaluation of a matrix-assisted laser desorption ionization-time of flight mass spectrometry system in a hospital clinical microbiology laboratory for identification of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identification and cost-effectiveness.

PubMed

Tan, K E; Ellis, B C; Lee, R; Stamper, P D; Zhang, S X; Carroll, K C

2012-10-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories.

Alkaloid profiling of the traditional Chinese medicine Rhizoma corydalis using high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry

PubMed Central

Sun, Mingqian; Liu, Jianxun; Lin, Chengren; Miao, Lan; Lin, Li

2014-01-01

Since alkaloids are the major active constituents of Rhizoma corydalis (RC), a convenient and accurate analytical method is needed for their identification and characterization. Here we report a method to profile the alkaloids in RC based on liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (LC–Q-TOF-MS/MS). A total of 16 alkaloids belonging to four different classes were identified by comparison with authentic standards. The fragmentation pathway of each class of alkaloid was clarified and their differences were elucidated. Furthermore, based on an analysis of fragmentation pathways and alkaloid profiling, a rapid and accurate method for the identification of unknown alkaloids in RC is proposed. The method could also be useful for the quality control of RC. PMID:26579385

Assessment of four protocols for rapid bacterial identification from positive blood culture pellets by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Vitek® MS).

PubMed

Thomin, Jean; Aubin, Guillaume Ghislain; Foubert, Fabrice; Corvec, Stéphane

2015-08-01

In this study, we developed and compared four protocols to prepare a bacterial pellet from 944 positive blood cultures for direct MALDI-TOF mass spectrometry Vitek® MS analysis. Protocol 4, tested on 200 monomicrobial samples, allowed 83% of bacterial identification. This easy, fast, cheap and accurate method is promising in daily practice, especially to limit broad range antibiotic treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

High-throughput molecular identification of Staphylococcus spp. isolated from a clean room facility in an environmental monitoring program

PubMed Central

2010-01-01

Background The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. Findings We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. Conclusions Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features. PMID:21047438

High-throughput molecular identification of Staphylococcus spp. isolated from a clean room facility in an environmental monitoring program.

PubMed

Sheraba, Norhan S; Yassin, Aymen S; Amin, Magdy A

2010-11-04

The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features.

Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y.; Drake, Steven K.; Gucek, Marjan; Sacks, David B.; Yu, Yi-Kuo

2018-06-01

Rapid and accurate identification and classification of microorganisms is of paramount importance to public health and safety. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is complicating correct microbial identification even in a simple sample due to the large number of candidates present. To properly untwine candidate microbes in samples containing one or more microbes, one needs to go beyond apparent morphology or simple "fingerprinting"; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptide-centric representations of microbes to better separate them and by augmenting our earlier analysis method that yields accurate statistical significance. Here, we present an updated analysis workflow that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000 MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. We have also shown that the proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry.

PubMed

Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y; Drake, Steven K; Gucek, Marjan; Sacks, David B; Yu, Yi-Kuo

2018-06-05

Rapid and accurate identification and classification of microorganisms is of paramount importance to public health and safety. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is complicating correct microbial identification even in a simple sample due to the large number of candidates present. To properly untwine candidate microbes in samples containing one or more microbes, one needs to go beyond apparent morphology or simple "fingerprinting"; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptide-centric representations of microbes to better separate them and by augmenting our earlier analysis method that yields accurate statistical significance. Here, we present an updated analysis workflow that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000 MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. We have also shown that the proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html . Graphical Abstract ᅟ.

Capillary electrophoresis for rapid identification of monoclonal antibodies for routine application in hospital.

PubMed

Jaccoulet, Emmanuel; Smadja, Claire; Prognon, Patrice; Taverna, Myriam

2015-09-01

mAbs are widely used in cancer therapy. Their compounding, performed just before their administration to patients, is executed in a production unit of the hospital. Identification of these drugs, individually prepared in bags for infusion before patient administration, is of paramount importance to detect potential mistakes during compounding stage. A fast and reliable analytical method based on CZE combined to a cationic capillary coating (hexadimethrine bromide) was developed for identification of the most widely used compounded therapeutic for cancer therapy (bevacizumab, cetuximab, rituximab, and trastuzumab). Considering the high structural and physico-chemical similarities of these mAbs, an extensive optimization of the BGE composition has been performed. The addition of perchlorate ions and polysorbate in the BGE greatly increased the resolution. To validate the method, an internal standard was used and the relative migration times (RTm) were estimated. Very satisfactory RSDs of the RTm for rituximab (0.76%), cetuximab (0.46%), bevacizumab (0.31%), and trastuzumab (0.60%) were obtained. The intraday and interday RSD of the method were less than 0.32 and 1.3%, respectively for RTm. Significant differences between theses RTms have been demonstrated allowing mAbs identification. Finally, accurate mAbs identification has been demonstrated by a blind test. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid identification of single microbes by various Raman spectroscopic techniques

NASA Astrophysics Data System (ADS)

Rösch, Petra; Harz, Michaela; Schmitt, Michael; Peschke, Klaus-Dieter; Ronneberger, Olaf; Burkhardt, Hans; Motzkus, Hans-Walter; Lankers, Markus; Hofer, Stefan; Thiele, Hans; Popp, Jürgen

2006-02-01

A fast and unambiguous identification of microorganisms is necessary not only for medical purposes but also in technical processes such as the production of pharmaceuticals. Conventional microbiological identification methods are based on the morphology and the ability of microbes to grow under different conditions on various cultivation media depending on their biochemical properties. These methods require pure cultures which need cultivation of at least 6 h but normally much longer. Recently also additional methods to identify bacteria are established e.g. mass spectroscopy, polymerase chain reaction (PCR), flow cytometry or fluorescence spectroscopy. Alternative approaches for the identification of microorganisms are vibrational spectroscopic techniques. With Raman spectroscopy a spectroscopic fingerprint of the microorganisms can be achieved. Using UV-resonance Raman spectroscopy (UVRR) macromolecules like DNA/RNA and proteins are resonantly enhanced. With an excitation wavelength of e.g. 244 nm it is possible to determine the ratio of guanine/cytosine to all DNA bases which allows a genotypic identification of microorganisms. The application of UVRR requires a large amount of microorganisms (> 10 6 cells) e.g. at least a micro colony. For the analysis of single cells micro-Raman spectroscopy with an excitation wavelength of 532 nm can be used. Here, the obtained information is from all type of molecules inside the cells which lead to a chemotaxonomic identification. In this contribution we show how wavelength dependent Raman spectroscopy yields significant molecular information applicable for the identification of microorganisms on a single cell level.

[Identification characters of leaf morphological and venation pattern of Houttuynia cordata with its confused herb Gymnotheca chinensis].

PubMed

Lu, Hai-Lin; Guo, Min; Liao, Yue-Kui; Huang, Ding-Ying; Huang, Chun-Ni; Wu, Xiao-Chen; He, Bao-Zuo

2012-11-01

To study the identification characters of Houttuynia cordata and its confused herb Gymnotheca chinensis and establish an identification method. LMVP (leaf morphological-venation pattern for identification Chinese herbs), and QAERM (quantitatively analyze and evaluate reliability for the method of identification Chinese herbs) were applied for the study. Both venations were brochidodromous-acrodromous and arising from the mid-petiole or the upper section of petiole. The main characteristic of the leaf of Houttuynia cordata: surface with small gray-white stoma protuberances; Ligulate process of stipule-petiole sheath were clear; Primary veins 7 or 5; The innermost pair of primary vein closed up the top of the sinus at blade base or above sinus, and the section of closed vein was straight; Emitted a smell of fish when fresh leaf was kneaded into pieces. The main feature of the leaf of Gymnotheca chinensis: no small gray-white stoma protuberances; Ligulate process of stipule-petiole sheath were not clear; Primary veins 5; The innermost pair of primary vein closed into the sinus at blade base, and the section of closed vein was slightly curve; No smell of fish. With the mentioned key differences, the both plants could be successfully identified from each other. The accuracy of identification results (AC) was 100%, the repeatability of identification results: agreement rate for observation (ARO) was 100% and Kappa value was 1.00. The established method is simple, rapid, economic and reliable.

VIP Barcoding: composition vector-based software for rapid species identification based on DNA barcoding.

PubMed

Fan, Long; Hui, Jerome H L; Yu, Zu Guo; Chu, Ka Hou

2014-07-01

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time-consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user-friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two-stage algorithm. First, an alignment-free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment-based K2P distance nearest-neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment-free methods and (ii) higher scalability than alignment-based distance methods and character-based methods. These results suggest that this platform is able to deal with both large-scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/. © 2014 John Wiley & Sons Ltd.

[Applications of MALDI-TOF-MS in clinical microbiology laboratory].

PubMed

Carbonnelle, Etienne; Nassif, Xavier

2011-10-01

For twenty years, mass spectrometry (MS) has emerged as a particularly powerful tool for analysis and characterization of proteins in research. It is only recently that this technology, especially MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization Time-Of-Flight) has entered the field of routine microbiology. This method has proven to be reliable and safe for the identification of bacteria, yeasts, filamentous fungi and dermatophytes. MALDI-TOF-MS is a rapid, precise and cost-effective method for identification, compared to conventional phenotypic techniques or molecular biology. Its ability to analyse whole microorganisms with few sample preparation has greatly reduced the time to identification (1-2 min). Furthermore, this technology can be used to identify bacteria directly from clinical samples as blood culture bottles or urines. Future applications will be developed in order to provide direct information concerning virulence or resistance protein markers. © 2011 médecine/sciences – Inserm / SRMS.

Use of constant wavelength synchronous spectrofluorimetry for identification of polycyclic aromatic hydrocarbons in air particulate samples

NASA Astrophysics Data System (ADS)

Sharma, Homdutt; Jain, V. K.; Khan, Zahid H.

2013-05-01

We have developed a simple, rapid, inexpensive method for the identification of fluoranthene (Flan), benz(a)anthracene (BaA), benzo(a)pyrene (BaP), benzo(k)fluoranthene (BkF), pyrene (Pyr), benz(ghi)perylene (BghiP) in suspended particulate matter in an urban environment of Delhi. Suspended particulate matter samples of 24 h duration were collected on glass fiber filter papers. Polycyclic aromatic hydrocarbons (PAHs) were extracted from the filter papers using dichloromethane (DCM) and hexane with ultrasonication method. Comparison of the characteristic emission of spectra of PAHs with standard spectra indicated the degree of condensation of aromatic compounds present in investigated mixtures. It was also possible to identify some individual compounds. However, this identification could be more effective with the use of the respective values of Δλ parameter for each particular component of the mixture.

Impact of Pharmaceutical Impurities in Ecstasy Tablets: Gas Chromatography-Mass Spectrometry Study

PubMed Central

Jalali, Amir; Hatamie, Amir; Saferpour, Tahere; Khajeamiri, Alireza; Safa, Tahere; Buazar, Foad

2016-01-01

In this study, a simple and reliable method by gas chromatograph–mass spectrometry (GC–MS) was developed for the fast and regular identification of 3, 4-MDMA impurities in ecstasy tablets. In so doing, 8 samples of impurities were extracted by diethyl ether under alkaline condition and then analyzed by GC–MS. The results revealed high MDMA levels ranging from 37.6% to 57.7%. The GC-MS method showed that unambiguous identification can be achieved for MDMA from 3, 4-methylenedioxyamphetamine (MDA), Amphetamine (AM), methamphetamine (MA) and ketamine (Keta) compounds, respectively. The experimental results indicated the acceptable time window without interfering peaks. It is found that GC-MS was provided a suitable and rapid identification approach for MDMA (Ecstacy) tablets, particularly in the Forensic labs. Consequently, the intense MDMA levels would support the police to develop a simple quantification of impurity in Ecstasy tablets. PMID:27610162

Application of SERS spectroscopy to the identification of (3,4-methylenedioxy)amphetamine in forensic samples utilizing matrix stabilized silver halides.

PubMed

Sägmüller, B; Schwarze, B; Brehm, G; Schneider, S

2001-11-01

A method based on surface-enhanced Raman scattering (SERS) spectroscopy was developed to meet the need for the reliable and rapid identification of illicit drugs such as the 'designer drug' XTC, preferably to increase the security of legal certificates. A matrix stabilized silver halide dispersion on a microtiter plate is used as the SERS-active substrate, providing an easy to use system for sample preparation and probing by means of a Raman microscope. The potential of the method is demonstrated by applying it to the identification of the psychoactive ingredients of drug containing tablets which were confiscated by the local police at techno-music events. The samples of interest were 26 different brands of XTC tablets and several pieces of evidence (powders) containing amphetamine. For reference, we show SERS and Raman spectra of pristine amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethamphetamine.

RAPD-PCR characterization of lactobacilli isolated from artisanal meat plants and traditional fermented sausages of Veneto region (Italy).

PubMed

Andrighetto, C; Zampese, L; Lombardi, A

2001-07-01

The study was carried out to evaluate the use of randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) as a method for the identification of lactobacilli isolated from meat products. RAPD-PCR with primers M13 and D8635 was applied to the identification and intraspecific differentiation of 53 lactobacilli isolates originating from traditional fermented sausages and artisanal meat plants of the Veneto region (Italy). Most of the isolates were assigned to the species Lactobacillus sakei and Lact. curvatus; differentiation of groups of strains within the species was also possible. RAPD-PCR could be applied to the identification of lactobacilli species most commonly found in meat products. The method, which is easy and rapid to perform, could be useful for the study of the lactobacilli populations present in fermented sausages, and could help in the selection of candidate strains to use as starter cultures in meat fermentation.

Data mining for the identification of metabolic syndrome status

PubMed Central

Worachartcheewan, Apilak; Schaduangrat, Nalini; Prachayasittikul, Virapong; Nantasenamat, Chanin

2018-01-01

Metabolic syndrome (MS) is a condition associated with metabolic abnormalities that are characterized by central obesity (e.g. waist circumference or body mass index), hypertension (e.g. systolic or diastolic blood pressure), hyperglycemia (e.g. fasting plasma glucose) and dyslipidemia (e.g. triglyceride and high-density lipoprotein cholesterol). It is also associated with the development of diabetes mellitus (DM) type 2 and cardiovascular disease (CVD). Therefore, the rapid identification of MS is required to prevent the occurrence of such diseases. Herein, we review the utilization of data mining approaches for MS identification. Furthermore, the concept of quantitative population-health relationship (QPHR) is also presented, which can be defined as the elucidation/understanding of the relationship that exists between health parameters and health status. The QPHR modeling uses data mining techniques such as artificial neural network (ANN), support vector machine (SVM), principal component analysis (PCA), decision tree (DT), random forest (RF) and association analysis (AA) for modeling and construction of predictive models for MS characterization. The DT method has been found to outperform other data mining techniques in the identification of MS status. Moreover, the AA technique has proved useful in the discovery of in-depth as well as frequently occurring health parameters that can be used for revealing the rules of MS development. This review presents the potential benefits on the applications of data mining as a rapid identification tool for classifying MS. PMID:29383020

Data mining for the identification of metabolic syndrome status.

PubMed

Worachartcheewan, Apilak; Schaduangrat, Nalini; Prachayasittikul, Virapong; Nantasenamat, Chanin

2018-01-01

Metabolic syndrome (MS) is a condition associated with metabolic abnormalities that are characterized by central obesity (e.g. waist circumference or body mass index), hypertension (e.g. systolic or diastolic blood pressure), hyperglycemia (e.g. fasting plasma glucose) and dyslipidemia (e.g. triglyceride and high-density lipoprotein cholesterol). It is also associated with the development of diabetes mellitus (DM) type 2 and cardiovascular disease (CVD). Therefore, the rapid identification of MS is required to prevent the occurrence of such diseases. Herein, we review the utilization of data mining approaches for MS identification. Furthermore, the concept of quantitative population-health relationship (QPHR) is also presented, which can be defined as the elucidation/understanding of the relationship that exists between health parameters and health status. The QPHR modeling uses data mining techniques such as artificial neural network (ANN), support vector machine (SVM), principal component analysis (PCA), decision tree (DT), random forest (RF) and association analysis (AA) for modeling and construction of predictive models for MS characterization. The DT method has been found to outperform other data mining techniques in the identification of MS status. Moreover, the AA technique has proved useful in the discovery of in-depth as well as frequently occurring health parameters that can be used for revealing the rules of MS development. This review presents the potential benefits on the applications of data mining as a rapid identification tool for classifying MS.

Disseminated histoplasmosis and AIDS: clinical aspects and diagnostic methods for early detection.

PubMed

Corti, M E; Cendoya, C A; Soto, I; Esquivel, P; Trione, N; Villafañe, M F; Corbera, K M; Helou, S; Negroni, R

2000-03-01

Disseminated histoplasmosis in AIDS patients is the focus of this paper. Cutaneous lesions are reported as a frequent clinical sign. Bone marrow aspiration and biopsy, blood cultures (lysis-centrifugation technique), bronchoalveolar lavage, and skin lesion scrapings are the most effective diagnostic methods. The identification of a specific antigen in blood and urine may be a rapid means of evaluation and follow-up of patients with this disease.

Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections.

PubMed

Pulcrano, Giovanna; Iula, Dora Vita; Vollaro, Antonio; Tucci, Alessandra; Cerullo, Monica; Esposito, Matilde; Rossano, Fabio; Catania, Maria Rosaria

2013-09-01

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has recently become an effective instrument for rapid microbiological diagnostics and in particular for identification of micro-organisms directly in a positive blood culture. The aim of the study was to evaluate a collection of 82 stored yeast isolates from bloodstream infection, by MALDI-TOF MS; 21 isolates were identified also directly from positive blood cultures and in the presence of other co-infecting micro-organisms. Of the 82 isolates grown on plates, 64 (76%) were correctly identified by the Vitek II system and 82 (100%) by MALDI-TOF MS; when the two methods gave different results, the isolate was identified by PCR. MALDI-TOF MS was unreliable in identifying two isolates (Candida glabrata and Candida parapsilosis) directly from blood culture; however, direct analysis from positive blood culture samples was fast and effective for the identification of yeast, which is of great importance for early and adequate treatment. © 2013. Published by Elsevier B.V. All rights reserved.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification.

PubMed

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64 °C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 10(3) cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

PubMed Central

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness. PMID:26208001

Performance evaluation of three automated identification systems in detecting carbapenem-resistant Enterobacteriaceae.

PubMed

He, Qingwen; Chen, Weiyuan; Huang, Liya; Lin, Qili; Zhang, Jingling; Liu, Rui; Li, Bin

2016-06-21

Carbapenem-resistant Enterobacteriaceae (CRE) is prevalent around the world. Rapid and accurate detection of CRE is urgently needed to provide effective treatment. Automated identification systems have been widely used in clinical microbiology laboratories for rapid and high-efficient identification of pathogenic bacteria. However, critical evaluation and comparison are needed to determine the specificity and accuracy of different systems. The aim of this study was to evaluate the performance of three commonly used automated identification systems on the detection of CRE. A total of 81 non-repetitive clinical CRE isolates were collected from August 2011 to August 2012 in a Chinese university hospital, and all the isolates were confirmed to be resistant to carbapenems by the agar dilution method. The potential presence of carbapenemase genotypes of the 81 isolates was detected by PCR and sequencing. Using 81 clinical CRE isolates, we evaluated and compared the performance of three automated identification systems, MicroScan WalkAway 96 Plus, Phoenix 100, and Vitek 2 Compact, which are commonly used in China. To identify CRE, the comparator methodology was agar dilution method, while the PCR and sequencing was the comparator one to identify CPE. PCR and sequencing analysis showed that 48 of the 81 CRE isolates carried carbapenemase genes, including 23 (28.4 %) IMP-4, 14 (17.3 %) IMP-8, 5 (6.2 %) NDM-1, and 8 (9.9 %) KPC-2. Notably, one Klebsiella pneumoniae isolate produced both IMP-4 and NDM-1. One Klebsiella oxytoca isolate produced both KPC-2 and IMP-8. Of the 81 clinical CRE isolates, 56 (69.1 %), 33 (40.7 %) and 77 (95.1 %) were identified as CRE by MicroScan WalkAway 96 Plus, Phoenix 100, and Vitek 2 Compact, respectively. The sensitivities/specificities of MicroScan WalkAway, Phoenix 100 and Vitek 2 were 93.8/42.4 %, 54.2/66.7 %, and 75.0/36.4 %, respectively. The MicroScan WalkAway and Viteck2 systems are more reliable in clinical identification of CRE, whereas additional tests are required for the Pheonix 100 system. Our study provides a useful guideline for using automated identification systems for CRE identification.

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

PubMed

Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang

2018-05-01

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

Comparative evaluation of the identification of rapidly growing non-tuberculous mycobacteria by mass spectrometry (MALDI-TOF MS), GenoType Mycobacterium CM/AS assay and partial sequencing of the rpoβ gene with phylogenetic analysis as a reference method.

PubMed

Costa-Alcalde, José Javier; Barbeito-Castiñeiras, Gema; González-Alba, José María; Aguilera, Antonio; Galán, Juan Carlos; Pérez-Del-Molino, María Luisa

2018-06-02

The American Thoracic Society and the Infectious Diseases Society of America recommend that clinically significant non-tuberculous mycobacteria (NTM) should be identified to the species level in order to determine their clinical significance. The aim of this study was to evaluate identification of rapidly growing NTM (RGM) isolated from clinical samples by using MALDI-TOF MS and a commercial molecular system. The results were compared with identification using a reference method. We included 46 clinical isolates of RGM and identified them using the commercial molecular system GenoType ® CM/AS (Hain, Lifescience, Germany), MALDI-TOF MS (Bruker) and, as reference method, partial rpoβ gene sequencing followed by BLAST and phylogenetic analysis with the 1093 sequences available in the GeneBank. The degree of agreement between GenoType ® and MALDI-TOF MS and the reference method, partial rpoβ sequencing, was 27/43 (62.8%) and 38/43 cases (88.3%) respectively. For all the samples correctly classified by GenoType ® , we obtained the same result with MALDI-TOF MS (27/27). However, MALDI-TOF MS also correctly identified 68.75% (11/16) of the samples that GenoType ® had misclassified (p=0.005). MALDI-TOF MS classified significantly better than GenoType ® . When a MALDI-TOF MS score >1.85 was achieved, MALDI-TOF MS and partial rpoβ gene sequencing were equivalent. GenoType ® was not able to distinguish between species belonging to the M. fortuitum complex. MALDI-TOF MS methodology is simple, rapid and associated with lower consumable costs than GenoType ® . The partial rpoβ sequencing methods with BLAST and phylogenetic analysis were not able to identify some RGM unequivocally. Therefore, sequencing of additional regions would be indicated in these cases. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Rapid screening for citrus canker resistance employing pattern-triggered immunity (PTI) responses

USDA-ARS?s Scientific Manuscript database

Citrus canker, caused by the bacterial pathogen Xanthomonas citri ssp. citri (Xcc), has been attributed to millions of dollars in loss or damage to commercial citrus crops in subtropical production areas of the world. Since identification of resistant plants is one of the most effective methods of d...

In-Flight Pitot-Static Calibration

NASA Technical Reports Server (NTRS)

Foster, John V. (Inventor); Cunningham, Kevin (Inventor)

2016-01-01

A GPS-based pitot-static calibration system uses global output-error optimization. High data rate measurements of static and total pressure, ambient air conditions, and GPS-based ground speed measurements are used to compute pitot-static pressure errors over a range of airspeed. System identification methods rapidly compute optimal pressure error models with defined confidence intervals.

Identification of Bacterial Populations in Drinking Water Using 16S rRNA-Based Sequence Analyses

EPA Science Inventory

Intracellular RNA is rapidly degraded in stressed cells and is more unstable outside of the cell than DNA. As a result, RNA-based methods have been suggested to study the active microbial fraction in environmental matrices. The aim of this study was to identify bacterial populati...

New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization▿

PubMed Central

Traversa, Donato; Iorio, Raffaella; Klei, Thomas R.; Kharchenko, Vitaliy A.; Gawor, Jakub; Otranto, Domenico; Sparagano, Olivier A. E.

2007-01-01

The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species. PMID:17626168

Studies on the methods of identification of irradiated food I. Seedling growth test

NASA Astrophysics Data System (ADS)

Qiongying, Liu; Yanhua, Kuang; Yuemei, Zheng

1993-07-01

A seedling growth test for the identification of gamma irradiated edible vegetable seeds was described. The identification of gamma irradiated grape and the other seeds has been investigated. The purpose of this study was to develop an easy, rapid and practical technique for the identification of irradiated edible vegetable seeds. Seven different irradiated edible vegetable seeds as: rice ( Oryza sativa), peanut ( Arachis hypogaea), maize ( Zeamays), soybean ( Glycine max), red bean ( Phaseolus angularis), mung bean ( Phaseolus aureus) and catjang cowpea ( Vigna cylindrica) were tested by using the method of seedling growth. All of the edible vegetable seeds were exposed to gamma radiation on different doses, O(CK), 0.5, 1.0, 1.5, 2.0, 3.0, 5.0 kGy. After treatment with above 1.0 kGy dose to the seeds, the seedling rate was less than 50% compared with the control. Although the seedling rate of rice seeds can reached 58%, the seedling growth was not normal and the seedling leaves appeared deformed. The results by this method were helpful to identify gamma treatment of the edible vegetable seeds with above 1.0 kGy dose.

Direct Identification of Enteroviruses in Cerebrospinal Fluid of Patients with Suspected Meningitis by Nested PCR Amplification.

PubMed

Krasota, Alexandr; Loginovskih, Natalia; Ivanova, Olga; Lipskaya, Galina

2016-01-06

Enteroviruses, the most common human viral pathogens worldwide, have been associated with serous meningitis, encephalitis, syndrome of acute flaccid paralysis, myocarditis and the onset of diabetes type 1. In the future, the rapid identification of the etiological agent would allow to adjust the therapy promptly and thereby improve the course of the disease and prognosis. We developed RT-nested PCR amplification of the genomic region coding viral structural protein VP1 for direct identification of enteroviruses in clinical specimens and compared it with the existing analogs. One-hundred-fifty-nine cerebrospinal fluids (CSF) from patients with suspected meningitis were studied. The amplification of VP1 genomic region using the new method was achieved for 86 (54.1%) patients compared with 75 (47.2%), 53 (33.3%) and 31 (19.5%) achieved with previously published methods. We identified 11 serotypes of the Enterovirus species B in 2012, including relatively rare echovirus 14 (E-14), E-15 and E-32, and eight serotypes of species B and 5 enteroviruses A71 (EV-A71) in 2013. The developed method can be useful for direct identification of enteroviruses in clinical material with the low virus loads such as CSF.

Direct Identification of Enteroviruses in Cerebrospinal Fluid of Patients with Suspected Meningitis by Nested PCR Amplification

PubMed Central

Krasota, Alexandr; Loginovskih, Natalia; Ivanova, Olga; Lipskaya, Galina

2016-01-01

Enteroviruses, the most common human viral pathogens worldwide, have been associated with serous meningitis, encephalitis, syndrome of acute flaccid paralysis, myocarditis and the onset of diabetes type 1. In the future, the rapid identification of the etiological agent would allow to adjust the therapy promptly and thereby improve the course of the disease and prognosis. We developed RT-nested PCR amplification of the genomic region coding viral structural protein VP1 for direct identification of enteroviruses in clinical specimens and compared it with the existing analogs. One-hundred-fifty-nine cerebrospinal fluids (CSF) from patients with suspected meningitis were studied. The amplification of VP1 genomic region using the new method was achieved for 86 (54.1%) patients compared with 75 (47.2%), 53 (33.3%) and 31 (19.5%) achieved with previously published methods. We identified 11 serotypes of the Enterovirus species B in 2012, including relatively rare echovirus 14 (E-14), E-15 and E-32, and eight serotypes of species B and 5 enteroviruses A71 (EV-A71) in 2013. The developed method can be useful for direct identification of enteroviruses in clinical material with the low virus loads such as CSF. PMID:26751470

Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

PubMed

Yang, Samuel; Rothman, Richard E; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A

2008-04-01

To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit™ for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital.

PubMed

Tadros, Manal; Petrich, Astrid

2013-01-01

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit - the Sepsityper Kit (Bruker Daltonik, Germany) - and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%. MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.

Identification of Enterococcus, Streptococcus, and Staphylococcus by Multivariate Analysis of Proton Magnetic Resonance Spectroscopic Data from Plate Cultures

PubMed Central

Bourne, Roger; Himmelreich, Uwe; Sharma, Ansuiya; Mountford, Carolyn; Sorrell, Tania

2001-01-01

A new fingerprinting technique with the potential for rapid identification of bacteria was developed by combining proton magnetic resonance spectroscopy (1H MRS) with multivariate statistical analysis. This resulted in an objective identification strategy for common clinical isolates belonging to the bacterial species Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and the Streptococcus milleri group. Duplicate cultures of 104 different isolates were examined one or more times using 1H MRS. A total of 312 cultures were examined. An optimized classifier was developed using a bootstrapping process and a seven-group linear discriminant analysis to provide objective classification of the spectra. Identification of isolates was based on consistent high-probability classification of spectra from duplicate cultures and achieved 92% agreement with conventional methods of identification. Fewer than 1% of isolates were identified incorrectly. Identification of the remaining 7% of isolates was defined as indeterminate. PMID:11474013

Method for rapid base sequencing in DNA and RNA

DOEpatents

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

1987-10-07

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

Method for rapid base sequencing in DNA and RNA

DOEpatents

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

1990-10-09

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

Method for rapid base sequencing in DNA and RNA

DOEpatents

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

1990-01-01

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

A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

PubMed

Handy, Sara M; Deeds, Jonathan R; Ivanova, Natalia V; Hebert, Paul D N; Hanner, Robert H; Ormos, Andrea; Weigt, Lee A; Moore, Michelle M; Yancy, Haile F

2011-01-01

The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use.

Development of a new protocol for rapid bacterial identification and susceptibility testing directly from urine samples.

PubMed

Zboromyrska, Y; Rubio, E; Alejo, I; Vergara, A; Mons, A; Campo, I; Bosch, J; Marco, F; Vila, J

2016-06-01

The current gold standard method for the diagnosis of urinary tract infections (UTI) is urine culture that requires 18-48 h for the identification of the causative microorganisms and an additional 24 h until the results of antimicrobial susceptibility testing (AST) are available. The aim of this study was to shorten the time of urine sample processing by a combination of flow cytometry for screening and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for bacterial identification followed by AST directly from urine. The study was divided into two parts. During the first part, 675 urine samples were processed by a flow cytometry device and a cut-off value of bacterial count was determined to select samples for direct identification by MALDI-TOF-MS at ≥5 × 10(6) bacteria/mL. During the second part, 163 of 1029 processed samples reached the cut-off value. The sample preparation protocol for direct identification included two centrifugation and two washing steps. Direct AST was performed by the disc diffusion method if a reliable direct identification was obtained. Direct MALDI-TOF-MS identification was performed in 140 urine samples; 125 of the samples were positive by urine culture, 12 were contaminated and 3 were negative. Reliable direct identification was obtained in 108 (86.4%) of the 125 positive samples. AST was performed in 102 identified samples, and the results were fully concordant with the routine method among 83 monomicrobial infections. In conclusion, the turnaround time of the protocol described to diagnose UTI was about 1 h for microbial identification and 18-24 h for AST. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A distributed computational search strategy for the identification of diagnostics targets: application to finding aptamer targets for methicillin-resistant staphylococci.

PubMed

Flanagan, Keith; Cockell, Simon; Harwood, Colin; Hallinan, Jennifer; Nakjang, Sirintra; Lawry, Beth; Wipat, Anil

2014-06-30

The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

A distributed computational search strategy for the identification of diagnostics targets: Application to finding aptamer targets for methicillin-resistant staphylococci.

PubMed

Flanagan, Keith; Cockell, Simon; Harwood, Colin; Hallinan, Jennifer; Nakjang, Sirintra; Lawry, Beth; Wipat, Anil

2014-06-01

The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization-time of flight mass spectrometry and cydB real-time quantitative PCR.

PubMed

Pizauro, Lucas J L; de Almeida, Camila C; Soltes, Glenn A; Slavic, Durda; Rossi-Junior, Oswaldo D; de Ávila, Fernando A; Zafalon, Luiz F; MacInnes, Janet I

2017-05-01

Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning and sequencing of the histidine decarboxylase genes of gram-negative, histamine-producing bacteria and their application in detection and identification of these organisms in fish.

PubMed

Takahashi, Hajime; Kimura, Bon; Yoshikawa, Miwako; Fujii, Tateo

2003-05-01

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

Rapid real-time diagnostic PCR for Trichophyton rubrum and Trichophyton mentagrophytes in patients with tinea unguium and tinea pedis using specific fluorescent probes.

PubMed

Miyajima, Yoshiharu; Satoh, Kazuo; Uchida, Takao; Yamada, Tsuyoshi; Abe, Michiko; Watanabe, Shin-ichi; Makimura, Miho; Makimura, Koichi

2013-03-01

Trichophyton rubrum and Trichophyton mentagrophytes human-type (synonym, Trichophyton interdigitale (anthropophilic)) are major causative pathogens of tinea unguium. For suitable diagnosis and treatment, rapid and accurate identification of etiologic agents in clinical samples using reliable molecular based method is required. For identification of organisms causing tinea unguium, we developed a new real-time polymerase chain reaction (PCR) with a pan-fungal primer set and probe, as well as specific primer sets and probes for T. rubrum and T. mentagrophytes human-type. We designed two sets of primers from the internal transcribed spacer 1 (ITS1) region of fungal ribosomal DNA (rDNA) and three quadruple fluorescent probes, one for detection wide range pathogenic fungi and two for classification of T. rubrum and T. mentagrophytes by specific binding to different sites in the ITS1 region. We investigated the specificity of these primer sets and probes using fungal genomic DNA, and also examined 42 clinical specimens with our real-time PCR. The primers and probes specifically detected T. rubrum, T. mentagrophytes, and a wide range of pathogenic fungi. The causative pathogens were identified in 42 nail and skin samples from 32 patients. The total time required for identification of fungal species in each clinical specimen was about 3h. The copy number of each fungal DNA in the clinical specimens was estimated from the intensity of fluorescence simultaneously. This PCR system is one of the most rapid and sensitive methods available for diagnosing dermatophytosis, including tinea unguium and tinea pedis. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Direct Analysis in Real Time-Mass Spectrometry & Kohonen Artificial Neural Networks for the Rapid Species Identification of Larvae, Pupae and Adult Life Stages of Carrion Insects.

PubMed

Beyramysoltan, Samira; Giffen, Justine E; Rosati, Jennifer Y; Musah, Rabi Ann

2018-06-20

Species determination of the various life stages of flies (order: Diptera) is challenging, particularly for the immature forms, because analogous life stages of different species are difficult to differentiate morphologically. It is demonstrated here that DART high-resolution mass spectrometry (DART-HRMS) combined with supervised Kohonen Self-Organizing Maps (SOM) enables accomplishment of species-level identification of larvae, pupae and adult life stages of carrion flies. DART-HRMS data for each life stage were acquired from analysis of ethanol suspensions representing Calliphoridae, Phoridae and Sarcophagidae families, without additional sample preparation. After preprocessing, the data were subjected to a combination of minimum Redundancy Maximal Relevance (mRMR) and Sparse Discriminant Analysis (SDA) methods to select the most significant variables for creating accurate SOM models. The resulting data were divided into training and validation sets, and then analyzed by the SOM method to define the proper discrimination models. The 5-fold venetian blind cross-validation misclassification error was below 7% for all life stages, and the validation samples were correctly identified in all cases. The multiclass SOM model also revealed which chemical components were the most significant markers for each species, with several of these being amino acids. The results show that processing of DART-HRMS data using artificial neural networks (ANNs) based on the Kohonen SOM approach enables rapid discrimination and identification of fly species even for the immature life stages. The ANNs can be continuously expanded to include a larger number of species, and can be used to screen DART-HRMS data from unknowns to rapidly determine species identity.

Proteome-based bacterial identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS): A revolutionary shift in clinical diagnostic microbiology.

PubMed

Nomura, Fumio

2015-06-01

Rapid and accurate identification of microorganisms, a prerequisite for appropriate patient care and infection control, is a critical function of any clinical microbiology laboratory. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a quick and reliable method for identification of microorganisms, including bacteria, yeast, molds, and mycobacteria. Indeed, there has been a revolutionary shift in clinical diagnostic microbiology. In the present review, the state of the art and advantages of MALDI-TOF MS-based bacterial identification are described. The potential of this innovative technology for use in strain typing and detection of antibiotic resistance is also discussed. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

[Rapid Identification of Epicarpium Citri Grandis via Infrared Spectroscopy and Fluorescence Spectrum Imaging Technology Combined with Neural Network].

PubMed

Pan, Sha-sha; Huang, Fu-rong; Xiao, Chi; Xian, Rui-yi; Ma, Zhi-guo

2015-10-01

To explore rapid reliable methods for detection of Epicarpium citri grandis (ECG), the experiment using Fourier Transform Attenuated Total Reflection Infrared Spectroscopy (FTIR/ATR) and Fluorescence Spectrum Imaging Technology combined with Multilayer Perceptron (MLP) Neural Network pattern recognition, for the identification of ECG, and the two methods are compared. Infrared spectra and fluorescence spectral images of 118 samples, 81 ECG and 37 other kinds of ECG, are collected. According to the differences in tspectrum, the spectra data in the 550-1 800 cm(-1) wavenumber range and 400-720 nm wavelength are regarded as the study objects of discriminant analysis. Then principal component analysis (PCA) is applied to reduce the dimension of spectroscopic data of ECG and MLP Neural Network is used in combination to classify them. During the experiment were compared the effects of different methods of data preprocessing on the model: multiplicative scatter correction (MSC), standard normal variable correction (SNV), first-order derivative(FD), second-order derivative(SD) and Savitzky-Golay (SG). The results showed that: after the infrared spectra data via the Savitzky-Golay (SG) pretreatment through the MLP Neural Network with the hidden layer function as sigmoid, we can get the best discrimination of ECG, the correct percent of training set and testing set are both 100%. Using fluorescence spectral imaging technology, corrected by the multiple scattering (MSC) results in the pretreatment is the most ideal. After data preprocessing, the three layers of the MLP Neural Network of the hidden layer function as sigmoid function can get 100% correct percent of training set and 96.7% correct percent of testing set. It was shown that the FTIR/ATR and fluorescent spectral imaging technology combined with MLP Neural Network can be used for the identification study of ECG and has the advantages of rapid, reliable effect.

MALDI-TOF mass spectrometry following short incubation on a solid medium is a valuable tool for rapid pathogen identification from positive blood cultures.

PubMed

Kohlmann, Rebekka; Hoffmann, Alexander; Geis, Gabriele; Gatermann, Sören

2015-01-01

Rapid identification of the causative microorganism is a key element in appropriate antimicrobial therapy of bloodstream infections. Whereas traditional analysis of positive blood cultures requires subculture over at least 16-24h prior to pathogen identification by, e.g. matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), sample preparation procedures enabling direct MALDI-TOF MS, i.e. without preceding subculture, are associated with additional effort and costs. Hence, we integrated an alternative MALDI-TOF MS approach in diagnostic routine using a short incubation on a solid medium. Positive blood cultures were routinely plated on chocolate agar plates and incubated for 4h (37 °C, 5% CO2). Subsequently, MALDI-TOF MS using a Microflex LT instrument (Bruker Daltonics) and direct smear method was performed once per sample. For successful identification of bacteria at species level, score cut-off values were used as proposed by the manufacturer (≥ 2.0) and in a modified form (≥ 1.5 for MALDI-TOF MS results referring to Gram-positive cocci and ≥ 1.7 for MALDI-TOF MS results referring to bacteria other than Gram-positive cocci). Further data analysis also included an assessment of the clinical impact of the MALDI-TOF MS result. Applying the modified score cut-off values, our approach led to an overall correct species identification in 69.5% with misidentification in 3.4% (original cut-offs: 49.2% and 1.8%, respectively); for Gram-positive cocci, correct identification in 68.4% (100% for Staphylococcus aureus and enterococci, 80% for beta-hemolytic streptococci), for Gram-negative bacteria, correct identification in 97.6%. In polymicrobial blood cultures, in 72.7% one of the pathogens was correctly identified. Results were not reliable for Gram-positive rods and yeasts. The approach was easy to implement in diagnostic routine. In cases with available clinical data and successful pathogen identification, in 51.1% our approach allowed an optimized treatment recommendation. MALDI-TOF MS following 4h pre-culture is a valuable tool for rapid pathogen identification from positive blood cultures, allowing easy integration in diagnostic routine and the opportunity of considerably earlier treatment adaptation. Copyright © 2015 Elsevier GmbH. All rights reserved.

A Rapid Diagnostic Technique to Discriminate between Two Pests of Palms, Brontispa longissima and Octodonta nipae (Coleoptera: Chrysomelidae), for Quarantine Applications.

PubMed

Zhang, X; Tang, B; Hou, Y

2015-02-01

Octodonta nipae (Maulik) is morphologically and biologically similar to Brontispa longissima (Gestro), one of the most damaging pests of coconut. The two species share several palm hosts and produce similar symptoms. They are easily confused when they invade a new area, and without professional taxonomic expertise accurate identification is arduous and time-consuming. Thus, a method of rapidly distinguishing these two invasive insects rapidly is critical for quarantine. Based on the first internal transcribed spacer and the mitochondrial cytochrome oxidase I gene, two pairs of special primers and corresponding polymerase chain reaction processes have been developed to enhance a single objective band only from the O. nipae DNA template. This will quickly discriminate between these two species. The present results provide a rapid method of distinguishing O. nipae from B. longissima to help avoid misidentification, and furthermore, to facilitate rapid and appropriate quarantine decisions and effective treatments to the pest. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Rapid identification of triterpenoid sulfates and hydroxy fatty acids including two new constituents from Tydemania expeditionis by liquid chromatography-mass spectrometry.

PubMed

Zhang, Jian-Long; Kubanek, Julia; Hay, Mark E; Aalbersberg, William; Ye, Wen-Cai; Jiang, Ren-Wang

2011-09-01

Tydemania expeditionis Weber-van Bosse (Udoteaceae) is a weakly calcified green alga. In the present paper, liquid chromatography coupled with photodiode array detection and electrospray mass spectrometry was developed to identify the fingerprint components. A total of four triterpenoid sulfates and three hydroxy fatty acids in the ethyl acetate fraction of the crude extract were structurally characterized on the basis of retention time, online UV spectrum, and mass fragmentation pattern. Furthermore, a detailed liquid chromatography-mass spectrometry analysis revealed two new hydroxy fatty acids, which were then prepared and characterized by extensive nuclear magnetic resonance (NMR) analyses. The proposed method provides a scientific and technical platform for the rapid identification of triterpenoid sulfates and hydroxy fatty acids in similar marine algae and terrestrial plants. Copyright © 2011 John Wiley & Sons, Ltd.

Identification of the five human Plasmodium species including P. knowlesi by real-time polymerase chain reaction.

PubMed

Oddoux, O; Debourgogne, A; Kantele, A; Kocken, C H; Jokiranta, T S; Vedy, S; Puyhardy, J M; Machouart, M

2011-04-01

Recently, Plasmodium knowlesi has been recognised as the fifth Plasmodium species causing malaria in humans. Hundreds of human cases infected with this originally simian Plasmodium species have been described in Asian countries and increasing numbers are reported in Europe from travellers. The growing impact of tourism and economic development in South and Southeast Asia are expected to subsequently lead to a further increase in cases both among locals and among travellers. P. knowlesi is easily misidentified in microscopy as P. malariae or P. falciparum. We developed new primers for the rapid and specific detection of this species by low-cost real-time polymerase chain reaction (PCR) and added this method to an already existing panel of primers used for the molecular identification of the other four species in one reaction. Reference laboratories should now be able to identify undisputably and rapidly P. knowlesi, as it is a potentially fatal pathogen.

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

PubMed Central

Bacconi, Andrea; Richmond, Gregory S.; Baroldi, Michelle A.; Laffler, Thomas G.; Blyn, Lawrence B.; Carolan, Heather E.; Frinder, Mark R.; Toleno, Donna M.; Metzgar, David; Gutierrez, Jose R.; Massire, Christian; Rounds, Megan; Kennel, Natalie J.; Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Wakefield, Teresa; Ecker, David J.

2014-01-01

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections. PMID:24951806

Evaluation of the Andromas Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Aerobically Growing Gram-Positive Bacilli

PubMed Central

Farfour, E.; Leto, J.; Barritault, M.; Barberis, C.; Meyer, J.; Dauphin, B.; Le Guern, A.-S.; Leflèche, A.; Badell, E.; Guiso, N.; Leclercq, A.; Le Monnier, A.; Lecuit, M.; Rodriguez-Nava, V.; Bergeron, E.; Raymond, J.; Vimont, S.; Bille, E.; Carbonnelle, E.; Guet-Revillet, H.; Lécuyer, H.; Beretti, J.-L.; Vay, C.; Berche, P.; Ferroni, A.; Nassif, X.

2012-01-01

Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry. PMID:22692743

Rapid Identification and Differentiation of Trichophyton Species, Based on Sequence Polymorphisms of the Ribosomal Internal Transcribed Spacer Regions, by Rolling-Circle Amplification▿

PubMed Central

Kong, Fanrong; Tong, Zhongsheng; Chen, Xiaoyou; Sorrell, Tania; Wang, Bin; Wu, Qixuan; Ellis, David; Chen, Sharon

2008-01-01

DNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense, T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophyton spp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanense but not T. rubrum. Signature sets of SNPs between T. rubrum and T. soudanense (4-bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified. The RCA assay correctly identified five Trichophyton species. Although the use of two “group-specific” probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense, the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophyton spp. PMID:18234865

Approach to identifying pollutant source and matching flow field

NASA Astrophysics Data System (ADS)

Liping, Pang; Yu, Zhang; Hongquan, Qu; Tao, Hu; Wei, Wang

2013-07-01

Accidental pollution events often threaten people's health and lives, and it is necessary to identify a pollutant source rapidly so that prompt actions can be taken to prevent the spread of pollution. But this identification process is one of the difficulties in the inverse problem areas. This paper carries out some studies on this issue. An approach using single sensor information with noise was developed to identify a sudden continuous emission trace pollutant source in a steady velocity field. This approach first compares the characteristic distance of the measured concentration sequence to the multiple hypothetical measured concentration sequences at the sensor position, which are obtained based on a source-three-parameter multiple hypotheses. Then we realize the source identification by globally searching the optimal values with the objective function of the maximum location probability. Considering the large amount of computation load resulting from this global searching, a local fine-mesh source search method based on priori coarse-mesh location probabilities is further used to improve the efficiency of identification. Studies have shown that the flow field has a very important influence on the source identification. Therefore, we also discuss the impact of non-matching flow fields with estimation deviation on identification. Based on this analysis, a method for matching accurate flow field is presented to improve the accuracy of identification. In order to verify the practical application of the above method, an experimental system simulating a sudden pollution process in a steady flow field was set up and some experiments were conducted when the diffusion coefficient was known. The studies showed that the three parameters (position, emission strength and initial emission time) of the pollutant source in the experiment can be estimated by using the method for matching flow field and source identification.

Sample preparation method influences direct identification of anaerobic bacteria from positive blood culture bottles using MALDI-TOF MS.

PubMed

Jeverica, Samo; Nagy, Elisabeth; Mueller-Premru, Manica; Papst, Lea

2018-05-15

Rapid detection and identification of anaerobic bacteria from blood is important to adjust antimicrobial therapy by including antibiotics with activity against anaerobic bacteria. Limited data is available about direct identification of anaerobes from positive blood culture bottles using MALDI-TOF mass spectrometry (MS). In this study, we evaluated the performance of two sample preparation protocols for direct identification of anaerobes from positive blood culture bottles, the MALDI Sepsityper kit (Sepsityper) and the in-house saponin (saponin) method. Additionally, we compared two blood culture bottle types designed to support the growth of anaerobic bacteria, the BacT/ALERT-FN Plus (FN Plus) and the BACTEC-Lytic (Lytic), and their influence on direct identification. A selection of 30 anaerobe strains belonging to 22 different anaerobic species (11 reference strains and 19 clinical isolates) were inoculated to 2 blood culture bottle types in duplicate. In total, 120 bottles were inoculated and 99.2% (n = 119) signalled growth within 5 days of incubation. The Sepsityper method correctly identified 56.3% (n = 67) of anaerobes, while the saponin method correctly identified 84.9% (n = 101) of anaerobes with at least log(score) ≥1.6 (low confidence correct identification), (p < 0.001). Gram negative anaerobes were better identified with the saponin method (100% vs. 46.5%; p < 0.001), while Gram positive anaerobes were better identified with the Sepsityper method (70.8% vs. 62.5%; p = 0.454). Average log(score) values among only those isolates that were correctly identified simultaneously by both sample preparation methods were 2.119 and 2.029 in favour of the Sepsityper method, (p = 0.019). The inoculated bottle type didn't influence the performance of the two sample preparation methods. We confirmed that direct identification from positive blood culture bottles with MALDI-TOF MS is reliable for anaerobic bacteria. However, the results are influenced by the sample preparation method used. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparison of CHROMagar, polymerase chain reaction-restriction fragment length polymorphism, and polymerase chain reaction-fragment size for the identification of Candida species.

PubMed

Jafari, Zahra; Motamedi, Marjan; Jalalizand, Nilufar; Shokoohi, Gholam R; Charsizadeh, Arezu; Mirhendi, Hossein

2017-09-01

The epidemiological alteration in the distribution of Candida species, as well as the significantly increasing trend of either intrinsic or acquired resistance of some of these fungi highlights the need for a reliable method for the identification of the species. Polymerase chain reaction (PCR) is one of the methods facilitating the quick and precise identification of Candida species. The aim of this study was to compare the efficiency of CHROMagar, PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-fragment size polymorphism (PCR-FSP) assays in the identification of Candida species to determine the benefits and limitations of these methods. This study was conducted on 107 Candida strains, including 20 standard strains and 87 clinical isolates. The identification of the isolates was accomplished by using CHROMagar as a conventional method. The PCR-RFLP assay was performed on the entire internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), and the consequent enzymatic digestion was compared with PCR-FSP results in which ITS1 and ITS2 regions were separately PCR amplified. In both molecular assays, yeast identification was carried out through the specific electrophoretic profiles of the PCR products. According to the results, the utilization of CHROMagar resulted in the identification of 29 (33.3%) Candida isolates, while the PCR-RFLP and PCR-FSP facilitated the identification of 83 (95.4%) and 80 (91.9%) clinical isolates, respectively. The obtained concordances between CHROMagar and PCR-RFLP, between CHROMagar and PCR-FSP, as well as between PCR-RFLP and PCR-FSP were 0.23, 0.20, and 0.77, respectively. The recognition of the benefits and limitations of PCR methods allows for the selection of the most efficient technique for a fast and correct differentiation. The PCR-RFLP and PCR-FSP assays had satisfactory concordance. The PCR-FSP provides a rapid, technically simple, and cost-effective method for the identification of Candida species. Nevertheless, to accurately differentiate among the taxonomically related species, PCR-RFLP should be implemented.

Performance of Kiestra Total Laboratory Automation Combined with MS in Clinical Microbiology Practice

PubMed Central

Hodiamont, Caspar J.; de Jong, Menno D.; Overmeijer, Hendri P. J.; van den Boogaard, Mandy; Visser, Caroline E.

2014-01-01

Background Microbiological laboratories seek technologically innovative solutions to cope with large numbers of samples and limited personnel and financial resources. One platform that has recently become available is the Kiestra Total Laboratory Automation (TLA) system (BD Kiestra B.V., the Netherlands). This fully automated sample processing system, equipped with digital imaging technology, allows superior detection of microbial growth. Combining this approach with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) (Bruker Daltonik, Germany) is expected to enable more rapid identification of pathogens. Methods Early growth detection by digital imaging using Kiestra TLA combined with MS was compared to conventional methods (CM) of detection. Accuracy and time taken for microbial identification were evaluated for the two methods in 219 clinical blood culture isolates. The possible clinical impact of earlier microbial identification was assessed according to antibiotic treatment prescription. Results Pathogen identification using Kiestra TLA combined with MS resulted in a 30.6 hr time gain per isolate compared to CM. Pathogens were successfully identified in 98.4% (249/253) of all tested isolates. Early microbial identification without susceptibility testing led to an adjustment of antibiotic regimen in 12% (24/200) of patients. Conclusions The requisite 24 hr incubation time for microbial pathogens to reach sufficient growth for susceptibility testing and identification would be shortened by the implementation of Kiestra TLA in combination with MS, compared to the use of CM. Not only can this method optimize workflow and reduce costs, but it can allow potentially life-saving switches in antibiotic regimen to be initiated sooner. PMID:24624346

Evaluation of methods for rapid determination of freezing point of aviation fuels

NASA Technical Reports Server (NTRS)

Mathiprakasam, B.

1982-01-01

Methods for identification of the more promising concepts for the development of a portable instrument to rapidly determine the freezing point of aviation fuels are described. The evaluation process consisted of: (1) collection of information on techniques previously used for the determination of the freezing point, (2) screening and selection of these techniques for further evaluation of their suitability in a portable unit for rapid measurement, and (3) an extensive experimental evaluation of the selected techniques and a final selection of the most promising technique. Test apparatuses employing differential thermal analysis and the change in optical transparency during phase change were evaluated and tested. A technique similar to differential thermal analysis using no reference fuel was investigated. In this method, the freezing point was obtained by digitizing the data and locating the point of inflection. Results obtained using this technique compare well with those obtained elsewhere using different techniques. A conceptual design of a portable instrument incorporating this technique is presented.

Interdigitated microelectrode based impedance biosensor for detection of salmonella enteritidis in food samples

NASA Astrophysics Data System (ADS)

Kim, G.; Morgan, M.; Hahm, B. K.; Bhunia, A.; Mun, J. H.; Om, A. S.

2008-03-01

Salmonella enteritidis outbreaks continue to occur, and S. enteritidis-related outbreaks from various food sources have increased public awareness of this pathogen. Conventional methods for pathogens detection and identification are labor-intensive and take days to complete. Some immunological rapid assays are developed, but these assays still require prolonged enrichment steps. Recently developed biosensors have shown great potential for the rapid detection of foodborne pathogens. To develop the biosensor, an interdigitated microelectrode (IME) was fabricated by using semiconductor fabrication process. Anti-Salmonella antibodies were immobilized based on avidin-biotin binding on the surface of the IME to form an active sensing layer. To increase the sensitivity of the sensor, three types of sensors that have different electrode gap sizes (2 μm, 5 μm, 10 μm) were fabricated and tested. The impedimetric biosensor could detect 103 CFU/mL of Salmonella in pork meat extract with an incubation time of 5 minutes. This method may provide a simple, rapid and sensitive method to detect foodborne pathogens.

Rapid method for identification and enumeration of oral Actinomyces.

PubMed Central

Marucha, P T; Keyes, P H; Wittenberger, C L; London, J

1978-01-01

Serotype-specific antisera prepared against whole cells of Actinomyces viscosus, A. naeslundii, and A. israeli were labeled with fluorescein dye and used to detect and quantitate antigenically related microorganisms in human dental plaque. By relating the DNA content of the dental plaque microflora to the number of Actinomyces present in the plaque samples, a reproducible method was developed for specifically enumerating five serotypic representatives of this genus found in human plaque. PMID:711333

Utility of a stressed-SNP real-time PCR assay for the rapid identification of measles vaccine strain in patient samples.

PubMed

Tran, Thomas; Kostecki, Renata; Catton, Michael; Druce, Julian

2018-05-09

Rapid differentiation of wild-type measles virus from measles vaccine strains is crucial during a measles outbreak and in a measles elimination setting. A real-time RT-PCR for the rapid detection of measles vaccine strains was developed with high specificity and greater sensitivity than when compared to traditional measles genotyping methods. The "stressed" minor grove binder TaqMan probe design approach achieves specificity to vaccine strains only, without compromising sensitivity. This assay has proven to be extremely useful in outbreak settings, without requiring sequence genotyping, for over 4 years at the Regional Measles Reference Laboratory for the Western Pacific Region. Copyright © 2018 Tran et al.

Pitfalls of Establishing DNA Barcoding Systems in Protists: The Cryptophyceae as a Test Case

PubMed Central

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed. PMID:22970104

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

PubMed

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections.

PubMed

Rudkjøbing, Vibeke Børsholt; Thomsen, Trine Rolighed; Xu, Yijuan; Melton-Kreft, Rachael; Ahmed, Azad; Eickhardt, Steffen; Bjarnsholt, Thomas; Poulsen, Steen Seier; Nielsen, Per Halkjær; Earl, Joshua P; Ehrlich, Garth D; Moser, Claus

2016-11-08

Necrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold standard for identification of pathogens is culture; however molecular methods for identification of microorganisms may provide a more rapid result and may be able to identify additional microorganisms that are not detected by culture. In this study, tissue samples (n = 20) obtained after debridement of 10 patients with NSTI were analyzed by standard culture, fluorescence in situ hybridization (FISH) and multiple molecular methods. The molecular methods included analysis of microbial diversity by 1) direct 16S and D2LSU rRNA gene Microseq 2) construction of near full-length 16S rRNA gene clone libraries with subsequent Sanger sequencing for most samples, 3) the Ibis T5000 biosensor and 4) 454-based pyrosequencing. Furthermore, quantitative PCR (qPCR) was used to verify and determine the relative abundance of Streptococcus pyogenes in samples. For 70 % of the surgical samples it was possible to identify microorganisms by culture. Some samples did not result in growth (presumably due to administration of antimicrobial therapy prior to sampling). The molecular methods identified microorganisms in 90 % of the samples, and frequently detected additional microorganisms when compared to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods. Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also made including infection by a) Acinetobacter baumannii, b) Streptococcus pneumoniae, and c) fungi, mycoplasma and Fusobacterium necrophorum. The study emphasizes that many pathogens can be involved in NSTIs, and that no specific "NSTI causing" combination of species exists. This means that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially for fulminant life-threatening infections such as NSTI.

Rapid screening for human-pathogenic Mucorales using rolling circle amplification.

PubMed

Dolatabadi, S; Najafzadeh, M J; de Hoog, G S

2014-12-01

Mucormycosis has emerged as a relatively common severe mycosis in patients with haematological and allogeneic stem cell transplantation. Source of transmission is from unidentified sources in the environment. Early diagnosis of infection and its source of contamination are paramount for rapid and appropriate therapy. In this study, rolling circle amplification (RCA) is introduced as a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of six of the most virulent species (Rhizopus microsporus, R. arrhizus var. arrhizus, R. arrhizus var. delemar, Mucor irregularis, Mucor circinelloides, Lichtheimia ramosa, Lichtheimia corymbifera). DNAs of target species were successfully amplified, with no cross reactivity between species. RCA can be considered as a rapid detection method with high specificity and sensitivity, suitable for large screening. © 2014 Blackwell Verlag GmbH.

Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization Allows for Enrichment-Independent Detection of Microcolony-Forming Soil Bacteria

PubMed Central

Ferrari, Belinda C.; Tujula, Niina; Stoner, Kate; Kjelleberg, Staffan

2006-01-01

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition. PMID:16391135

New technologies in predicting, preventing and controlling emerging infectious diseases.

PubMed

Christaki, Eirini

2015-01-01

Surveillance of emerging infectious diseases is vital for the early identification of public health threats. Emergence of novel infections is linked to human factors such as population density, travel and trade and ecological factors like climate change and agricultural practices. A wealth of new technologies is becoming increasingly available for the rapid molecular identification of pathogens but also for the more accurate monitoring of infectious disease activity. Web-based surveillance tools and epidemic intelligence methods, used by all major public health institutions, are intended to facilitate risk assessment and timely outbreak detection. In this review, we present new methods for regional and global infectious disease surveillance and advances in epidemic modeling aimed to predict and prevent future infectious diseases threats.

New technologies in predicting, preventing and controlling emerging infectious diseases

PubMed Central

Christaki, Eirini

2015-01-01

Surveillance of emerging infectious diseases is vital for the early identification of public health threats. Emergence of novel infections is linked to human factors such as population density, travel and trade and ecological factors like climate change and agricultural practices. A wealth of new technologies is becoming increasingly available for the rapid molecular identification of pathogens but also for the more accurate monitoring of infectious disease activity. Web-based surveillance tools and epidemic intelligence methods, used by all major public health institutions, are intended to facilitate risk assessment and timely outbreak detection. In this review, we present new methods for regional and global infectious disease surveillance and advances in epidemic modeling aimed to predict and prevent future infectious diseases threats. PMID:26068569

Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods.

PubMed

Kulkarni, Sughosh S; Madalgi, Radhika; Ajantha, Ganavalli S; Kulkarni, Raghavendra D

2017-01-01

Acinetobacter is grouped under nonfermenting Gram-negative bacilli. It is increasingly isolated from pathological samples. The ability of this genus to acquire drug resistance and spread in the hospital settings is posing a grave problem in healthcare. Specific treatment protocols are advocated for Acinetobacter infections. Hence, rapid identification and drug susceptibility profiling are critical in the management of these infections. To standardize an in-house polymerase chain reaction (PCR) for identification of genus Acinetobacter and to compare PCR with two protocols for its phenotypic identification. A total of 96 clinical isolates of Acinetobacter were included in the study. An in-house PCR for genus level identification of Acinetobacter was standardized. All the isolates were phenotypically identified by two protocols. The results of PCR and phenotypic identification protocols were compared. The in-house PCR standardized was highly sensitive and specific for the genus Acinetobacter . There was 100% agreement between the phenotypic and molecular identification of the genus. The preliminary identification tests routinely used in clinical laboratories were also in complete agreement with phenotypic and molecular identification. The in-house PCR for genus level identification is specific and sensitive. However, it may not be essential for routine identification as the preliminary phenotypic identification tests used in the clinical laboratory reliably identify the genus Acinetobacter .

The RAPID method for blood processing yields new insight in plasma concentrations and molecular forms of circulating gut peptides.

PubMed

Stengel, Andreas; Keire, David; Goebel, Miriam; Evilevitch, Lena; Wiggins, Brian; Taché, Yvette; Reeve, Joseph R

2009-11-01

The correct identification of circulating molecular forms and measurement of peptide levels in blood entails that the endocrine peptide being studied is stable and recovered in good yields during blood processing. However, it is not clear whether this is achieved in studies using standard blood processing. Therefore, we compared peptide concentration and form of 12 (125)I-labeled peptides using the standard procedure (EDTA-blood on ice) and a new method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls, and Dilution (RAPID). During standard processing there was at least 80% loss for calcitonin-gene-related peptide and cholecystokinin-58 (CCK-58) and more than 35% loss for amylin, insulin, peptide YY forms (PYY((1-36)) and PYY((3-36))), and somatostatin-28. In contrast, the RAPID method significantly improved the recovery for 11 of 12 peptides (P < 0.05) and eliminated the breakdown of endocrine peptides occurring after standard processing as reflected in radically changed molecular forms for CCK-58, gastrin-releasing peptide, somatostatin-28, and ghrelin. For endogenous ghrelin, this led to an acyl/total ghrelin ratio of 1:5 instead of 1:19 by the standard method. These results show that the RAPID method enables accurate assessment of circulating gut peptide concentrations and forms such as CCK-58, acylated ghrelin, and somatostatin-28. Therefore, the RAPID method represents an efficacious means to detect circulating variations in peptide concentrations and form relevant to the understanding of physiological function of endocrine peptides.

Evaluation of the MIT RMID 1000 system for the identification of Listeria species.

PubMed

Ricardi, John; Haavig, David; Cruz, Lasaunta; Paoli, George; Gehring, Andrew

2010-01-01

The Micro Imaging Technology (MIT) 1000 Rapid Microbial Identification (RMID) System is a device that uses the principles of light scattering coupled with proprietary algorithms to identify bacteria after being cultured and placed in a vial of filtered water. This specific method is for pure culture identification of Listeria spp. A total of 81 microorganisms (55 isolates) were tested by the MIT 1000 System, of which 25 were Listeria spp. and 30 a variety of other bacterial species. In addition, a total of 406 tests over seven different ruggedness parameters were tested by the MIT 1000 System to determine its flexibility to the specifications stated in the MIT 1000 System User Guide in areas where they might be deviated by a user to shorten the test cycle. Overall, MIT concluded that the MIT 1000 System had an accuracy performance that should certify this Performance Test Method for the identification of Listeria spp. This report discusses the tests performed, results achieved, and conclusions, along with several reference documents to enable a higher understanding of the technology used by the MIT 1000 System.

A mass graph-based approach for the identification of modified proteoforms using top-down tandem mass spectra

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

Kou, Qiang; Wu, Si; Tolić, Nikola

Motivation: Although proteomics has rapidly developed in the past decade, researchers are still in the early stage of exploring the world of complex proteoforms, which are protein products with various primary structure alterations resulting from gene mutations, alternative splicing, post-translational modifications, and other biological processes. Proteoform identification is essential to mapping proteoforms to their biological functions as well as discovering novel proteoforms and new protein functions. Top-down mass spectrometry is the method of choice for identifying complex proteoforms because it provides a “bird’s eye view” of intact proteoforms. The combinatorial explosion of various alterations on a protein may result inmore » billions of possible proteoforms, making proteoform identification a challenging computational problem. Results: We propose a new data structure, called the mass graph, for efficient representation of proteoforms and design mass graph alignment algorithms. We developed TopMG, a mass graph-based software tool for proteoform identification by top-down mass spectrometry. Experiments on top-down mass spectrometry data sets showed that TopMG outperformed existing methods in identifying complex proteoforms.« less

Rapid Identification of Mycobacterial Whole Cells in Solid and Liquid Culture Media by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry ▿

PubMed Central

Lotz, Aurélie; Ferroni, Agnès; Beretti, Jean-Luc; Dauphin, Brunhilde; Carbonnelle, Etienne; Guet-Revillet, Hélène; Veziris, Nicolas; Heym, Béate; Jarlier, Vincent; Gaillard, Jean-Louis; Pierre-Audigier, Catherine; Frapy, Eric; Berche, Patrick; Nassif, Xavier; Bille, Emmanuelle

2010-01-01

Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species. PMID:20943874

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens.

PubMed

Ruppitsch, W; Stöger, A; Indra, A; Grif, K; Schabereiter-Gurtner, C; Hirschl, A; Allerberger, F

2007-03-01

In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.

Rapid identification and quantification of tumor cells using an electrocatalytic method based on gold nanoparticles.

PubMed

de la Escosura-Muñiz, Alfredo; Sánchez-Espinel, Christian; Díaz-Freitas, Belén; González-Fernández, Africa; Maltez-da Costa, Marisa; Merkoçi, Arben

2009-12-15

There is a high demand for simple, rapid, efficient, and user-friendly alternative methods for the detection of cells in general and, in particular, for the detection of cancer cells. A biosensor able to detect cells would be an all-in-one dream device for such applications. The successful integration of nanoparticles into cell detection assays could allow for the development of this novel class of cell sensors. Indeed, their application could well have a great future in diagnostics, as well as other fields. As an example of a novel biosensor, we report here an electrocatalytic device for the specific identification of tumor cells that quantifies gold nanoparticles (AuNPs) coupled with an electrotransducing platform/sensor. Proliferation and adherence of tumor cells are achieved on the electrotransducer/detector, which consists of a mass-produced screen-printed carbon electrode (SPCE). In situ identification/quantification of tumor cells is achieved with a detection limit of 4000 cells per 700 microL of suspension. This novel and selective cell-sensing device is based on the reaction of cell surface proteins with specific antibodies conjugated with AuNPs. Final detection requires only a couple of minutes, taking advantage of the catalytic properties of AuNPs on hydrogen evolution. The proposed detection method does not require the chemical agents used in most existing assays for the detection of AuNPs. It allows for the miniaturization of the system and is much cheaper than other expensive and sophisticated methods used for tumor cell detection. We envisage that this device could operate in a simple way as an immunosensor or DNA sensor. Moreover, it could be used, even by inexperienced staff, for the detection of protein molecules or DNA strands.

Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF.

PubMed

Tanner, Hannah; Evans, Jason T; Gossain, Savita; Hussain, Abid

2017-01-18

Patient mortality is significantly reduced by rapid identification of bacteria from sterile sites. MALDI-TOF can identify bacteria directly from positive blood cultures and multiple sample preparation methods are available. We evaluated three sample preparation methods and two MALDI-TOF score cut-off values. Positive blood culture bottles with organisms present in Gram stains were prospectively analysed by MALDI-TOF. Three lysis reagents (Saponin, SDS, and SepsiTyper lysis bufer) were applied to each positive culture followed by centrifugation, washing and protein extraction steps. Methods were compared using the McNemar test and 16S rDNA sequencing was used to assess discordant results. In 144 monomicrobial cultures, using ≥2.000 as the cut-off value, species level identifications were obtained from 69/144 (48%) samples using Saponin, 86/144 (60%) using SDS, and 91/144 (63%) using SepsiTyper. The difference between SDS and SepsiTyper was not statistically significant (P = 0.228). Differences between Saponin and the other two reagents were significant (P < 0.01). Using ≥1.700 plus top three results matching as the cut-off value, species level identifications were obtained from 100/144 (69%) samples using Saponin, 103/144 (72%) using SDS, and 106/144 (74%) using SepsiTyper and there was no statistical difference between the methods. No true discordances between culture and direct MALDI-TOF identification were observed in monomicrobial cultures. In 32 polymicrobial cultures, MALDI-TOF identified one organism in 34-75% of samples depending on the method. This study demonstrates two inexpensive in-house detergent lysis methods are non-inferior to a commercial kit for analysis of positive blood cultures by direct MALDI-TOF in a clinical diagnostic microbiology laboratory.

Plant seed species identification from chemical fingerprints: a high-throughput application of direct analysis in real time mass spectrometry.

PubMed

Lesiak, Ashton D; Cody, Robert B; Dane, A John; Musah, Rabi A

2015-09-01

Plant species identification based on the morphological features of plant parts is a well-established science in botany. However, species identification from seeds has largely been unexplored, despite the fact that the seeds contain all of the genetic information that distinguishes one plant from another. Using seeds of genus Datura plants, we show here that the mass spectrum-derived chemical fingerprints for seeds of the same species are similar. On the other hand, seeds from different species within the same genus display distinct chemical signatures, even though they may contain similar characteristic biomarkers. The intraspecies chemical signature similarities on the one hand, and interspecies fingerprint differences on the other, can be processed by multivariate statistical analysis methods to enable rapid species-level identification and differentiation. The chemical fingerprints can be acquired rapidly and in a high-throughput manner by direct analysis in real time mass spectrometry (DART-MS) analysis of the seeds in their native form, without use of a solvent extract. Importantly, knowledge of the identity of the detected molecules is not required for species level identification. However, confirmation of the presence within the seeds of various characteristic tropane and other alkaloids, including atropine, scopolamine, scopoline, tropine, tropinone, and tyramine, was accomplished by comparison of the in-source collision-induced dissociation (CID) fragmentation patterns of authentic standards, to the fragmentation patterns observed in the seeds when analyzed under similar in-source CID conditions. The advantages, applications, and implications of the chemometric processing of DART-MS derived seed chemical signatures for species level identification and differentiation are discussed.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and database for identification of Legionella species.

PubMed

He, Ying; Chang, Tsung C; Li, Haijing; Shi, Gongyi; Tang, Yi-Wei

2011-07-01

More than 20 species of Legionella have been identified in relation to human infections. Rapid detection and identification of Legionella isolates is clinically useful to differentiate between infection and contamination and to determine treatment regimens. We explored the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonik GmbH, Bremen, Germany) for the identification of Legionella species. The MALDI MS spectra were generated and compared with the Biotyper database, which includes 25 Legionella strains covering 22 species and four Legionella pneumophila serogroups. A total of 83 blind-coded Legionella strains, consisting of 54 reference and 29 clinical strains, were analyzed in the study. Overall, the Biotyper system correctly identified 51 (61.4%) of all strains and isolates to the species level. For species included in the Biotyper database, the method identified 51 (86.4%) strains out of 59 Legionella strains to the correct species level, including 24 (100%) L. pneumophila and 27 (77.1%) non-L. pneumophila strains. The remaining 24 Legionella strains, belonging to species not covered by the Biotyper database, were either identified to the Legionella genus level or had no reliable identification. The Biotyper system produces constant and reproducible MALDI MS spectra for Legionella strains and can be used for rapid and accurate Legionella identification. More Legionella strains, especially the non-L. pneumophila strains, need to be included in the current Biotyper database to cover varieties of Legionella species and to increase identification accuracy.

Confirmation and Identification of Listeria monocytogenes, Listeria spp. and Other Gram-Positive Organisms by the Bruker MALDI Biotyper Method: Collaborative Study, First Action 2017.10.

PubMed

Bastin, Benjamin; Bird, Patrick; Crowley, Erin; Benzinger, M Joseph; Agin, James; Goins, David; Sohier, Daniele; Timke, Markus; Awad, Marian; Kostrzewa, Markus

2018-04-27

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate confirmation and identification of Gram-positive bacteria from select media types. This alternative method was evaluated using nonselective and selective agar plates to identify and confirm Listeria monocytogenes, Listeria species, and select Gram-positive bacteria. Results obtained by the Bruker MALDI Biotyper were compared with the traditional biochemical methods as prescribed in the appropriate reference method standards. Sixteen collaborators from 16 different laboratories located within the European Union participated in the collaborative study. A total of 36 blind-coded isolates were evaluated by each collaborator. In each set of 36 organisms, there were 16 L. monocytogenes strains, 12 non- monocytogenes Listeria species strains, and 8 additional Gram-positive exclusivity strains. After testing was completed, the total percentage of correct identifications (to both genus and species level) and confirmation from each agar type for each strain was determined at a percentage of 99.9% to the genus level and 98.8% to the species level. The results indicated that the alternative method produced equivalent results when compared with the confirmatory procedures specified by each reference method.

Toluidine blue: rapid and simple malaria parasite screening and species identification.

PubMed

Awale, Rupali; Maji, Ratnaprabha; Patil, Parag; Lingiah, Raghavendra; Mukhopadhyay, Ashok Kumar; Sharma, Subhadra

2017-01-01

Malaria, a febrile illness mostly confined to the tropical countries is transmitted by bite of infected female Anopheles mosquito. In 2015 alone, 88% of the malaria burden and 90% deaths due to malaria were confined to the African and Asian countries. Although number of tests are available for rapid diagnosis and screening for malaria, peripheral blood smear examination remains the gold standard. Leishman stain is recommended by WHO however herein we evaluate one of the alternative methods of staining which is simple and rapid. Fifty patients attending the various outpatient departments of the tertiary care hospital with fever and suspected to have malaria were selected. Two thin-air dried smears prepared from the peripheral venous blood from these subjects were stained by Leishman and Toluidine blue method. The findings of the slides by two independent qualified professionals were noted and the results were analyzed. A total of 14% (7/50) cases were diagnosed to have malaria. All the malaria cases which were positive in Leishman stain were also detected in Toluidine blue stain. Malarial parasites were clearly visible against the homogenously light green background in Toluidine blue. The detection of malarial parasite by Toluidine blue was quick, easy and confirmative. Toluidine blue stained peripheral blood smear allows for easy identification and speciation of malarial parasite at low magnification and in shorter period of time.

Identification of Cronobacter species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with an optimized analysis method.

PubMed

Wang, Qi; Zhao, Xiao-Juan; Wang, Zi-Wei; Liu, Li; Wei, Yong-Xin; Han, Xiao; Zeng, Jing; Liao, Wan-Jin

2017-08-01

Rapid and precise identification of Cronobacter species is important for foodborne pathogen detection, however, commercial biochemical methods can only identify Cronobacter strains to genus level in most cases. To evaluate the power of mass spectrometry based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) for Cronobacter species identification, 51 Cronobacter strains (eight reference and 43 wild strains) were identified by both MALDI-TOF MS and 16S rRNA gene sequencing. Biotyper RTC provided by Bruker identified all eight reference and 43 wild strains as Cronobacter species, which demonstrated the power of MALDI-TOF MS to identify Cronobacter strains to genus level. However, using the Bruker's database (6903 main spectra products) and Biotyper software, the MALDI-TOF MS analysis could not identify the investigated strains to species level. When MALDI-TOF MS analysis was performed using the combined in-house Cronobacter database and Bruker's database, bin setting, and unweighted pair group method with arithmetic mean (UPGMA) clustering, all the 51 strains were clearly identified into six Cronobacter species and the identification accuracy increased from 60% to 100%. We demonstrated that MALDI-TOF MS was reliable and easy-to-use for Cronobacter species identification and highlighted the importance of establishing a reliable database and improving the current data analysis methods by integrating the bin setting and UPGMA clustering. Copyright © 2017. Published by Elsevier B.V.

MALDI-TOF MS versus VITEK 2 ANC card for identification of anaerobic bacteria.

PubMed

Li, Yang; Gu, Bing; Liu, Genyan; Xia, Wenying; Fan, Kun; Mei, Yaning; Huang, Peijun; Pan, Shiyang

2014-05-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid and inexpensive technique that has initiated a revolution in the clinical microbiology laboratory for identification of pathogens. The Vitek 2 anaerobe and Corynebacterium (ANC) identification card is a newly developed method for identification of corynebacteria and anaerobic species. The aim of this study was to evaluate the effectiveness of the ANC card and MALDI-TOF MS techniques for identification of clinical anaerobic isolates. Five reference strains and a total of 50 anaerobic bacteria clinical isolates comprising ten different genera and 14 species were identified and analyzed by the ANC card together with Vitek 2 identification system and Vitek MS together with version 2.0 database respectively. 16S rRNA gene sequencing was used as reference method for accuracy in the identification. Vitek 2 ANC card and Vitek MS provided comparable results at species level for the five reference strains. Of 50 clinical strains, the Vitek MS provided identification for 46 strains (92%) to the species level, 47 (94%) to genus level, one (2%) low discrimination, two (4%) no identification and one (2%) misidentification. The Vitek 2 ANC card provided identification for 43 strains (86%) correct to the species level, 47 (94%) correct to the genus level, three (6%) low discrimination, three (6%) no identification and one (2%) misidentification. Both Vitek MS and Vitek 2 ANC card can be used for accurate routine clinical anaerobe identification. Comparing to the Vitek 2 ANC card, Vitek MS is easier, faster and more economic for each test. The databases currently available for both systems should be updated and further developed to enhance performance.

[Automated RNA amplification for the rapid identification of Mycobacterium tuberculosis complex in respiratory specimens].

PubMed

Drouillon, V; Houriez, F; Buze, M; Lagrange, P; Herrmann, J-L

2006-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly on clinical respiratory specimens is essential for a correct management of patients suspected of tuberculosis. For this purpose PCR-based kits are available to detect MTB in respiratory specimen but most of them need at least 4 hours to be completed. New methods, based on TRC method (TRC: Transcription Reverse transcription Concerted--TRCRapid M. Tuberculosis--Tosoh Bioscience, Tokyo, Japon) and dedicated monitor have been developed. A new kit (TRC Rapid M. tuberculosis and Real-time monitor TRCRapid-160, Tosoh Corporation, Japan) enabling one step amplification and real-time detection of MTB 16S rRNA by a combination of intercalative dye oxazole yellow-linked DNA probe and isothermal RNA amplification directly on respiratory specimens has been tested in our laboratory. 319 respiratory specimens were tested in this preliminary study and results were compared to smear and culture. Fourteen had a positive culture for MTB. Among theses samples, smear was positive in 11 cases (78.6%) and TRC process was positive in 8 cases (57.1%). Overall sensitivity of TRC compared to smear positive samples is 73%. Theses first results demonstrated that a rapid identification of MTB was possible (less than 2 processing hours for 14 specimens and about 1 hour for 1 specimen) in most cases of smear positive samples using ready to use reagents for real time detection of MTB rRNA in clinical samples. New pretreatment and extraction reagents kits to increase the stability of the sputum RNA and the extraction efficiency are now tested in our laboratory.

Novel aptamer-linked nanoconjugate approach for detection of waterborne bacterial pathogens: an update

NASA Astrophysics Data System (ADS)

Singh, Gulshan; Manohar, Murli; Adegoke, Anthony Ayodeji; Stenström, Thor Axel; Shanker, Rishi

2017-01-01

The lack of microbiologically safe water in underdeveloped nations is the prime cause of infectious disease outbreaks. The need for the specific identification and detection of microorganisms encourages the development of advanced, rapid, sensitive and highly specific methods for the monitoring of pathogens and management of potential risk to human health. The rapid molecular assays based on detection of specific molecular signatures offer advantages over conventional methods in terms of specificity and sensitivity but require complex instrumentation and skilled personnel. Nanotechnology is an emerging area and provides a robust approach for the identification of pathogenic microorganism utilizing the peculiar properties of nanomaterials, i.e. small size (1-100 nm) and large surface area. This emerging technology promises to fulfill the urgent need of a novel strategy to enhance the bacterial identification and quantitation in the environment. In this context, the peculiar properties of gold nanoparticles, their plasmonic shifts, and changes in magnetic properties have been utilized for the simple and cost-effective detection of bacterial nucleic acids, antigens and toxins with quite improved sensitivity. One of the promising leads to develop an advance detection method might be the coupling of nucleic acid aptamers (capable of interacting specifically with bacteria, protozoa, and viruses) with nanomaterials. Such aptamer-nano conjugate can be used for the specific recognition of infectious agents in different environmental matrices. This review summarizes the application of nanotechnology in the area of pathogen detection and discusses the prospects of coupling nucleic acid aptamers with nanoparticles for the specific detection of targeted pathogens.

Rapid screening and identification of ACE inhibitors in snake venoms using at-line nanofractionation LC-MS.

PubMed

Mladic, Marija; de Waal, Tessa; Burggraaff, Lindsey; Slagboom, Julien; Somsen, Govert W; Niessen, Wilfried M A; Manjunatha Kini, R; Kool, Jeroen

2017-10-01

This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC 50  = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC 50  = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.

Forensic Facial Reconstruction: The Final Frontier.

PubMed

Gupta, Sonia; Gupta, Vineeta; Vij, Hitesh; Vij, Ruchieka; Tyagi, Nutan

2015-09-01

Forensic facial reconstruction can be used to identify unknown human remains when other techniques fail. Through this article, we attempt to review the different methods of facial reconstruction reported in literature. There are several techniques of doing facial reconstruction, which vary from two dimensional drawings to three dimensional clay models. With the advancement in 3D technology, a rapid, efficient and cost effective computerized 3D forensic facial reconstruction method has been developed which has brought down the degree of error previously encountered. There are several methods of manual facial reconstruction but the combination Manchester method has been reported to be the best and most accurate method for the positive recognition of an individual. Recognition allows the involved government agencies to make a list of suspected victims'. This list can then be narrowed down and a positive identification may be given by the more conventional method of forensic medicine. Facial reconstruction allows visual identification by the individual's family and associates to become easy and more definite.

Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens.

PubMed

Rivera, Vanessa; Gaviria, Marcela; Muñoz-Cadavid, Cesar; Cano, Luz; Naranjo, Tonny

2015-01-01

The diagnosis of cryptococcosis is usually performed based on cultures of tissue or body fluids and isolation of the fungus, but this method may require several days. Direct microscopic examination, although rapid, is relatively insensitive. Biochemical and immunodiagnostic rapid tests are also used. However, all of these methods have limitations that may hinder final diagnosis. The increasing incidence of fungal infections has focused attention on tools for rapid and accurate diagnosis using molecular biological techniques. Currently, PCR-based methods, particularly nested, multiplex and real-time PCR, provide both high sensitivity and specificity. In the present study, we evaluated a nested PCR targeting the gene encoding the ITS-1 and ITS-2 regions of rDNA in samples from a cohort of patients diagnosed with cryptococcosis. The results showed that in our hands, this Cryptococcus nested PCR assay has 100% specificity and 100% sensitivity and was able to detect until 2 femtograms of Cryptococcus DNA. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

DNA barcoding and development of species-specific markers for the identification of tea mosquito bugs (Miridae: Heteroptera) in India.

PubMed

Rebijith, K B; Asokan, R; Kumar, N K Krishna; Srikumar, K K; Ramamurthy, V V; Bhat, P Shivarama

2012-10-01

Rapid, accurate, and timely identification of insects as a group is important and challenging worldwide, as they outnumber all other animals in number and diversity. DNA barcoding is a method for the identification of species in a wide range of animal taxa, which uses the 5' region of the mitochondrial cytochrome c oxidase-I (CO-I). Yet another easy, accurate, and economical method of species discrimination is by developing species-specific markers, which produce specific amplicon for the species in question. The method is handy because it is not limited by life stages, sex, polymorphism, and other factors. Herein, we measured the usefulness of CO-I for the species discrimination of mirids in India viz. Helopeltis antonii Signoret, H. thievora Waterhouse, H. bradyi Waterhouse, and Pachypeltis maesarum Kirkaldy in their various life stages. Furthermore, our study showed the utility of species-specific markers in differentiating H. antonii (295) and H. bradyi (514) regardless of their life stages. Analysis of CO-I gene revealed <1% intraspecific divergence for all four species examined, whereas the interspecific distances ranged from 7 to 13%. This study showed that the DNA barcode and species-specific markers will aid the identification of mirids in India and will stand as a decisive tool in formulating integrated pest management (IPM) strategy, quick identification of invasive and cryptic species, haplotypes, biotypes, and other factors, if any.

Identification of Staphylococcus saprophyticus isolated from patients with urinary tract infection using a simple set of biochemical tests correlating with 16S-23S interspace region molecular weight patterns.

PubMed

Ferreira, Adriano Martison; Bonesso, Mariana Fávero; Mondelli, Alessandro Lia; da Cunha, Maria de Lourdes Ribeiro de Souza

2012-12-01

The emergence of Staphylococcus spp. not only as human pathogens, but also as reservoirs of antibiotic resistance determinants, requires the development of methods for their rapid and reliable identification in medically important samples. The aim of this study was to compare three phenotypic methods for the identification of Staphylococcus spp. isolated from patients with urinary tract infection using the PCR of the 16S-23S interspace region generating molecular weight patterns (ITR-PCR) as reference. All 57 S. saprophyticus studied were correctly identified using only the novobiocin disk. A rate of agreement of 98.0% was obtained for the simplified battery of biochemical tests in relation to ITR-PCR, whereas the Vitek I system and novobiocin disk showed 81.2% and 89.1% agreement, respectively. No other novobiocin-resistant non-S. saprophyticus strain was identified. Thus, the novobiocin disk is a feasible alternative for the identification of S. saprophyticus in urine samples in laboratories with limited resources. ITR-PCR and the simplified battery of biochemical tests were more reliable than the commercial systems currently available. This study confirms that automated systems are still unable to correctly differentiate CoNS species and that simple, reliable and inexpensive methods can be used for routine identification. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure.

PubMed

Pinot, C; Deredjian, A; Nazaret, S; Brothier, E; Cournoyer, B; Segonds, C; Favre-Bonté, S

2011-11-01

Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples. Recovery of Sten. maltophilia-like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar medium enabled distinction of various morphotype colonies. A set of soil and clinical isolates was tested for species identification using different methods. 16S rDNA analyses showed the dark green with a blue halo morphotype to be typical Sten. maltophilia strains. The API-20NE, Vitek-2 and Biolog phenotypic analyses typically used for the identification of clinical isolates did not perform well on these soil isolates. The species-specific PCR screening targeting Sten. maltophilia 23S rDNA and the multiplex smeD/ggpS PCR, differentiating Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all isolates tested in this study, whatever be their origin. Isolation on VIA medium and confirmation of Sten. maltophilia species membership by smeD PCR is proposed to identify environmental and clinical isolates of Sten. maltophilia. The proposed approach enables isolation and identification of Sten. maltophilia from different environments in an easy and rapid way. This approach will be useful to accurately manage studies on the abundance and distribution of Sten. maltophilia in hospital and nonhospital environments. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis.

PubMed

Wang, Jun; Chen, Wen Feng; Li, Qing X

2012-02-24

The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4-99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9-99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid identification of antibiotic-resistant corynebacteria with the API 20S system.

PubMed Central

Kelly, M C; Smith, I D; Anstey, R J; Thornley, J H; Rennie, R P

1984-01-01

The API 20S system (Analytab Products, Plainview, N.Y.) was evaluated for the rapid identification of multiply antibiotic-resistant aerobic diphtheroids. Sixty-eight clinical isolates of multiply resistant Centers for Disease Control group JK and group D2 corynebacteria had API 20S profiles which were clearly different from those of a number of strains of other Corynebacterium species which were tested. The API 20S system allowed more rapid identification of antibiotic-resistant diphtheroids than conventional biochemical tests. Its use for corynebacteria other than group JK and group D2 is not recommended at this time. PMID:6699150

Differentiation of five enterohepatic Helicobacter species by nested PCR with high-resolution melting curve analysis.

PubMed

Wu, Miaoli; Rao, Dan; Zhu, Yujun; Wang, Jing; Yuan, Wen; Zhang, Yu; Huang, Ren; Guo, Pengju

2017-04-01

Enterohepatic Helicobacter species (EHS) are widespread in rodent species around the world. Several studies have demonstrated that infection with EHS can interfere with the outcomes of animal experiments in cancer research and significantly influence the study results. Therefore, it is essential to establish a rapid detection and identification of EHS for biomedical research using laboratory rodents. Our study aimed to develop a rapid and sensitive method to detect and distinguish five enterohepatic Helicobacter species. Nested PCR followed by high-resolution melting curve analysis (HRM) was developed for identification of H. bilis, H. rodentium, H. muridarum, H. typhlonius, as well as H. hepaticus. To validate the accuracy of nested PCR-HRM analysis, quantitative real-time PCR methods for five different enterohepatic Helicobacter species were developed. A total of 50 cecal samples were tested using both nested PCR-HRM analysis and qPCR method. The nested PCR-HRM method could distinguish five enterohepatic Helicobacter species by different melting temperatures. The melting curve were characterized by peaks of 78.7 ± 0.12°C for H. rodentium, 80.51 ± 0.09°C for H. bilis, 81.6 ± 0.1°C for H. typhlonius, 82.11 ± 0.18°C for H. muridarum, and 82.95 ± 0.09°C for H. hepaticus. The nested PCR-HRM assay is a simple, rapid, and cost-effective assay. This assay could be a useful tool for molecular epidemiology study of enterohepatic Helicobacter infection and an attractive alternative for genotyping of enterohepatic Helicobacter species. © 2016 John Wiley & Sons Ltd.

Rapid identification of apolipoprotein E genotypes by high-resolution melting analysis in Chinese Han and African Fang populations.

PubMed

Zhan, Xiu-Hui; Zha, Guang-Cai; Jiao, Ji-Wei; Yang, Li-Ye; Zhan, Xiao-Fen; Chen, Jiang-Tao; Xie, Dong-DE; Eyi, Urbano Monsuy; Matesa, Rocio Apicante; Obono, Maximo Miko Ondo; Ehapo, Carlos Sala; Wei, Er-Jia; Zheng, Yu-Zhong; Yang, Hui; Lin, Min

2015-02-01

Apolipoprotein E (APOE) gene polymorphism can affect APOE gene transcription, serum lipid levels and repair of tissue damage, which could place individuals at serious risk of cardiovascular disease or certain infectious diseases. Recently, high-resolution melting (HRM) analysis was reported to be a simple, inexpensive, accurate and sensitive method for the genotyping or/and scanning of rare mutations. For this reason, an HRM analysis was used in the present study for APOE genotyping in the Southern Chinese Han and African Fang populations. A total of 100 healthy Southern Chinese Han and 175 healthy African Fang individuals attended the study. Polymerase chain reaction-DNA sequencing was used as a reference method for the genotyping of these samples. The six APOE genotypes could all be rapidly and efficiently identified by HRM analysis, and 100% concordance was found between the HRM analysis and the reference method. The allele frequencies of APOE in the Southern Chinese Han population were 7.0, 87.5 and 5.5% for ɛ2, ɛ3 and ɛ4, respectively. In the African Fang population, the allele frequencies of APOE were 24.3, 65.7 and 10.0% for ɛ2, ɛ3 and ɛ4, respectively. A statistically significant difference was found between the allele frequencies between the populations (P<0.05). In conclusion, the present study revealed the molecular characterization of APOE gene polymorphism in the Han population from the Chaozhou region of Southern China and the Fang population from Equatorial Guinea. The findings of the study indicated that HRM analysis could be used as an accurate and sensitive method for the rapid screening and identification of APOE genotypes in prospective clinical and population genetic analyses.

Vitek 2 ANC card versus BBL Crystal Anaerobe and RapID ANA II for identification of clinical anaerobic bacteria.

PubMed

Blairon, Laurent; Maza, Mengi L; Wybo, Ingrid; Piérard, Denis; Dediste, Anne; Vandenberg, Olivier

2010-08-01

The Vitek 2 Anaerobe and Corynebacterium Identification Card (ANC) was recently evaluated in a multicentre study. In the present work, this system was compared with the BBL Crystal Anaerobe and RapID ANA II panels. These kits were tested using 196 strains of anaerobes that had been previously identified by gas-liquid chromatography. Identification to the species or to the genus level was 75.0%, 81.1% and 70.9% for Crystal, RapID and Vitek, respectively. Vitek ANC failed to provide any identification in 20.4% of the strains, but it had fewer misidentifications than RapID. The confidence factors provided on the results report of each kit were not always correlated with a lower risk of major errors, with the exception of Vitek 2 in which a confidence factor higher than 0.86 excluded the risk of misidentification in more than 87% of isolates. The lower rate of identification by the Vitek and Crystal panels is mostly due the lower ability of these systems to identify the Clostridia. Overall, the three panels are comparable but need improvement to a better accuracy. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Peptide Peak Detection for Low Resolution MALDI-TOF Mass Spectrometry.

PubMed

Yao, Jingwen; Utsunomiya, Shin-Ichi; Kajihara, Shigeki; Tabata, Tsuyoshi; Aoshima, Ken; Oda, Yoshiya; Tanaka, Koichi

2014-01-01

A new peak detection method has been developed for rapid selection of peptide and its fragment ion peaks for protein identification using tandem mass spectrometry. The algorithm applies classification of peak intensities present in the defined mass range to determine the noise level. A threshold is then given to select ion peaks according to the determined noise level in each mass range. This algorithm was initially designed for the peak detection of low resolution peptide mass spectra, such as matrix-assisted laser desorption/ionization Time-of-Flight (MALDI-TOF) mass spectra. But it can also be applied to other type of mass spectra. This method has demonstrated obtaining a good rate of number of real ions to noises for even poorly fragmented peptide spectra. The effect of using peak lists generated from this method produces improved protein scores in database search results. The reliability of the protein identifications is increased by finding more peptide identifications. This software tool is freely available at the Mass++ home page (http://www.first-ms3d.jp/english/achievement/software/).

Peptide Peak Detection for Low Resolution MALDI-TOF Mass Spectrometry

PubMed Central

Yao, Jingwen; Utsunomiya, Shin-ichi; Kajihara, Shigeki; Tabata, Tsuyoshi; Aoshima, Ken; Oda, Yoshiya; Tanaka, Koichi

2014-01-01

A new peak detection method has been developed for rapid selection of peptide and its fragment ion peaks for protein identification using tandem mass spectrometry. The algorithm applies classification of peak intensities present in the defined mass range to determine the noise level. A threshold is then given to select ion peaks according to the determined noise level in each mass range. This algorithm was initially designed for the peak detection of low resolution peptide mass spectra, such as matrix-assisted laser desorption/ionization Time-of-Flight (MALDI-TOF) mass spectra. But it can also be applied to other type of mass spectra. This method has demonstrated obtaining a good rate of number of real ions to noises for even poorly fragmented peptide spectra. The effect of using peak lists generated from this method produces improved protein scores in database search results. The reliability of the protein identifications is increased by finding more peptide identifications. This software tool is freely available at the Mass++ home page (http://www.first-ms3d.jp/english/achievement/software/). PMID:26819872

Mapping Challenging Mutations by Whole-Genome Sequencing

PubMed Central

Smith, Harold E.; Fabritius, Amy S.; Jaramillo-Lambert, Aimee; Golden, Andy

2016-01-01

Whole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homozygous variants). However, such approaches are not entirely suitable for the characterization of a variety of more challenging mutations, such as dominant and semidominant alleles or multigenic traits. Therefore, we have developed strategies for the identification of those classes of mutations, using polymorphism mapping in Caenorhabditis elegans as our model for validation. We also report an alternative approach for mutation identification from traditional recombinant crosses, and a solution to the technical challenge of sequencing sterile or terminally arrested strains where population size is limiting. The methods described herein extend the applicability of whole-genome sequencing to a broader spectrum of mutations, including classes that are difficult to map by traditional means. PMID:26945029

Rapid identification of regulated organic chemical compounds in toys using ambient ionization and a miniature mass spectrometry system.

PubMed

Guo, Xiangyu; Bai, Hua; Lv, Yueguang; Xi, Guangcheng; Li, Junfang; Ma, Xiaoxiao; Ren, Yue; Ouyang, Zheng; Ma, Qiang

2018-04-01

Rapid, on-site analysis was achieved through significantly simplified operation procedures for a wide variety of toy samples (crayon, temporary tattoo sticker, finger paint, modeling clay, and bubble solution) using a miniature mass spectrometry system with ambient ionization capability. The labor-intensive analytical protocols involving sample workup and chemical separation, traditionally required for MS-based analysis, were replaced by direct sampling analysis using ambient ionization methods. A Mini β ion trap miniature mass spectrometer was coupled with versatile ambient ionization methods, e.g. paper spray, extraction spray and slug-flow microextraction nanoESI for direct identification of prohibited colorants, carcinogenic primary aromatic amines, allergenic fragrances, preservatives and plasticizers from raw toy samples. The use of paper substrates coated with Co 3 O 4 nanoparticles allowed a great increase in sensitivity for paper spray. Limits of detection as low as 5μgkg -1 were obtained for target analytes. The methods being developed based on the integration of ambient ionization with miniature mass spectrometer represent alternatives to current in-lab MS analysis operation, and would enable fast, outside-the-lab screening of toy products to ensure children's safety and health. Copyright © 2017 Elsevier B.V. All rights reserved.

Buccal microbiology analyzed by infrared spectroscopy

NASA Astrophysics Data System (ADS)

de Abreu, Geraldo Magno Alves; da Silva, Gislene Rodrigues; Khouri, Sônia; Favero, Priscila Pereira; Raniero, Leandro; Martin, Airton Abrahão

2012-01-01

Rapid microbiological identification and characterization are very important in dentistry and medicine. In addition to dental diseases, pathogens are directly linked to cases of endocarditis, premature delivery, low birth weight, and loss of organ transplants. Fourier Transform Infrared Spectroscopy (FTIR) was used to analyze oral pathogens Aggregatibacter actinomycetemcomitans ATCC 29523, Aggregatibacter actinomycetemcomitans-JP2, and Aggregatibacter actinomycetemcomitans which was clinically isolated from the human blood-CI. Significant spectra differences were found among each organism allowing the identification and characterization of each bacterial species. Vibrational modes in the regions of 3500-2800 cm-1, the 1484-1420 cm-1, and 1000-750 cm-1 were used in this differentiation. The identification and classification of each strain were performed by cluster analysis achieving 100% separation of strains. This study demonstrated that FTIR can be used to decrease the identification time, compared to the traditional methods, of fastidious buccal microorganisms associated with the etiology of the manifestation of periodontitis.

Computational inverse methods of heat source in fatigue damage problems

NASA Astrophysics Data System (ADS)

Chen, Aizhou; Li, Yuan; Yan, Bo

2018-04-01

Fatigue dissipation energy is the research focus in field of fatigue damage at present. It is a new idea to solve the problem of calculating fatigue dissipation energy by introducing inverse method of heat source into parameter identification of fatigue dissipation energy model. This paper introduces the research advances on computational inverse method of heat source and regularization technique to solve inverse problem, as well as the existing heat source solution method in fatigue process, prospects inverse method of heat source applying in fatigue damage field, lays the foundation for further improving the effectiveness of fatigue dissipation energy rapid prediction.

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

PubMed Central

Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R.

2009-01-01

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids. PMID:19956680

PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis.

PubMed Central

Ceron, J; Covarrubias, L; Quintero, R; Ortiz, A; Ortiz, M; Aranda, E; Lina, L; Bravo, A

1994-01-01

A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology. Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B. thuringiensis CryI proteins were described. Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products. B. thuringiensis strains, isolated from soil samples, were analyzed by PCR technology. Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers. This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects. Images PMID:8117089

[Applications of near-infrared spectroscopy to analysis of traditional Chinese herbal medicine].

PubMed

Li, Yan-Zhou; Min, Shun-Geng; Liu, Xia

2008-07-01

Analysis of traditional Chinese herbal medicine is of great importance to its quality control Conventional analysis methods can not meet the requirement of rapid and on-line analysis because of complex process more experiences or needed. In recent years, near-infrared spectroscopy technique has been used for rapid determination of active components, on-line quality control, identification of counterfeit and discrimination of geographical origins of herbal medicines and so on, due to its advantages of simple pretreatment, high efficiency, convenience to use solid diffuse reflection spectroscopy and fiber. In the present paper, the principles and methods of near-infrared spectroscopy technique are introduced concisely. Especially, the applications of this technique in quantitative analysis and qualitative analysis of traditional Chinese herbal medicine are reviewed.

Culture-independent diagnostics for health security

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

Doggett, Norman A.; Mukundan, Harshini; Lefkowitz, Elliot J.

The past decade has seen considerable development in the diagnostic application of nonculture methods, including nucleic acid amplification-based methods and mass spectrometry, for the diagnosis of infectious diseases. The implications of these new culture-independent diagnostic tests (CIDTs) include bypassing the need to culture organisms, thus potentially affecting public health surveillance systems, which continue to use isolates as the basis of their surveillance programs and to assess phenotypic resistance to antimicrobial agents. CIDTs may also affect the way public health practitioners detect and respond to a bioterrorism event. In response to a request from the Department of Homeland Security, Los Alamosmore » National Laboratory and the Centers for Disease Control and Prevention cosponsored a workshop to review the impact of CIDTs on the rapid detection and identification of biothreat agents. Four panel discussions were held that covered nucleic acid amplification–based diagnostics, mass spectrometry, antibody-based diagnostics, and next-generation sequencing. Exploiting the extensive expertise available at this workshop, we identified the key features, benefits, and limitations of the various CIDT methods for providing rapid pathogen identification that are critical to the response and mitigation of a bioterrorism event. After the workshop we conducted a thorough review of the literature, investigating the current state of these 4 culture-independent diagnostic methods. Furthermore, this article combines information from the literature review and the insights obtained at the workshop.« less

Rapid identification of drug resistant Candida species causing recurrent vulvovaginal candidiasis.

PubMed

Diba, Kambiz; Namaki, Atefeh; Ayatolahi, Haleh; Hanifian, Haleh

2012-01-01

Some yeast agents including Candida albicans, Candida tropicalis and Candida glabrata have a role in recurrent vulvovaginal candidiasis. We studied the frequency of both common and recurrent vulvovaginal candidiasis in symptomatic cases which were referred to Urmia Medical Sciences University related gynecology clinics using morphologic and molecular methods. The aim of this study was the identification of Candida species isolated from recurrent vulvovaginal candidiasis cases using a rapid and reliable molecular method. Vaginal swabs obtained from each case, were cultured on differential media including cornmeal agar and CHROM agar Candida. After 48 hours at 37℃, the cultures were studied for growth characteristics and color production respectively. All isolates were identified using the molecular method of PCR - restriction fragment length polymorphism. Among all clinical specimens, we detected 19 ( 16 % ) non fungal agents, 87 ( 82.1 % ) yeasts and 2 ( 1.9 % ) multiple infections. The yeast isolates identified morphologically included Candida albicans ( n = 62 ), Candida glabrata ( n = 9 ), Candida tropicalis ( n = 8 ), Candida parapsilosis ( n = 8 ) and Candida guilliermondii and Candida krusei ( n = 1 each ). We also obtained very similar results for Candida albicans, Candida glabrata and Candida tropicalis as the most common clinical isolates, by using PCR - Restriction Fragment Length Polymorphism. Use of two differential methods, morphologic and molecular, enabled us to identify most medically important Candida species which particularly cause recurrent vulvovaginal candidiasis.

Methods for Kinetic and Thermodynamic Analysis of Aminoacyl-tRNA Synthetases

PubMed Central

Francklyn, Christopher S.; First, Eric A.; Perona, John J.; Hou, Ya-Ming

2008-01-01

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion. PMID:18241792

Culture-independent diagnostics for health security

DOE PAGES

Doggett, Norman A.; Mukundan, Harshini; Lefkowitz, Elliot J.; ...

2016-06-17

The past decade has seen considerable development in the diagnostic application of nonculture methods, including nucleic acid amplification-based methods and mass spectrometry, for the diagnosis of infectious diseases. The implications of these new culture-independent diagnostic tests (CIDTs) include bypassing the need to culture organisms, thus potentially affecting public health surveillance systems, which continue to use isolates as the basis of their surveillance programs and to assess phenotypic resistance to antimicrobial agents. CIDTs may also affect the way public health practitioners detect and respond to a bioterrorism event. In response to a request from the Department of Homeland Security, Los Alamosmore » National Laboratory and the Centers for Disease Control and Prevention cosponsored a workshop to review the impact of CIDTs on the rapid detection and identification of biothreat agents. Four panel discussions were held that covered nucleic acid amplification–based diagnostics, mass spectrometry, antibody-based diagnostics, and next-generation sequencing. Exploiting the extensive expertise available at this workshop, we identified the key features, benefits, and limitations of the various CIDT methods for providing rapid pathogen identification that are critical to the response and mitigation of a bioterrorism event. After the workshop we conducted a thorough review of the literature, investigating the current state of these 4 culture-independent diagnostic methods. Furthermore, this article combines information from the literature review and the insights obtained at the workshop.« less

Culture-Independent Diagnostics for Health Security.

PubMed

Doggett, Norman A; Mukundan, Harshini; Lefkowitz, Elliot J; Slezak, Tom R; Chain, Patrick S; Morse, Stephen; Anderson, Kevin; Hodge, David R; Pillai, Segaran

2016-01-01

The past decade has seen considerable development in the diagnostic application of nonculture methods, including nucleic acid amplification-based methods and mass spectrometry, for the diagnosis of infectious diseases. The implications of these new culture-independent diagnostic tests (CIDTs) include bypassing the need to culture organisms, thus potentially affecting public health surveillance systems, which continue to use isolates as the basis of their surveillance programs and to assess phenotypic resistance to antimicrobial agents. CIDTs may also affect the way public health practitioners detect and respond to a bioterrorism event. In response to a request from the Department of Homeland Security, Los Alamos National Laboratory and the Centers for Disease Control and Prevention cosponsored a workshop to review the impact of CIDTs on the rapid detection and identification of biothreat agents. Four panel discussions were held that covered nucleic acid amplification-based diagnostics, mass spectrometry, antibody-based diagnostics, and next-generation sequencing. Exploiting the extensive expertise available at this workshop, we identified the key features, benefits, and limitations of the various CIDT methods for providing rapid pathogen identification that are critical to the response and mitigation of a bioterrorism event. After the workshop we conducted a thorough review of the literature, investigating the current state of these 4 culture-independent diagnostic methods. This article combines information from the literature review and the insights obtained at the workshop.

A novel radio-tolerant astaxanthin-producing bacterium reveals a new astaxanthin derivative: astaxanthin dirhamnoside.

PubMed

Asker, Dalal; Awad, Tarek S; Beppu, Teruhiko; Ueda, Kenji

2012-01-01

Astaxanthin is a red ketocarotenoid that exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster. The recent discovery of the beneficial roles of astaxanthin against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue has raised its market demand as a nutraceutical and medicinal ingredient in aquaculture, food, and pharmaceutical industries. To satisfy the growing demand for this high-value nutraceuticals ingredient and consumer interest in natural products, many research efforts are being made to discover novel microbial producers with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diodearray-MS methods for carotenoids analysis, we succeeded to isolate a unique astaxanthin-producing bacterium (strain TDMA-17(T)) that belongs to the family Sphingomonadaceae (Asker et al., Appl Microbiol Biotechnol 77: 383-392, 2007). In this chapter, we provide a detailed description of effective HPLC-Diodearray-MS methods for rapid analysis and identification of the carotenoids produced by strain TDMA-17(T). We also describe the methods of isolation and identification for a novel bacterial carotenoid (astaxanthin derivative), a major carotenoid that is produced by strain TDMA-17(T). Finally, we describe the polyphasic taxonomic analysis of strain TDMA-17(T) and the description of a novel species belonging to genus Sphingomonas.

Rapid identification of bacteria from bioMérieux BacT/ALERT blood culture bottles by MALDI-TOF MS.

PubMed

Haigh, J D; Green, I M; Ball, D; Eydmann, M; Millar, M; Wilks, M

2013-01-01

Several studies have reported poor results when trying to identify microorganisms directly from the bioMérieux BacT/ALERT blood culture system using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry. The aim of this study is to evaluate two new methods, Sepsityper and an enrichment method for direct identification of microorganisms from this system. For both methods the samples were processed using the Bruker Microflex LT mass spectrometer (Biotyper) using the Microflex Control software to obtain spectra. The results from direct analysis were compared with those obtained by subculture and subsequent identification. A total of 350 positive blood cultures were processed simultaneously by the two methods. Fifty-three cultures were polymocrobial or failed to grow any organism on subculture, and these results were not included as there was either no subculture result, or for polymicrobial cultures it was known that the Biotyper would not be able to distinguish the constituent organisms correctly. Overall, the results showed that, contrary to previous reports, it is possible to identify bacteria directly from bioMérieux blood culture bottles, as 219/297 (74%) correct identifications were obtained using the Bruker Sepsityper method and 228/297 (77%) were obtained for the enrichment method when there is only one organism was present. Although the enrichment method was simpler, the reagent costs for the Sepsityper method were approximately pound 4.00 per sample compared to pound 0.50. An even simpler and cheaper method, which was less labour-intensive and did not require further reagents, was investigated. Seventy-seven specimens from positive signalled blood cultures were analysed by inoculating prewarmed blood agar plates and analysing any growth after 1-, 2- and 4-h periods of incubation at 37 degrees C, by either direct transfer or alcohol extraction. This method gave the highest number of correct identifications, 66/77 (86%), and was cheaper and less labour-intensive than either of the two above methods.

Evaluation of the Vitek 2 ANC card for identification of clinical isolates of anaerobic bacteria.

PubMed

Lee, E H L; Degener, J E; Welling, G W; Veloo, A C M

2011-05-01

An evaluation of the Vitek 2 ANC card (bioMérieux, Marcy l'Etoile, France) was performed with 301 anaerobic isolates. Each strain was identified by 16S rRNA gene sequencing, which is considered to be the reference method. The Vitek 2 ANC card correctly identified 239 (79.4%) of the 301 clinical isolates to the genus level, including 100 species that were not represented in the database. Correct species identification was obtained for 60.1% (181/301) of the clinical isolates. For the isolates not identified to the species level, a correct genus identification was obtained for 47.0% of them (47/100), and 16 were accurately designated not identified. Although the Vitek 2 ANC card allows the rapid and acceptable identification of the most common clinically important anaerobic bacteria within 6 h, improvement is required for the identification of members of the genera Fusobacterium, Prevotella, and Actinomyces and certain Gram-positive anaerobic cocci (GPAC).

Analysis of blind identification methods for estimation of kinetic parameters in dynamic medical imaging

NASA Astrophysics Data System (ADS)

Riabkov, Dmitri

Compartment modeling of dynamic medical image data implies that the concentration of the tracer over time in a particular region of the organ of interest is well-modeled as a convolution of the tissue response with the tracer concentration in the blood stream. The tissue response is different for different tissues while the blood input is assumed to be the same for different tissues. The kinetic parameters characterizing the tissue responses can be estimated by blind identification methods. These algorithms use the simultaneous measurements of concentration in separate regions of the organ; if the regions have different responses, the measurement of the blood input function may not be required. In this work it is shown that the blind identification problem has a unique solution for two-compartment model tissue response. For two-compartment model tissue responses in dynamic cardiac MRI imaging conditions with gadolinium-DTPA contrast agent, three blind identification algorithms are analyzed here to assess their utility: Eigenvector-based Algorithm for Multichannel Blind Deconvolution (EVAM), Cross Relations (CR), and Iterative Quadratic Maximum Likelihood (IQML). Comparisons of accuracy with conventional (not blind) identification techniques where the blood input is known are made as well. The statistical accuracies of estimation for the three methods are evaluated and compared for multiple parameter sets. The results show that the IQML method gives more accurate estimates than the other two blind identification methods. A proof is presented here that three-compartment model blind identification is not unique in the case of only two regions. It is shown that it is likely unique for the case of more than two regions, but this has not been proved analytically. For the three-compartment model the tissue responses in dynamic FDG PET imaging conditions are analyzed with the blind identification algorithms EVAM and Separable variables Least Squares (SLS). A method of identification that assumes that FDG blood input in the brain can be modeled as a function of time and several parameters (IFM) is analyzed also. Nonuniform sampling SLS (NSLS) is developed due to the rapid change of the FDG concentration in the blood during the early postinjection stage. Comparisons of accuracy of EVAM, SLS, NSLS and IFM identification techniques are made.

Sequence Characterized Amplified Regions (SCAR) that differentiate New World screwworms from other potential wound inhabiting flies

USDA-ARS?s Scientific Manuscript database

Guarding against the introduction of screwworms, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), to North America or any other screwworm free area relies on rapid, reliable identification of suspected cases. Identification of first instars suspected to be C. hominivorax can be rapidly v...

Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

USDA-ARS?s Scientific Manuscript database

Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that el...

Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

USDA-ARS?s Scientific Manuscript database

Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that eli...

Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

DTIC Science & Technology

2010-08-25

or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic ...genetic changes conferring antibiotic resistance can be deciphered rapidly and accurately using WGS. We demonstrate the utility of Roche 454...Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing Peter E. Chen1

Flanking sequence determination and event-specific detection of genetically modified wheat B73-6-1.

PubMed

Xu, Junyi; Cao, Jijuan; Cao, Dongmei; Zhao, Tongtong; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2013-05-01

In order to establish a specific identification method for genetically modified (GM) wheat, exogenous insert DNA and flanking sequence between exogenous fragment and recombinant chromosome of GM wheat B73-6-1 were successfully acquired by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, herbicide-resistant bar, ubiquitin promoter, and high-molecular-weight gluten subunit. The flanking sequence between insert DNA revealed high similarity with Triticum turgidum A gene (GenBank: AY494981.1). A specific PCR detection method for GM wheat B73-6-1 was established on the basis of primers designed according to the flanking sequence. This specific PCR method was validated by GM wheat, GM corn, GM soybean, GM rice, and non-GM wheat. The specifically amplified target band was observed only in GM wheat B73-6-1. This method is of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of GM wheat B73-6-1.

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 ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

PubMed

Taira, Chiaki; Matsuda, Kazuyuki; Takeichi, Naoya; Furukawa, Satomi; Sugano, Mitsutoshi; Uehara, Takeshi; Okumura, Nobuo; Honda, Takayuki

2018-01-01

ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care. © 2017 Wiley Periodicals, Inc.

Comparative analysis of storage conditions and homogenization methods for tick and flea species for identification by MALDI-TOF MS.

PubMed

Nebbak, A; El Hamzaoui, B; Berenger, J-M; Bitam, I; Raoult, D; Almeras, L; Parola, P

2017-12-01

Ticks and fleas are vectors for numerous human and animal pathogens. Controlling them, which is important in combating such diseases, requires accurate identification, to distinguish between vector and non-vector species. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was applied to the rapid identification of arthropods. The growth of this promising tool, however, requires guidelines to be established. To this end, standardization protocols were applied to species of Rhipicephalus sanguineus (Ixodida: Ixodidae) Latreille and Ctenocephalides felis felis (Siphonaptera: Pulicidae) Bouché, including the automation of sample homogenization using two homogenizer devices, and varied sample preservation modes for a period of 1-6 months. The MS spectra were then compared with those obtained from manual pestle grinding, the standard homogenization method. Both automated methods generated intense, reproducible MS spectra from fresh specimens. Frozen storage methods appeared to represent the best preservation mode, for up to 6 months, while storage in ethanol is also possible, with some caveats for tick specimens. Carnoy's buffer, however, was shown to be less compatible with MS analysis for the purpose of identifying ticks or fleas. These standard protocols for MALDI-TOF MS arthropod identification should be complemented by additional MS spectrum quality controls, to generalize their use in monitoring arthropods of medical interest. © 2017 The Royal Entomological Society.

Identification of species origin of meat and meat products on the DNA basis: a review.

PubMed

Kumar, Arun; Kumar, Rajiv Ranjan; Sharma, Brahm Deo; Gokulakrishnan, Palanisamy; Mendiratta, Sanjod Kumar; Sharma, Deepak

2015-01-01

The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness, and unhealthy competition in meat market. Consumer should be protected from these malicious practices of meat adulterations by quick, precise, and specific identification of meat animal species. Several analytical methodologies have been employed for meat speciation based on anatomical, histological, microscopic, organoleptic, chemical, electrophoretic, chromatographic, or immunological principles. However, by virtue of their inherent limitations, most of these techniques have been replaced by the recent DNA-based molecular techniques. In the last decades, several methods based on polymerase chain reaction have been proposed as useful means for identifying the species origin in meat and meat products, due to their high specificity and sensitivity, as well as rapid processing time and low cost. This review intends to provide an updated and extensive overview on the DNA-based methods for species identification in meat and meat products.

Fingerprinting Green Curry: An Electrochemical Approach to Food Quality Control.

PubMed

Chaibun, Thanyarat; La-O-Vorakiat, Chan; O'Mullane, Anthony P; Lertanantawong, Benchaporn; Surareungchai, Werasak

2018-06-07

The detection and identification of multiple components in a complex sample such as food in a cost-effective way is an ongoing challenge. The development of on-site and rapid detection methods to ensure food quality and composition is of significant interest to the food industry. Here we report that an electrochemical method can be used with an unmodified glassy carbon electrode for the identification of the key ingredients found within Thai green curries. It was found that green curry presents a fingerprint electrochemical response that contains four distinct peaks when differential pulse voltammetry is performed. The reproducibility of the sensor is excellent as no surface modification is required and therefore storage is not an issue. By employing particle swarm optimization algorithms the identification of ingredients within a green curry could be obtained. In addition, the quality and freshness of the sample could be monitored by detecting a change in the intensity of the peaks in the fingerprint response.

Identification of three internal feeders from Korla fragrant pears by quantitative real-time polymerase chain reaction.

PubMed

Wang, Fengjun; Feng, Junli; Ye, Sudan; Li, Xin; Huang, Hannian; Hua, Peng

2018-04-01

Cydia pomonella, Euzophera pyriella Yang, and Grapholitha molesta are destructive pest species of Korla fragrant pears in Xinjiang. They are also quarantine pests of concern in a number of countries. Identification of these small pest larvae by morphological characters is difficult, and misidentifications will influence appropriate quarantine decisions. Here, a 520 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was first amplified and sequenced from each species, and a diagnostic region was observed. Subsequently, the species-specific primer and probe sets of quantitative real-time PCR (qPCR) were designed, which amplified a 114-116 bp fragment of COI genes. This method was validated by amplification DNA extracted from single, multiple, and mixed pest samples. Results indicated that this method allows rapid discrimination and reliable identification of larvae, pupae, and adult specimens of all three species, which could help the international export trading of Korla fragrant pears and related products.

Roles and methods of performance evaluation of hospital academic leadership.

PubMed

Zhou, Ying; Yuan, Huikang; Li, Yang; Zhao, Xia; Yi, Lihua

2016-01-01

The rapidly advancing implementation of public hospital reform urgently requires the identification and classification of a pool of exceptional medical specialists, corresponding with incentives to attract and retain them, providing a nucleus of distinguished expertise to ensure public hospital preeminence. This paper examines the significance of academic leadership, from a strategic management perspective, including various tools, methods and mechanisms used in the theory and practice of performance evaluation, and employed in the selection, training and appointment of academic leaders. Objective methods of assessing leadership performance are also provided for reference.

Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture.

PubMed

Wagner, K; Springer, B; Pires, V P; Keller, P M

2018-05-03

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows.

PubMed

Marques, S; Huss, V A R; Pfisterer, K; Grosse, C; Thompson, G

2015-05-01

The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture.

PubMed

Keane, O M; Budd, K E; Flynn, J; McCoy, F

2013-09-21

Rapid and accurate identification of mastitis pathogens is important for disease control. Bacterial culture and isolate identification is considered the gold standard in mastitis diagnosis but is time consuming and results in many culture-negative samples. Identification of mastitis pathogens by PCR has been proposed as a fast and sensitive alternative to bacterial culture. The results of bacterial culture and PCR for the identification of the aetiological agent of clinical mastitis were compared. The pathogen identified by traditional culture methods was also detected by PCR in 98 per cent of cases indicating good agreement between the positive results of bacterial culture and PCR. A mastitis pathogen could not be recovered from approximately 30 per cent of samples by bacterial culture, however, an aetiological agent was identified by PCR in 79 per cent of these samples. Therefore, a mastitis pathogen was detected in significantly more milk samples by PCR than by bacterial culture (92 per cent and 70 per cent, respectively) although the clinical relevance of PCR-positive culture-negative results remains controversial. A mixed infection of two or more mastitis pathogens was also detected more commonly by PCR. Culture-negative samples due to undetected Staphylococcus aureus infections were rare. The use of PCR technology may assist in rapid mastitis diagnosis, however, accurate interpretation of PCR results in the absence of bacterial culture remains problematic.

Direct identification of bacteria in blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new methodological approach.

PubMed

Kroumova, Vesselina; Gobbato, Elisa; Basso, Elisa; Mucedola, Luca; Giani, Tommaso; Fortina, Giacomo

2011-08-15

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently been demonstrated to be a powerful tool for the rapid identification of bacteria from growing colonies. In order to speed up the identification of bacteria, several authors have evaluated the usefulness of this MALDI-TOF MS technology for the direct and quick identification bacteria from positive blood cultures. The results obtained so far have been encouraging but have also shown some limitations, mainly related to the bacterial growth and to the presence of interference substances belonging to the blood cultures. In this paper, we present a new methodological approach that we have developed to overcome these limitations, based mainly on an enrichment of the sample into a growing medium before the extraction process, prior to mass spectrometric analysis. The proposed method shows important advantages for the identification of bacterial strains, yielding an increased identification score, which gives higher confidence in the results. Copyright © 2011 John Wiley & Sons, Ltd.

Identification of Legionella Species by Random Amplified Polymorphic DNA Profiles

PubMed Central

Lo Presti, François; Riffard, Serge; Vandenesch, François; Etienne, Jerome

1998-01-01

Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42 Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified Legionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of Legionella isolates to the species level without further restriction or hybridization. PMID:9774564