A TaqMan real-time PCR-based assay for the identification of Fasciola spp.

PubMed

Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Jowers, Michael J; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

2011-06-30

Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

PubMed

Yang, Litao; Pan, Aihu; Zhang, Kewei; Guo, Jinchao; Yin, Changsong; Chen, Jianxiu; Huang, Cheng; Zhang, Dabing

2005-08-10

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

PubMed

Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

2015-12-01

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget.

PubMed

Rahman, Md Mahfujur; Hamid, Sharifah Bee Abd; Basirun, Wan Jefrey; Bhassu, Subha; Rashid, Nur Raifana Abdul; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Ali, Md Eaqub

2016-01-01

This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

Detection of KIT Genotype in Pigs by TaqMan MGB Real-Time Quantitative Polymerase Chain Reaction.

PubMed

Li, Xiuxiu; Li, Xiaoning; Luo, Rongrong; Wang, Wenwen; Wang, Tao; Tang, Hui

2018-05-01

The dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene: a 450 kb duplication containing the entire KIT gene together with flanking sequences and one splice mutation with a G:A substitution in intron 17. The purpose of this study was to establish a simple, rapid method to determine KIT genotype in pigs. First, to detect KIT copy number variation (CNV), primers for exon 2 of the KIT gene, along with a TaqMan minor groove binder (MGB) probe, were designed. The single-copy gene, estrogen receptor (ESR), was used as an internal control. A real-time fluorescence-based quantitative PCR (FQ-PCR) protocol was developed to accurately detect KIT CNVs. Second, to detect the splice mutation ratio of the G:A substitution in intron 17, a 175 bp region, including the target mutation, was amplified from genomic DNA. Based on the sequence of the resulting amplified fragment, an MGB probe set was designed to detect the ratio of splice mutation to normal using FQ-PCR. A series of parallel amplification curves with the same internal distances were obtained using gradually diluted DNA as templates. The CT values among dilutions were significantly different (p < 0.001) and the coefficients of variation from each dilution were low (from 0.13% to 0.26%). The amplification efficiencies for KIT and ESR were approximately equal, indicating ESR was an appropriate control gene. Furthermore, use of the MGB probe set resulted in detection of the target mutation at a high resolution and stability; standard curves illustrated that the amplification efficiencies of KIT1 (G) and KIT2 (A) were approximately equal (98.8% and 97.2%). In conclusion, a simple, rapid method, with high specificity and stability, for the detection of the KIT genotype in pigs was established using TaqMan MGB probe real-time quantitative PCR.

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide.

PubMed

Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao

2015-09-01

Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10 3  CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10 0  CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.

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

PubMed

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

2017-06-01

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

Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.

PubMed

Henritzi, Dinah; Zhao, Na; Starick, Elke; Simon, Gaelle; Krog, Jesper S; Larsen, Lars Erik; Reid, Scott M; Brown, Ian H; Chiapponi, Chiara; Foni, Emanuela; Wacheck, Silke; Schmid, Peter; Beer, Martin; Hoffmann, Bernd; Harder, Timm C

2016-11-01

A diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. New SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples. A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages "av" (European avian-derived), "hu" (European human-derived) and "pdm" (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage "pdm" is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping. These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe. © 2016 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Detection of rabbit and hare processed material in compound feeds by TaqMan real-time PCR.

PubMed

Pegels, N; López-Calleja, I; García, T; Martín, R; González, I

2013-01-01

Food and feed traceability has become a priority for governments due to consumer demand for comprehensive and integrated safety policies. In the present work, a TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for specific detection of rabbit and hare material in animal feeds and pet foods. The technique is based on the use of three species-specific primer/probe detection systems targeting three 12S rRNA gene fragments: one from rabbit species, another one from hare species and a third fragment common to rabbit and hare (62, 102 and 75 bp length, respectively). A nuclear 18S rRNA PCR system, detecting a 77-bp amplicon, was used as positive amplification control. Assay performance and sensitivity were assessed through the analysis of a batch of laboratory-scale feeds treated at 133°C at 3 bar for 20 min to reproduce feed processing conditions dictated by European regulations. Successful detection of highly degraded rabbit and hare material was achieved at the lowest target concentration assayed (0.1%). Furthermore, the method was applied to 96 processed commercial pet food products to determine whether correct labelling had been used at the market level. The reported real-time PCR technique detected the presence of rabbit tissues in 80 of the 96 samples analysed (83.3%), indicating a possible labelling fraud in some pet foods. The real-time PCR method reported may be a useful tool for traceability purposes within the framework of feed control.

Comparative evaluation of two Rickettsia typhi-specific quantitative real-time PCRs for research and diagnostic purposes.

PubMed

Papp, Stefanie; Rauch, Jessica; Kuehl, Svenja; Richardt, Ulricke; Keller, Christian; Osterloh, Anke

2017-02-01

Rickettsioses are caused by intracellular bacteria of the family of Rickettsiaceae. Rickettsia (R.) typhi is the causative agent of endemic typhus. The disease occurs worldwide and is one of the most prevalent rickettsioses. Rickettsial diseases, however, are generally underdiagnosed which is mainly due to the lack of sensitive and specific methods. In addition, methods for quantitative detection of the bacteria for research purposes are rare. We established two qPCRs for the detection of R. typhi by amplification of the outer membrane protein B (ompB) and parvulin-type PPIase (prsA) genes. Both qPCRs are specific and exclusively recognize R. typhi but no other rickettsiae including the closest relative, R. prowazekii. The prsA-based qPCR revealed to be much more sensitive than the amplification of ompB and provided highly reproducible results in the detection of R. typhi in organs of infected mice. Furthermore, as a nested PCR the prsA qPCR was applicable for the detection of R. typhi in human blood samples. Collectively, the prsA-based qPCR represents a reliable method for the quantitative detection of R. typhi for research purposes and is a promising candidate for differential diagnosis.

A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples.

PubMed

Campos, Maria Doroteia; Valadas, Vera; Campos, Catarina; Morello, Laura; Braglia, Luca; Breviario, Diego; Cardoso, Hélia G

2018-01-01

Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and species-specific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.

Development and validation of a real-time TaqMan assay for the detection and enumeration of Pseudomonas fluorescens ATCC 13525 used as a challenge organism in testing of food equipments.

PubMed

Saha, Ratul; Bestervelt, Lorelle L; Donofrio, Robert S

2012-02-01

Pseudomonas fluorescens ATCC 13525 is used as the challenge organism to evaluate the efficacy of the clean-in-place (CIP) process of food equipment (automatic ice-maker) as per NSF/ANSI Standard 12. Traditional culturing methodology is presently used to determine the concentration of the challenge organism, which takes 48 h to confirm the cell density. Storage of the challenge preparation in the refrigerator might alter the cell density as P. fluorescens is capable of growing at 4 °C. Also, background organism can grow on the Pseudomonas F agar (PFA) used for the recovery of P. fluorescens thus affecting the results of the test. Real-time TaqMan assay targeting the cpn60 gene was developed for the enumeration and the identification of P. fluorescens because of its specificity, accuracy, and shorter turnaround time. The TaqMan primer-probe pair developed using the Allele ID® 7.0 probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10 colony forming units (CFU)/mL. The assay was also successful in determining the concentration of the challenge preparation within 2 h. Based on these observations, TaqMan assay targeting the cpn60 gene can be efficiently used for strain level identification and enumeration of bacteria. Pseudomonas fluorescens ATCC 13525 is used as a challenge organism in the efficacy testing of clean-in-place process of food equipments. Currently, culturing technique is used for its identification and estimation, which is not only time-consuming but also prone to error. Real-time TaqMan assay is more specific, sensitive, and accurate along with a shorter turnaround time compared to culturing techniques, thereby increasing the overall quality of the testing methodology to evaluate the clean-in-place process critical for the food industry to protect public health and safety. © 2012 Institute of Food Technologists®

Results of the Abbott RealTime HIV-1 assay for specimens yielding "target not detected" results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test.

PubMed

Babady, N Esther; Germer, Jeffrey J; Yao, Joseph D C

2010-03-01

No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding "target not detected" results by CTM.

Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6

PubMed Central

Wakeley, P. R.; Johnson, N.; McElhinney, L. M.; Marston, D.; Sawyer, J.; Fooks, A. R.

2005-01-01

Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus. PMID:15956398

Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR.

PubMed

Liu, Ya-Mei; Qiu, Liang; Sheng, An-Zhi; Wan, Xiao-Yuan; Cheng, Dong-Yuan; Huang, Jie

2018-01-01

A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 10 2 to 4 × 10 8 copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 10 1 copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass.

PubMed

López-Mondéjar, Rubén; Antón, Anabel; Raidl, Stefan; Ros, Margarita; Pascual, José Antonio

2010-04-01

The species of the genus Trichoderma are used successfully as biocontrol agents against a wide range of phytopathogenic fungi. Among them, Trichoderma harzianum is especially effective. However, to develop more effective fungal biocontrol strategies in organic substrates and soil, tools for monitoring the control agents are required. Real-time PCR is potentially an effective tool for the quantification of fungi in environmental samples. The aim of this study consisted of the development and application of a real-time PCR-based method to the quantification of T. harzianum, and the extrapolation of these data to fungal biomass values. A set of primers and a TaqMan probe for the ITS region of the fungal genome were designed and tested, and amplification was correlated to biomass measurements obtained with optical microscopy and image analysis, of the hyphal length of the mycelium of the colony. A correlation of 0.76 between ITS copies and biomass was obtained. The extrapolation of the quantity of ITS copies, calculated based on real-time PCR data, into quantities of fungal biomass provides potentially a more accurate value of the quantity of soil fungi. Copyright 2009 Elsevier Ltd. All rights reserved.

Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.

PubMed

Saha, Ratul; Donofrio, Robert S; Bagley, Susan T

2010-08-01

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.

High resolution TaqMan real-time PCR approach to detect hazelnut DNA encoding for ITS rDNA in foods.

PubMed

López-Calleja, Inés María; de la Cruz, Silvia; Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario

2013-12-01

A broad range of foods have been described as causing allergies, but the majority of allergic reactions can be ascribed to a limited number of food components. Recent extensive surveys showed how tree nuts, particularly hazelnut (Corylus avellana L.) seeds, rank amongst the most important sources of food allergy. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a real-time polymerase chain reaction (PCR) for detection of hazelnut in commercial food products. In this way a specific hazelnut primer pair based on the ITS marker (70 bp) and a nuclease (TaqMan) probe labelled with FAM and BHQ were designed. Sensibility of real-time PCR was determined by analysis of raw and heat treated hazelnut-wheat flour mixtures with a range of detection of 0.1-100,000 ppm. Practical applicability of the real-time PCR assay developed for determining hazelnut in different food matrices was investigated by analyzing 179 commercial foodstuffs comprising snacks, biscuits, chocolates, bonbons, creams, nut bars, ice creams, precooked meals, breads, beverages, yogurts, cereals, meat products, rice cake and nougat. From the total of samples analyzed, 40 commercial food products that didn't declare hazelnut nor traces on the label were found to contain hazelnut. The real-time PCR method proposed herein due to its high sensitivity facilitates the detection of hazelnut traces in commercial food products and can also be useful for monitoring the effectiveness of cleaning processes and as consequence, can help to prevent the food allergic consumer from unintentional ingestion of hidden allergens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development a of multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic virus

USDA-ARS?s Scientific Manuscript database

Asian prunus viruses (APV 1, APV 2 and APV 3) and Plum bark necrosis stem pitting associated virus (PBNSPaV) are two recently described viruses infecting Prunus spp., and Peach latent mosaic viroid (PLMVd) is a viroid that infects the same species. A single-tube multiplex, TaqMan real-time RT-PCR as...

New design, development, and optimization of an in-house quantitative TaqMan Real-time PCR assay for HIV-1 viral load measurement.

PubMed

Noorbazargan, Hassan; Nadji, Seyed Alireza; Samiee, Siamak Mirab; Paryan, Mahdi; Mohammadi-Yeganeh, Samira

2018-04-01

Background Viral load measurement is commonly applicable to monitor HIV infection in patients to determine the number of HIV-RNA in serum samples of individuals. The aim of the present study was to set up a highly specific, sensitive, and reproducible home-brewed Real-time PCR assay based on TaqMan chemistry to quantify HIV-1 RNA genome. Methods In this study, three sets of primer pairs and a TaqMan probe were designed for HIV subtypes conserved sequences. An internal control was included in this assay to evaluate the presence of inhibition. Standard curve and threshold cycle values were determined using in vitro transcribed RNA from int region of HIV-1. A serial dilution of RNA standards was generated by in vitro transcription, from 10 to 10 9 copies/ml to find the sensitivity and the limit of detection (LOD) of the assay and to evaluate its performance in a quantitative RT-PCR assay. Results The assay has a low LOD equivalent to 33.13 copies/ml of HIV-1 RNA and a linear range of detection from 10 to 10 9 copies/ml. The coefficient of variation (CV) for Inter and Intra-assay precision of this in-house HIV Real-time RT-PCR ranged from 0.28 to 2.49% and 0.72 to 4.47%, respectively. The analytical and clinical specificity was 100%. Conclusions The results indicate that the developed method has a suitable specificity and sensitivity and is highly reproducible and cost-benefit. Therefore, it will be useful to monitor HIV infection in plasma samples of individuals.

Quantification of rice brown leaf spot through Taqman real-time PCR specific to the unigene encoding Cochliobolus miyabeanus SCYTALONE DEHYDRATASE1 involved in fungal melanin biosynthesis.

PubMed

Su'udi, Mukhamad; Park, Jong-Mi; Kang, Woo-Ri; Park, Sang-Ryeol; Hwang, Duk-Ju; Ahn, Il-Pyung

2012-12-01

Rice brown leaf spot is a major disease in the rice paddy field. The causal agent Cochliobolus miyabeanus is an ascomycete fungus and a representative necrotrophic pathogen in the investigation of rice-microbe interactions. The aims of this research were to identify a quantitative evaluation method to determine the amount of C. miyabeanus proliferation in planta and determine the method's sensitivity. Real-time polymerase chain reaction (PCR) was employed in combination with the primer pair and Taqman probe specific to CmSCD1, a C. miyabeanus unigene encoding SCYTALONE DEHYDRATASE, which is involved in fungal melanin biosynthesis. Comparative analysis of the nucleotide sequences of CmSCD1 from Korean strains with those from the Japanese and Taiwanese strains revealed some sequence differences. Based on the crossing point (CP) values from Taqman real-time PCR containing a series of increasing concentrations of cloned amplicon or fungal genomic DNA, linear regressions with a high level of reliability (R(2)>0.997) were constructed. This system was able to estimate fungal genomic DNA at the picogram level. The reliability of this equation was further confirmed using DNA samples from both resistant and susceptible cultivars infected with C. miyabeanus. In summary, our quantitative system is a powerful alternative in brown leaf spot forecasting and in the consistent evaluation of disease progression.

TaqMan real-time RT-PCR detection of infectious salmon anaemia virus (ISAV) from formalin-fixed paraffin-embedded Atlantic salmon Salmo salar tissues.

PubMed

Godoy, M G; Kibenge, F S; Kibenge, M J; Olmos, P; Ovalle, L; Yañez, A J; Avendaño-Herrera, R

2010-05-18

The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.

Taqman Real-Time PCR Detects Avipoxvirus DNA in Blood of Hawaìi `Amakihi (Hemignathus virens)

PubMed Central

Farias, Margaret E. M.; LaPointe, Dennis A.; Atkinson, Carter T.; Czerwonka, Christopher; Shrestha, Rajesh; Jarvi, Susan I.

2010-01-01

Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Methodology/Principal Findings Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study. Conclusions/Significance We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus. PMID:20523726

Comparison of the Roche COBAS Amplicor Monitor, Roche COBAS Ampliprep/COBAS Taqman and Abbott RealTime Test assays for quantification of hepatitis C virus and HIV RNA.

PubMed

Wolff, Dietmar; Gerritzen, Andreas

2007-01-01

We have evaluated the performance of two newly developed automated real-time PCR assays, the COBAS Ampliprep/COBAS TaqMan (CAP/CTM) and the Abbott RealTime tests, in the quantification of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA. The widely used semi-automated COBAS Amplicor Monitor (CAM) assay served as the reference test. Several specimens were analyzed, including 102 plasma samples from HCV patients and 109 from HIV patients and 10 samples from negative donors, as well as Quality Control in Molecular Diagnostics (QCMD) and National Institute for Biological Standards and Controls (NIBSC) proficiency program panels. Good correlation was observed among the three assays, with correlation coefficients (R2) of 0.8 (CAM-CAP/CTM), 0.89 (CAM-RealTime) and 0.91 (CAP/CTM-RealTime) for HCV and 0.83 (CAM-RealTime), 0.85 (CAM-CAP/CTM) and 0.89 (CAP/CTM-RealTime) for HIV. The overall concordance for negative/positive results was 100% for HCV and 98% for HIV. All assays were equally able to quantify HCV genotypes 1, 3, 5 and HIV group M (subtypes A-H) and N from QCMD and NIBSC panels. In terms of workflow, the RealTime assay requires more hands-on-time than the CAP/CTM assay. The results indicate that real-time PCR assays can improve the efficiency of end-point PCR tests by better covering viral dynamic ranges and providing higher throughput and automation.

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Bai, Jian-Shan; Tang, Jian-Dong; Hu, Xu-Chu; Chen, Ding-Hu; Zhang, Ren-Li; Chen, Mu-Xin; Ai, Lin; Zhu, Xing-Quan

2012-03-01

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1pg of purified genomic DNA, 5EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A real time genotyping PCR assay for polyomavirus BK.

PubMed

Gard, Lilli; Niesters, Hubert G M; Riezebos-Brilman, Annelies

2015-09-01

Polyomavirus BK (BKV) may cause nephropathy in renal transplant recipients and hemorrhagic cystitis in bone marrow recipients. We developed real-time PCRs (RT-PCR) to determine easily and rapidly the different BKV genotypes (BKGT) (I-IV). On the VP1 gene a duplex of RT-PCRs was developed and validated to differentiate the four main BKGT. 212 BKV positive samples (21 plasma, 191 urine) were tested with these specific PCRs. Of these 212 samples, 55 PCR results were additionally confirmed by sequencing a VP1 gene fragment (nucleotide 1630-1956). For every genotype, a highly specific, precise and internally controlled assay was developed with a limit of detection of log 3 copies per ml. In 18 (8.5%) of these samples genotyping was not successful due to a low viral load. By sequence analysis, the genotype of 46 out of 55 and 2 out of 4 samples with double infection could be confirmed. This study describes RT-PCRs for detection of the main BKGT. It proved to be rapid, cheap and sensitive compared to sequencing. Double infections can also be detected. This method will be of value to investigate the role of BKV infection in relation to the genotype. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR.

PubMed

Monjezi, Razieh; Tan, Sheau Wei; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2013-01-01

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Two alternative multiplex PCRs for the identification of the seven species of anglerfish (Lophius spp.) using an end-point or a melting curve analysis real-time protocol.

PubMed

Castigliego, Lorenzo; Armani, Andrea; Tinacci, Lara; Gianfaldoni, Daniela; Guidi, Alessandra

2015-01-01

Anglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular analysis is the only possible mean to verify traceability and counteract fraud. We developed two multiplex PCRs, one end-point and one real-time with melting curve post-amplification analysis, which can even be run with the simplest two-channel thermocyclers. The two methods were tested on seventy-five reference samples. Their specificity was checked in twenty more species of those most commonly available on the market and in other species of the Lophiidae family. Both methods, the choice of which depends on the equipment and budget of the lab, provide a rapid and easy-to-read response, improving both the simplicity and cost-effectiveness of existing methods for identifying Lophius species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Detection of Bordetella avium by TaqMan real-time PCR in tracheal swabs from wildlife birds.

PubMed

Stenzel, T; Pestka, D; Tykałowski, B; Śmiałek, M; Koncicki, A; Bancerz-Kisiel, A

2017-03-28

Bordetella avium, the causing agent of bordetellosis, a highly contagious infection of the respiratory tract in young poultry, causes significant losses in poultry farming throughout the world. Wildlife birds can be a reservoir of various pathogens that infect farm animals. For this reason the studies were conducted to estimate the prevalence of Bordetella avium in wildlife birds in Poland. Tracheal swab samples were collected from 650 birds representing 27 species. The bacterial DNA was isolated directly from the swabs and screened for Bordetella avium by TaqMan real-time PCR. The assay specificity was evaluated by testing DNA isolated from 8 other bacteria that can be present in avian respiratory tract, and there was no amplification from non-Bordetella avium agents. Test sensitivity was determined by preparing standard tenfold serial dilutions of DNA isolated from positive control. The assay revealed to be sensitive, with detection limit of approximately 4.07x10^2 copies of Bordetella avium DNA. The genetic material of Bordetella avium was found in 54.54% of common pheasants, in 9.09% of Eurasian coots, in 3.22% of black-headed gulls and in 2.77% of mallard ducks. The results of this study point to low prevalence of Bordetella avium infections in wildlife birds. The results also show that described molecular assay proved to be suitable for the rapid diagnosis of bordetellosis in the routine diagnostic laboratory.

Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis, Enterococcus faecium and their vanA and vanB genotypes.

PubMed

Naserpour Farivar, Taghi; Najafipour, Reza; Johari, Pouran; Aslanimehr, Masoumeh; Peymani, Amir; Jahani Hashemi, Hoasan; Mirzaui, Baman

2014-10-01

We developed and evaluated the utility of a quadruplex Taqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a Quadruplex Taqman real-time PCR assay and melting curve analysis. Representative Quadruplex Taqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis. Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods. Vancomycin resistant enterococci (VRE) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.

Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

PubMed

Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

2017-05-17

Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

Molecular Detection and Species-Specific Identification of Medically Important Aspergillus Species by Real-Time PCR in Experimental Invasive Pulmonary Aspergillosis ▿

PubMed Central

Walsh, Thomas J.; Wissel, Mark C.; Grantham, Kevin J.; Petraitiene, Ruta; Petraitis, Vidmantas; Kasai, Miki; Francesconi, Andrea; Cotton, Margaret P.; Hughes, Johanna E.; Greene, Lora; Bacher, John D.; Manna, Pradip; Salomoni, Martin; Kleiboeker, Steven B.; Reddy, Sushruth K.

2011-01-01

Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients. PMID:21976757

DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD

EPA Science Inventory

In spite of their importance in public health, the detection of fecal enterococci is performed via culturing methods that are time consuming and that are subject to inaccuracies that relate to their culturable status. In order to address these problems, a real time PCR (TaqMan) ...

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

PubMed

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

2012-08-01

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

Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy.

PubMed

Hoffmann, B; Freuling, C M; Wakeley, P R; Rasmussen, T B; Leech, S; Fooks, A R; Beer, M; Müller, T

2010-11-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease.

PubMed

Bohuski, Elizabeth; Lorch, Jeffrey M; Griffin, Kathryn M; Blehert, David S

2015-04-15

Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease. We developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targets the internal transcribed spacer region (ITS) of the fungal genome while the other targets the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required. These assays represent the first published methods to detect O. ophiodiicola by real-time PCR. The ITS assay has great utility for assisting with SFD diagnoses whereas the IGS assay offers a valuable tool for research-based applications.

Use of the Genomic Subtractive Hybridization Technique To Develop a Real-Time PCR Assay for Quantitative Detection of Prevotella spp. in Oral Biofilm Samples

PubMed Central

Nagashima, Shiori; Yoshida, Akihiro; Suzuki, Nao; Ansai, Toshihiro; Takehara, Tadamichi

2005-01-01

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species. PMID:15956428

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

PubMed

Davidson, Irit; Raibshtein, I; Al-Touri, A

2013-06-01

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

Real-time PCR for type-specific identification of herpes simplex in clinical samples: evaluation of type-specific results in the context of CNS diseases.

PubMed

Meylan, Sylvain; Robert, Daniel; Estrade, Christine; Grimbuehler, Valérie; Péter, Olivier; Meylan, Pascal R; Sahli, Roland

2008-02-01

HSV-1 and HSV-2 cause CNS infections of dissimilar clinico-pathological characteristics with prognostic and therapeutic implications. To validate a type-specific real-time PCR that uses MGB/LNA Taqman probes and to review the virologico-clinical data of 25 eligible patients with non-neonatal CNS infections. This real-time PCR was evaluated against conventional PCR (26 CSF and 20 quality controls), and LightCycler assay (51 mucocutaneous, 8 CSF and 32 quality controls) and culture/immunofluorescence (75 mucocutaneous) to assess typing with independent methods. Taqman real-time PCR detected 240 HSV genomes per ml CSF, a level appropriate for the management of patients, and provided unambiguous typing for the 104 positive (62 HSV-1 and 42 HSV-2) out the 160 independent clinical samples tested. HSV type diagnosed by Taqman real-time PCR predicted final diagnosis (meningitis versus encephalitis/meningoencephalitis, p<0.001) in 24/25 patients at time of presentation, in contrast to clinical evaluation. Our real-time PCR, as a sensitive and specific means for type-specific HSV diagnosis, provided rapid prognostic information for patient management.

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay.

PubMed

Röder, Martin; Vieths, Stefan; Holzhauser, Thomas

2011-01-24

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(®) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(®) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil.

PubMed

Fischer, Carlo; Torres, Maria C; Patel, Pranav; Moreira-Soto, Andres; Gould, Ernest A; Charrel, Rémi N; de Lamballerie, Xavier; Nogueira, Rita Maria Ribeiro; Sequeira, Patricia C; Rodrigues, Cintia D S; Kümmerer, Beate M; Drosten, Christian; Landt, Olfert; Bispo de Filippis, Ana Maria; Drexler, Jan Felix

2017-11-01

The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy▿ †

PubMed Central

Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.; Rasmussen, T. B.; Leech, S.; Fooks, A. R.; Beer, M.; Müller, T.

2010-01-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories. PMID:20739489

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

[Mission oriented diagnostic real-time PCR].

PubMed

Tomaso, Herbert; Scholz, Holger C; Al Dahouk, Sascha; Splettstoesser, Wolf D; Neubauer, Heinrich; Pfeffer, Martin; Straube, Eberhard

2007-01-01

In out of area military missions soldiers are potentially exposed to bacteria that are endemic in tropical areas and can be used as biological agents. It can be difficult to culture these bacteria due to sample contamination, low number of bacteria or pretreatment with antibiotics. Commercial biochemical identification systems are not optimized for these agents which can result in misidentification. Immunological assays are often not commercially available or not specific. Real-time PCR assays are very specific and sensitive and can shorten the time required to establish a diagnosis markedly. Therefore, real-time PCRs for the identification of Bacillus anthracis, Brucella spp., Burkholderia mallei und Burkholderia pseudomallei, Francisella tularensis und Yersinia pestis have been developed. PCR results can be false negative due to inadequate clinical samples, low number of bacteria in samples, DNA degradation, inhibitory substances and inappropriate DNA preparation. Hence, it is crucial to cultivate the organisms as a prerequisite for adequate antibiotic therapy and typing of the agent. In a bioterrorist scenario samples have to be treated according to rules applied in forensic medicine and documentation has to be flawless.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

PubMed

Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

2010-03-01

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

PubMed

Ponchel, Frederique; Toomes, Carmel; Bransfield, Kieran; Leong, Fong T; Douglas, Susan H; Field, Sarah L; Bell, Sandra M; Combaret, Valerie; Puisieux, Alain; Mighell, Alan J; Robinson, Philip A; Inglehearn, Chris F; Isaacs, John D; Markham, Alex F

2003-10-13

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

PubMed

Cura, Carolina I; Duffy, Tomas; Lucero, Raúl H; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J; Valencia Ayala, Edward; Kjos, Sonia A; Santalla, José; Mahaney, Susan M; Cayo, Nelly M; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S; Acosta Viana, Karla Y; Brutus, Laurent; Ocampo, Susana B; Aznar, Christine; Cuba Cuba, Cesar A; Gürtler, Ricardo E; Ramsey, Janine M; Ribeiro, Isabela; VandeBerg, John L; Yadon, Zaida E; Osuna, Antonio; Schijman, Alejandro G

2015-05-01

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

A real-time TaqMan polymerase chain reaction for the identification of Culex vectors of West Nile and Saint Louis encephalitis viruses in North America.

PubMed

Sanogo, Yibayiri O; Kim, Chang-Hyun; Lampman, Richard; Novak, Robert J

2007-07-01

In North America, West Nile and St. Louis encephalitis viruses have been detected in a wide range of vector species, but the majority of isolations continue to be from pools of mixed mosquitoes in the Culex subgenus Culex. Unfortunately, the morphologic identification of these important disease vectors is often difficult, particularly in regions of sympatry. We developed a sensitive real-time TaqMan polymerase chain reaction assay that allows reliable identification of Culex mosquitoes including Culex pipiens pipiens, Cx. p. quinquefasciatus, Cx. restuans, Cx. salinarius, Cx. nigripalpus, and Cx. tarsalis. Primers and fluorogenic probes specific to each species were designed based on sequences of the acetylcholinesterase gene (Ace2). Both immature and adult mosquitoes were successfully identified as individuals and as mixed species pools. This identification technique provides the basis for a rapid, sensitive, and high-throughput method for expounding the species-specific contribution of vectors to various phases of arbovirus transmission.

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds.

PubMed

Heuvelink, Annet; Hassan, Abdulwahed Ahmed; van Weering, Hilmar; van Engelen, Erik; Bülte, Michael; Akineden, Ömer

2017-05-01

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne's disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold's egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne's) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.

Real-time PCR detection chemistry.

PubMed

Navarro, E; Serrano-Heras, G; Castaño, M J; Solera, J

2015-01-15

Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review. Copyright © 2014 Elsevier B.V. All rights reserved.

EQUAL-quant: an international external quality assessment scheme for real-time PCR.

PubMed

Ramsden, Simon C; Daly, Sarah; Geilenkeuser, Wolf-Jochen; Duncan, Graeme; Hermitte, Fabienne; Marubini, Ettore; Neumaier, Michael; Orlando, Claudio; Palicka, Vladimir; Paradiso, Angelo; Pazzagli, Mario; Pizzamiglio, Sara; Verderio, Paolo

2006-08-01

Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR. The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number. The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants. This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

PubMed Central

Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.

2013-01-01

Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002

Searching for the best real-time RT-PCRs to detect Zika virus infections: the importance of comparing several protocols.

PubMed

de Moraes, F M; Espósito, D L A; Klein, T M; da Fonseca, B A L

2018-01-01

Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.

EVALUATION OF RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

EPA Science Inventory

Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan (trademark)) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glab...

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

PubMed Central

Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

2015-01-01

Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of Herpes simplex virus types 1 and 2 and Varicella-zoster virus.

PubMed

Weidmann, Manfred; Armbruster, Katrin; Hufert, Frank T

2008-08-01

To optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (IPC) concentration on the quantitative results of HSV1, HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1, HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays.

The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

PubMed

Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

2017-07-01

Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

PubMed

Salvi, Sergio; D'Orso, Fabio; Morelli, Giorgio

2008-06-25

Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.

A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

PubMed

Thomson, Kirsty; Yaaran, Tal; Belshaw, Alex; Curson, Lucia; Tisi, Laurence; Maurice, Sarah; Kiddle, Guy

2018-06-18

Ehrlichiosis is an important emerging infectious disease of the canid family and humans worldwide. To date, no extensive evaluation or validation of a molecular diagnostic test for ehrlichiosis has been published. Here, we present data for a newly designed TaqMan assay and compare its performance to a commercial technology (PCRun®). Both of these real-time methods of analysis were evaluated using a comprehensive number of prospective and retrospective samples collected from dogs exhibiting symptoms of ehrlichiosis. Whole blood samples collected from dogs, retrospectively in the United Kingdom and prospectively in Israel, were analysed for the presence of Ehrlichia canis and Ehrlichia minasensis DNA using the TaqMan PCR, developed specifically for this study. The results were compared to those of a real time commercial isothermal amplification method (PCRun® system developed by Biogal Galed Labs ACS, Galed, Israel). The sensitivity and specificity (CI: 95%) of the TaqMan PCR and PCRun® were both determined to be 100% and absolute, for all of the samples tested. Interestingly, both tests were demonstrated to be highly comparable, irrespective of differences in amplification chemistry or sequences targeted. Host differences, incidence of disease and geographical location of the isolates had little impact on the positivity recorded by each of the diagnostic methods. It was evident that both amplification methods were equally suited for diagnosing canine ehrlichiosis and while the PCRun® clearly amplified all clinically relevant Ehrlichia species known to infect dogs and humans, the TaqMan method was more specific for E. canis and E. minasensis. This work demonstrates that despite good analytical sensitivities and specificities for Ehrlichia spp. neither method could fully account for the clinical diagnosis of thrombocytopenia.

Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets.

PubMed

Babu, Binoy; Jeyaprakash, Ayyamperumal; Jones, Debra; Schubert, Timothy S; Baker, Carlye; Washburn, Brian K; Miller, Steven H; Poduch, Kristina; Knox, Gary W; Ochoa-Corona, Francisco M; Paret, Mathews L

2016-09-01

Rose rosette virus (RRV), belonging to the genus Emaravirus, is a highly destructive pathogen that causes rose rosette disease. The disease is a major concern for the rose industry in the U.S. due to the lack of highly sensitive methods for early detection of RRV. This is critical, as early identification of the infected plants and eradication is necessary in minimizing the risks associated with the spread of the disease. A highly reliable, specific and sensitive detection assay is thus required to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes; two of them (RRV_2-1 and RRV_2-2) based on the consensus sequences of the glycoprotein gene (RNA2) and the other two (RRV_3-2 and RRV_3-5) based on the nucleocapsid gene (RNA3) were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, and Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays using the in vitro transcripts (spiked with total RNA from healthy plants, and non-spiked) showed that all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility, with the primer/probe RRV_3-5 showing 100% positive detection, while RRV_2-1, RRV_2-2 and RRV_3-2 showed 90% positive detection. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV. Copyright © 2016 Elsevier B.V. All rights reserved.

EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

EPA Science Inventory

Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

Application of TaqMan fluorescent probe-based quantitative real-time PCR assay for the environmental survey of Legionella spp. and Legionella pneumophila in drinking water reservoirs in Taiwan.

PubMed

Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Ji, Wen-Tsai; Huang, Po-Hsiang; Hsueh, Chih-Jen; Chiang, Chuen-Sheue; Huang, Shih-Wei; Huang, Yu-Li

2014-08-15

In this study, TaqMan fluorescent quantitative real-time PCR was performed to quantify Legionella species in reservoirs. Water samples were collected from 19 main reservoirs in Taiwan, and 12 (63.2%) were found to contain Legionella spp. The identified species included uncultured Legionella spp., L. pneumophila, L. jordanis, and L. drancourtii. The concentrations of Legionella spp. and L. pneumophila in the water samples were in the range of 1.8×10(2)-2.6×10(3) and 1.6×10(2)-2.4×10(2) cells/L, respectively. The presence and absence of Legionella spp. in the reservoir differed significantly in pH values. These results highlight the importance that L. pneumophila, L. jordanis, and L. drancourtii are potential pathogens in the reservoirs. The presence of L. pneumophila in reservoirs may be a potential public health concern that must be further examined. Copyright © 2014 Elsevier B.V. All rights reserved.

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

PubMed Central

DeShields, Joseph B.; Bomberger, Rachel A.; Woodhall, James W.; Wheeler, David L.; Moroz, Natalia; Johnson, Dennis A.; Tanaka, Kiwamu

2018-01-01

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis. PMID:29553557

Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

USDA-ARS?s Scientific Manuscript database

TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another prim...

Real-time PCR and its application to mumps rapid diagnosis.

PubMed

Jin, L; Feng, Y; Parry, R; Cui, A; Lu, Y

2007-11-01

A real-time polymerase chain reaction assay was initially developed in China to detect mumps genome. The primers and TaqMan-MGB probe were selected from regions of the hemagglutinin gene of mumps virus. The primers and probe for the real-time PCR were evaluated by both laboratories in China and in the UK using three different pieces of equipment, LightCycler (Roche), MJ DNA Engine Option 2 (BIO-RAD) and TaqMan (ABI Prism) on different samples. The reaction was performed with either a one-step (China) or two-step (UK) process. The sensitivity (10 copies) was estimated using a serial dilution of constructed mumps-plasmid DNA and a linear standard curve was obtained between 10 and 10(7) DNA copies/reaction, which can be used to quantify viral loads. The detection limit on cell culture-grown virus was approximately 2 pfu/ml with a two-step assay on TaqMan, which was equivalent to the sensitivity of the nested PCR routinely used in the UK. The specificity was proved by testing a range of respiratory viruses and several genotypes of mumps strains. The concentration of primers and probe is 22 pmol and 6.25 or 7 pmol respectively for a 25 microl reaction. The assay took 3 hr from viral RNA extraction to complete the detection using any of the three pieces of equipment. Three hundred forty-one (35 in China and 306 in the UK) clinical specimens were tested, the results showing that this real-time PCR assay is suitable for rapid and accurate detection of mumps virus RNA in various types of clinical specimens. (c) 2007 Wiley-Liss, Inc.

Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ.

PubMed

Koponen, Jonna K; Turunen, Anna-Mari; Ylä-Herttuala, Seppo

2002-03-01

Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.

Evaluation of the Abbott RealTime HCV assay for quantitative detection of hepatitis C virus RNA.

PubMed

Michelin, Birgit D A; Muller, Zsofia; Stelzl, Evelyn; Marth, Egon; Kessler, Harald H

2007-02-01

The Abbott RealTime HCV assay for quantitative detection of HCV RNA has recently been introduced. In this study, the performance of the Abbott RealTime HCV assay was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HCV test. Accuracy, linearity, interassay and intra-assay variations were determined, and a total of 243 routine clinical samples were investigated. When accuracy of the new assay was tested, the majority of results were found to be within +/-0.5 log(10) unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve up to 1.0 x 10(6)IU/ml. The interassay variation ranged from 15% to 32%, and the intra-assay variation ranged from 5% to 8%. When clinical samples were tested by the Abbott RealTime HCV assay and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HCV test, the results for 93% of all samples with positive results by both tests were found to be within +/-1.0 log(10) unit. The viral loads for all patients measured by the Abbott and Roche assays showed a high correlation (R(2)=0.93); quantitative results obtained by the Abbott assay were found to be lower than those obtained by the Roche assay. The Abbott RealTime HCV assay proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.

TaqMan RT-PCR and VERSANT HIV-1 RNA 3.0 (bDNA) assay Quantification of HIV-1 RNA viral load in breast milk.

PubMed

Israel-Ballard, Kiersten; Ziermann, Rainer; Leutenegger, Christian; Di Canzio, James; Leung, Kimmy; Strom, Lynn; Abrams, Barbara; Chantry, Caroline

2005-12-01

Transmission of HIV via breast milk is a primary cause of pediatric HIV infection in developing countries. Reliable methods to detect breast milk viral load are important. To correlate the ability of the VERSANT HIV 3.0 (bDNA) assay to real-time (RT) TaqMan PCR in quantifying breast milk HIV-1 RNA. Forty-six breast milk samples that had been spiked with cell-free HIV-1 and eight samples spiked with cell-associated HIV-1 were assayed for HIV-1 RNA by both VERSANT HIV 3.0 and TaqMan RNA assays. Only assays on the cell-free samples were statistically compared. Both a Deming regression slope and a Bland-Altman slope indicated a linear relationship between the two assays. TaqMan quantitations were on average 2.6 times higher than those of HIV 3.0. A linear relationship was observed between serial dilutions of spiked cell-free HIV-1 and both the VERSANT HIV 3.0 and the TaqMan RNA assays. The two methods correlated well although the VERSANT HIV 3.0 research protocol quantified HIV-1 RNA slightly lower than TaqMan.

The L2b real-time PCR targeting the pmpH gene of Chlamydia trachomatis used for the diagnosis of lymphogranuloma venereum is not specific to L2b strains.

PubMed

Touati, A; Peuchant, O; Hénin, N; Bébéar, C; de Barbeyrac, B

2016-06-01

The French Reference Centre for chlamydiae uses two real-time PCRs targeting the pmpH gene of Chlamydia trachomatis to differentiate between L strains and variant L2b, responsible for a lymphogranuloma venereum outbreak in Europe. We compared the results obtained for 122 L2b C. trachomatis-positive specimens, using the two real-time PCRs, with the sequencing of the ompA gene. Only 91 specimens were confirmed as L2b. Our results demonstrate that the lymphogranuloma venereum outbreak is no longer dominated by the variant L2b, and that many L-positive specimens were misidentified as L2b with the method used, which raises the question of its specificity. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Comparison of the Abbott RealTime HCV and Roche COBAS Ampliprep/COBAS TaqMan HCV assays for the monitoring of sofosbuvir-based therapy.

PubMed

Ogawa, Eiichi; Furusyo, Norihiro; Murata, Masayuki; Shimizu, Motohiro; Toyoda, Kazuhiro; Hotta, Taeko; Uchiumi, Takeshi; Hayashi, Jun

2017-01-01

On-treatment HCV kinetics play an invaluable role in evaluating the efficacy of interferon-based therapies. However, the importance of HCV RNA monitoring has not been well discussed concerning treatment with sofosbuvir (SOF)-based regimens, especially for the utility of the Abbott RealTime HCV (ART) assay. This study consisted of 151 patients infected with HCV genotype-1 or -2, including patients with prior treatment-experience or cirrhosis. HCV genotype-1 patients were treated with SOF/ledipasvir and genotype-2 patients with SOF/ribavirin, both for 12 weeks. Serial measurements of HCV RNA were performed with both the ART and COBAS AmpliPrep/COBAS TaqMan v2.0 (CAP/CTM) assays simultaneously at weeks 0, 1, 2, 4, 6, 8, 10 and 12 of treatment. The rates of HCV RNA target not detected (TND) by ART were significantly lower than those by CAP/CTM between weeks 2 and 12 (end of treatment [EOT]), irrespective of prior treatment-experience or cirrhosis. 11 (11.6%) genotype-1 and 8 (14.3%) genotype-2 patients did not achieve HCV RNA TND by ART at EOT, in contrast to all having HCV RNA TND by CAP/CTM; however, all achieved sustained virological response. The time at which HCV RNA became TND or unquantifiable was not associated with treatment outcome by either the ART or CAP/CTM assay. Over 10% of the patients continued to have detectable HCV RNA by ART at EOT, irrespective of HCV genotype, prior treatment-experience and/or cirrhosis. However, prolonged residual HCV RNA was not associated with treatment failure.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

PubMed Central

Ramírez, Juan Carlos; Cura, Carolina Inés; Moreira, Otacilio da Cruz; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Guedes, Paulo Marcos da Matta; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Galvão, Lúcia Maria da Cunha; da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; de Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

2015-01-01

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. PMID:26320872

Detection of the free living amoeba Naegleria fowleri by using conventional and real-time PCR based on a single copy DNA sequence.

PubMed

Régoudis, Estelle; Pélandakis, Michel

2016-02-01

The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify the N. fowleri cells. The real-time PCR results give a detection limit of 1 copy per reaction with high reproducibility without the need of a Taqman probe. This procedure is of interest as compared to other procedures which are mostly based on the detection of multi-copy DNA associated with a Taqman probe. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece

PubMed Central

2011-01-01

Background HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. Methods In this study, the Abbott RealTime HIV-1 (Abbott RealTime) assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan) and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0) assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. Results A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD) difference of -0.206 (0.298) log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log10 copies/ml, respectively. Overall, differences were less than 0.5 log10 for 85% of the samples, and >1 log10 in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD) difference: 0.160 (0.287) log10 copies/ml). The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log10 copies/ml, respectively. Overall, the majority of samples (86%) differed by less than 0.5 log10, while none of the samples showed a deviation of more than 1.0 log10. Conclusions In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3.0 assays, for all

Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

USDA-ARS?s Scientific Manuscript database

The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

PubMed

Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

2017-07-01

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for

Novel PCRs for differential diagnosis of cestodes.

PubMed

Roelfsema, Jeroen H; Nozari, Nahid; Pinelli, Elena; Kortbeek, Laetitia M

2016-02-01

Cestodes or tapeworms belong to a diverse group of helminths. The adult Taenia saginata and Taenia solium tapeworm can infest the human gut and the larval stage of Echinococcus spp. and T. solium can infect tissues of the human body, causing serious disease. Molecular diagnostics can be performed on proglottids, eggs and on cyst fluids taken by biopsy. Detection of cestodes when a helminthic infection is suspected is of vital importance and species determination is required for appropriate patient care. For routine diagnostics a single test that is able to detect and type a range of cestodes is preferable. We sought to improve our diagnostic procedure that used to rely on PCR and subsequent sequencing of the Cox1 and Nad1 genes. We have compared these PCRs with novel PCRs on the 12S rRNA and Nad5 gene and established the sensitivity and specificity. A single PCR on the 12S gene proved to be very suitable for detection and specification of Taenia sp. and Echinococcus sp. Both targets harbour enough polymorphic sites to determine the various Echinococcus species. The 12S PCR was most sensitive of all tested. Copyright © 2015 Elsevier Inc. All rights reserved.

Development and first evaluation of a novel multiplex real-time PCR on whole blood samples for rapid pathogen identification in critically ill patients with sepsis.

PubMed

van de Groep, Kirsten; Bos, Martine P; Savelkoul, Paul H M; Rubenjan, Anna; Gazenbeek, Christel; Melchers, Willem J G; van der Poll, Tom; Juffermans, Nicole P; Ong, David S Y; Bonten, Marc J M; Cremer, Olaf L

2018-04-26

Molecular tests may enable early adjustment of antimicrobial therapy and be complementary to blood culture (BC) which has imperfect sensitivity in critically ill patients. We evaluated a novel multiplex real-time PCR assay to diagnose bloodstream pathogens directly in whole blood samples (BSI-PCR). BSI-PCR included 11 species- and four genus-specific PCRs, a molecular Gram-stain PCR, and two antibiotic resistance markers. We collected 5 mL blood from critically ill patients simultaneously with clinically indicated BC. Microbial DNA was isolated using the Polaris method followed by automated DNA extraction. Sensitivity and specificity were calculated using BC as reference. BSI-PCR was evaluated in 347 BC-positive samples (representing up to 50 instances of each pathogen covered by the test) and 200 BC-negative samples. Bacterial species-specific PCR sensitivities ranged from 65 to 100%. Sensitivity was 26% for the Gram-positive PCR, 32% for the Gram-negative PCR, and ranged 0 to 7% for yeast PCRs. Yeast detection was improved to 40% in a smaller set-up. There was no overall association between BSI-PCR sensitivity and time-to-positivity of BC (which was highly variable), yet Ct-values were lower for true-positive versus false-positive PCR results. False-positive results were observed in 84 (4%) of the 2200 species-specific PCRs in 200 culture-negative samples, and ranged from 0 to 6% for generic PCRs. Sensitivity of BSI-PCR was promising for individual bacterial pathogens, but still insufficient for yeasts and generic PCRs. Further development of BSI-PCR will focus on improving sensitivity by increasing input volumes and on subsequent implementation as a bedside test.

Investigating the cost implications of including all respiratory medicines in PCRS schemes.

PubMed

O'Dwyer, Jackie; Murphy, Aileen

2018-02-01

This study estimates the additional cost to the State to pay for all respiratory medicines through the Primary Care Reimbursement Service (PCRS) schemes, reducing cost barriers to medication as a complement to existing chronic disease management programmes. Previous literature found higher medication adherence rates amongst medical card patients than those that had to pay or co-pay themselves. A review of medication expenditure on the PCRS schemes from 2005 to 2015. Data on medicines sold into and out of pharmacies was used to estimate the proportion to PCRS schemes or private. Scenario analyses were conducted to estimate what the cost to the State would be to provide funding for all respiratory medicines. Trend analysis findings showed that respiratory medicines have been less than 10% of total PCRS medicine expenditure for the years reviewed. The largest portion of the respiratory medicine expenditure is allocated to 'drugs for obstructive pulmonary disorder' (OPD), ranging from 90% in 2005 to 69% in 2015. Eighty-seven per cent of drugs to treat OPD are dispensed publicly and 13% privately. A scenario analysis estimated that the extra cost to the State to be €20.2 m. Respiratory disease is included in the Irish Government's chronic disease management programme. This aims to deliver optimal care in the most appropriate setting so as to improve health outcomes and quality of life. Medication adherence is imperative to achieving these aims. Reducing cost barriers as a complement to other initiatives may improve medicine adherence thereby improving the effectiveness of disease management and patient outcomes.

A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.

PubMed

Jothikumar, Narayanan; Cromeans, Theresa L; Robertson, Betty H; Meng, X J; Hill, Vincent R

2006-01-01

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.

Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

PubMed

Nguyen, T L; Lim, Y J; Kim, D-H; Austin, B

2016-01-01

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples. © 2014 John Wiley & Sons Ltd.

Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR.

PubMed

Donia, D; Divizia, M; Pana', A

2005-06-01

Armored Enterovirus RNA was used to standardize a real-time reverse transcription (RT)-PCR for environmental testing. Armored technology is a system to produce a robust and stable RNA standard, trapped into phage proteins, to be used as internal control. The Armored Enterovirus RNA protected sequence includes 263 bp of highly conserved sequences in 5' UTR region. During these tests, Armored RNA has been used to produce a calibration curve, comparing three different fluorogenic chemistry: TaqMan system, Syber Green I and Lux-primers. The effective evaluation of three amplifying commercial reagent kits, in use to carry out real-time RT-PCR, and several extraction procedures of protected viral RNA have been carried out. The highest Armored RNA recovery was obtained by heat treatment while chemical extraction may decrease the quantity of RNA. The best sensitivity and specificity was obtained using the Syber Green I technique since it is a reproducible test, easy to use and the cheapest one. TaqMan and Lux-primer assays provide good RT-PCR efficiency in relationship to the several extraction methods used, since labelled probe or primer request in these chemistry strategies, increases the cost of testing.

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.

PubMed

Sankuntaw, Nipaporn; Sukprasert, Saovaluk; Engchanil, Chulapan; Kaewkes, Wanlop; Chantratita, Wasun; Pairoj, Vantanit; Lulitanond, Viraphong

2011-01-01

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of a real-time clinical decision support system upon the Web MVC-based architecture for prostate cancer treatment.

PubMed

Lin, Hsueh-Chun; Wu, Hsi-Chin; Chang, Chih-Hung; Li, Tsai-Chung; Liang, Wen-Miin; Wang, Jong-Yi Wang

2011-03-08

A real-time clinical decision support system (RTCDSS) with interactive diagrams enables clinicians to instantly and efficiently track patients' clinical records (PCRs) and improve their quality of clinical care. We propose a RTCDSS to process online clinical informatics from multiple databases for clinical decision making in the treatment of prostate cancer based on Web Model-View-Controller (MVC) architecture, by which the system can easily be adapted to different diseases and applications. We designed a framework upon the Web MVC-based architecture in which the reusable and extractable models can be conveniently adapted to other hospital information systems and which allows for efficient database integration. Then, we determined the clinical variables of the prostate cancer treatment based on participating clinicians' opinions and developed a computational model to determine the pretreatment parameters. Furthermore, the components of the RTCDSS integrated PCRs and decision factors for real-time analysis to provide evidence-based diagrams upon the clinician-oriented interface for visualization of treatment guidance and health risk assessment. The resulting system can improve quality of clinical treatment by allowing clinicians to concurrently analyze and evaluate the clinical markers of prostate cancer patients with instantaneous clinical data and evidence-based diagrams which can automatically identify pretreatment parameters. Moreover, the proposed RTCDSS can aid interactions between patients and clinicians. Our proposed framework supports online clinical informatics, evaluates treatment risks, offers interactive guidance, and provides real-time reference for decision making in the treatment of prostate cancer. The developed clinician-oriented interface can assist clinicians in conveniently presenting evidence-based information to patients and can be readily adapted to an existing hospital information system and be easily applied in other chronic diseases.

Design and Development of a Quantitative TaqMan Real-Time PCR Assay for Evaluation of HIV-1 (group M) Viral Load in Plasma Using Armored RNA Standard.

PubMed

Gholami, Mohammad; Baesi, Kazem; Rouzbahani, Negin H; Mohraz, Minoo

2018-06-01

Human immunodeficiency virus-1 (HIV-1) is a viral infectious agent that gradually extinguishes the immune system, resulting in the acquired immune deficiency syndrome (AIDS). The aim of this study was to develop a TaqMan based detection assay to evaluate HIV-1 plasma viral load and to construct a stable internal positive control (IPC) and external positive control (EPC) RNA based on Armored RNA (AR) technology. The MS2 maturase, coat protein gene and HIV-1 pol gene were cloned in pET-32a plasmid. The recently fabricated recombinant plasmid was transformed into Escherichia coli strain BL2 (DE3) and protein expression and Armored RNA was fabricated in presence of isopropyl-L-thio-D-galactopyranoside (IPTG). The Armored RNA was precipitated and purified by polyethylene glycol (PEG) and sephacryl S-200 chromatography. The stability of Armored RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy (TEM) and gel agarose electrophoresis. The specificity, sensitivity, inter- and intra-day precision, and the dynamic range of the assay were experimentally determined. The AR was stable in presence of ribonuclease, and the assay had a dynamic detection range from 101 to 105 copies of AR. The coefficient of variation (CV) was 4.8% for intra-assay and 5.8% for inter-assay precision. Clinical specificity and sensitivity of the assay were assessed at 100% and 96.66%, respectively. The linear regression analysis confirmed a high correlation between the in-house and the commercial assay, Real Star HIV-1-qRTPCR, respectively (R2 = 0.888). The AR standard is non-infectious and highly resistant in the presence of ribonuclease. The TaqMan assay developed is able to quantify HIV viral load based on a novel conserved region of HIV-1 pol gene which has minimal sequence inconsistency.

Real-Time PCR Analysis of Vibrio vulnificus from Oysters

PubMed Central

Campbell, Mark S.; Wright, Anita C.

2003-01-01

Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (102 to 108 CFU ml−1), with a lower limit of 72 fg of genomic DNA μl of PCR mixture−1 or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r2 = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood. PMID:14660359

One step screening of retroviral producer clones by real time quantitative PCR.

PubMed

Towers, G J; Stockholm, D; Labrousse-Najburg, V; Carlier, F; Danos, O; Pagès, J C

1999-01-01

Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.

Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.

PubMed

de Wit, C; Fautz, C; Xu, Y

2000-09-01

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell

Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

PubMed

Kho, S L; Chua, K H; George, E; Tan, J A M A

2013-07-15

Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Förster resonance energy transfer probes in 5′-nuclease assays

PubMed Central

Kutyavin, Igor V.

2010-01-01

The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5′-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem–loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5′-nuclease activity of Taq. The stem–loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5′-flap sequence. It was found that at a certain length of these 5′-flap sequences the folded Snake amplicons have very little, if any, effect on PCR yield but benefit many aspects of the detection process, particularly the signal productivity. Unlike Taqman, the Snake system favors the use of short FRET probes with improved fluorescence background. The head-to-head comparison study of Snake and Taqman revealed that these two technologies have more differences than similarities with respect to their responses to changes in PCR protocol, e.g. the variations in primer concentration, annealing time, PCR asymmetry. The optimal PCR protocol for Snake has been identified. The technology’s real-time performance was compared to a number of conventional assays including Taqman, 3′-MGB-Taqman, Molecular Beacon and Scorpion primers. The test trial showed that Snake supersedes the conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphisms. Due to the assay’s cost-effectiveness and simplicity of design, the technology is anticipated to quickly replace all known conventional methods currently used for real-time nucleic acid detection

Development of a real-time clinical decision support system upon the web mvc-based architecture for prostate cancer treatment

PubMed Central

2011-01-01

Background A real-time clinical decision support system (RTCDSS) with interactive diagrams enables clinicians to instantly and efficiently track patients' clinical records (PCRs) and improve their quality of clinical care. We propose a RTCDSS to process online clinical informatics from multiple databases for clinical decision making in the treatment of prostate cancer based on Web Model-View-Controller (MVC) architecture, by which the system can easily be adapted to different diseases and applications. Methods We designed a framework upon the Web MVC-based architecture in which the reusable and extractable models can be conveniently adapted to other hospital information systems and which allows for efficient database integration. Then, we determined the clinical variables of the prostate cancer treatment based on participating clinicians' opinions and developed a computational model to determine the pretreatment parameters. Furthermore, the components of the RTCDSS integrated PCRs and decision factors for real-time analysis to provide evidence-based diagrams upon the clinician-oriented interface for visualization of treatment guidance and health risk assessment. Results The resulting system can improve quality of clinical treatment by allowing clinicians to concurrently analyze and evaluate the clinical markers of prostate cancer patients with instantaneous clinical data and evidence-based diagrams which can automatically identify pretreatment parameters. Moreover, the proposed RTCDSS can aid interactions between patients and clinicians. Conclusions Our proposed framework supports online clinical informatics, evaluates treatment risks, offers interactive guidance, and provides real-time reference for decision making in the treatment of prostate cancer. The developed clinician-oriented interface can assist clinicians in conveniently presenting evidence-based information to patients and can be readily adapted to an existing hospital information system and be easily

Concordance of HIV-1 RNA Values by Amplicor and TaqMan 2.0 in Patients With Confirmed Suppression in Clinical Trials

PubMed Central

Garner, Will; White, Kirsten; Szwarcberg, Javier; McCallister, Scott; Zhong, Lijie; Wulfsohn, Mike

2016-01-01

Background. The COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5 (Amplicor) has been replaced with the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0 (TaqMan 2.0), a real-time polymerase chain reaction human immunodeficiency virus type 1 (HIV-1) assay with higher sensitivity and broader dynamic range. HIV-1 RNA values at the 50 copies/mL cutoff drive major patient management decisions and clinical study outcomes. Methods. A total of 2217 samples were collected from 1922 HIV-1–infected subjects taking antiretroviral therapy for at least 48 weeks and had at least 2 consecutive samples with HIV-1 RNA <50 copies/mL by Amplicor from 7 recent clinical trials. HIV-1 RNA results were obtained from the Amplicor and TaqMan 2.0 assays in parallel by a reference laboratory. Results. The overall concordance between assay results was 96% at the cutoff of 50 copies/mL. However, statistically significant discordance at the 50 copies/mL cutoff was found between the assays for 3.9% of samples (n = 87). By TaqMan 2.0, virologic failure defined as HIV-1 RNA ≥50 copies/mL was reported for 2.8% more samples than Amplicor. Of these 87 samples, 68 samples fell within the predicted range of assay variability. Retesting of HIV-1 RNA by TaqMan 2.0 confirmed the discordance in only 28 of the 87 samples. Conclusions. The TaqMan 2.0 assay reports fewer subjects below the clinical endpoint of HIV-1 RNA <50 copies/mL in HIV clinical trials than the Amplicor assay. This difference must be considered when assessing disease progression, designing clinical trials, and comparisons with historical trials that used the Amplicor assay. PMID:26689956

Clinical evaluation of β-tubulin real-time PCR for rapid diagnosis of dermatophytosis, a comparison with mycological methods.

PubMed

Motamedi, Marjan; Mirhendi, Hossein; Zomorodian, Kamiar; Khodadadi, Hossein; Kharazi, Mahboobeh; Ghasemi, Zeinab; Shidfar, Mohammad Reza; Makimura, Koichi

2017-10-01

Following our previous report on evaluation of the beta tubulin real-time PCR for detection of dermatophytosis, this study aimed to compare the real-time PCR assay with conventional methods for the clinical assessment of its diagnostic performance. Samples from a total of 853 patients with suspected dermatophyte lesions were subjected to direct examination (all samples), culture (499 samples) and real-time PCR (all samples). Fungal DNA was extracted directly from clinical samples using a conical steel bullet, followed by purification with a commercial kit and subjected to the Taq-Man probe-based real-time PCR. The study showed that among the 499 specimens for which all three methods were used, 156 (31.2%), 128 (25.6%) and 205 (41.0%) were found to be positive by direct microscopy, culture and real-time PCR respectively. Real-time PCR significantly increased the detection rate of dermatophytes compared with microscopy (288 vs 229) with 87% concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of the real-time PCR was 87.5%, 85%, 66.5% and 95.2% respectively. Although real-time PCR performed better on skin than on nail samples, it should not yet fully replace conventional diagnosis. © 2017 Blackwell Verlag GmbH.

Validation of endogenous internal real-time PCR controls in renal tissues.

PubMed

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R; Mrug, Michal

2009-01-01

Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. Copyright 2009 S. Karger AG, Basel.

Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual Test in Kenya

PubMed Central

Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

2016-01-01

Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0–100.0) and 100.0% (95% CI: 96.0–100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0–100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9 copies/mL at 95% CIs (p = 0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants. PMID:24726703

Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya.

PubMed

Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

2014-08-01

Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0-100.0) and 100.0% (95% CI: 96.0-100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0-100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9copies/mL at 95% CIs (p=0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants. Published by Elsevier B.V.

Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen, Erwinia amylovora

PubMed Central

Barbé, Silvia; Bertolini, Edson; Roselló, Montserrat; Llop, Pablo

2014-01-01

Erwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 103 cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 102 cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora. PMID:24509928

Conventional and real-time PCRs for detection of Erwinia piriflorinigrans allow its distinction from the fire blight pathogen, Erwinia amylovora.

PubMed

Barbé, Silvia; Bertolini, Edson; Roselló, Montserrat; Llop, Pablo; López, María M

2014-04-01

Erwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 10(3) cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10(2) cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora.

Avian-specific real-time PCR assay for authenticity control in farm animal feeds and pet foods.

PubMed

Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario

2014-01-01

A highly sensitive TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for detection of an avian-specific DNA fragment (68bp) in farm animal and pet feeds. The specificity of the assay was verified against a wide representation of animal and plant species. Applicability assessment of the avian real-time PCR was conducted through representative analysis of two types of compound feeds: industrial farm animal feeds (n=60) subjected to extreme temperatures, and commercial dog and cat feeds (n=210). Results obtained demonstrated the suitability of the real-time PCR assay to detect the presence of low percentages of highly processed avian material in the feed samples analysed. Although quantification results were well reproducible under the experimental conditions tested, an accurate estimation of the target content in feeds is impossible in practice. Nevertheless, the method may be useful as an alternative tool for traceability purposes within the framework of feed control. Copyright © 2013 Elsevier Ltd. All rights reserved.

Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.

PubMed

Pitetti, Raymond D; Laus, Stella; Wadowsky, Robert M

2003-08-01

Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all

Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods.

PubMed

Vaïtilingom, M; Pijnenburg, H; Gendre, F; Brignon, P

1999-12-01

A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

PubMed

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

Single reaction, real time RT-PCR detection of all known avian and human metapneumoviruses.

PubMed

Lemaitre, E; Allée, C; Vabret, A; Eterradossi, N; Brown, P A

2018-01-01

Current molecular methods for the detection of avian and human metapneumovirus (AMPV, HMPV) are specifically targeted towards each virus species or individual subgroups of these. Here a broad range SYBR Green I real time RT-PCR was developed which amplified a highly conserved fragment of sequence in the N open reading frame. This method was sufficiently efficient and specific in detecting all MPVs. Its validation according to the NF U47-600 norm for the four AMPV subgroups estimated low limits of detection between 1000 and 10copies/μL, similar with detection levels described previously for real time RT-PCRs targeting specific subgroups. RNA viruses present a challenge for the design of durable molecular diagnostic test due to the rate of change in their genome sequences which can vary substantially in different areas and over time. The fact that the regions of sequence for primer hybridization in the described method have remained sufficiently conserved since the AMPV and HMPV diverged, should give the best chance of continued detection of current subgroups and of potential unknown or future emerging MPV strains. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

PubMed

van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

2016-01-01

Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels. © 2015 John Wiley & Sons Ltd.

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

PubMed

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

PubMed Central

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna MY; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease. PMID:26011746

Multicenter Comparative Evaluation of Five Commercial Methods for Toxoplasma DNA Extraction from Amniotic Fluid▿

PubMed Central

Yera, H.; Filisetti, D.; Bastien, P.; Ancelle, T.; Thulliez, P.; Delhaes, L.

2009-01-01

Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis. PMID:19846633

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.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

PubMed

Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

2016-03-01

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Real-Time PCR for the Detection of Precise Transgene Copy Number in Wheat.

PubMed

Giancaspro, Angelica; Gadaleta, Agata; Blanco, Antonio

2017-01-01

Despite the unceasing advances in genetic transformation techniques, the success of common delivery methods still lies on the behavior of the integrated transgenes in the host genome. Stability and expression of the introduced genes are influenced by several factors such as chromosomal location, transgene copy number and interaction with the host genotype. Such factors are traditionally characterized by Southern blot analysis, which can be time-consuming, laborious, and often unable to detect the exact copy number of rearranged transgenes. Recent research in crop field suggests real-time PCR as an effective and reliable tool for the precise quantification and characterization of transgene loci. This technique overcomes most problems linked to phenotypic segregation analysis and can analyze hundreds of samples in a day, making it an efficient method for estimating a gene copy number integrated in a transgenic line. This protocol describes the use of real-time PCR for the detection of transgene copy number in durum wheat transgenic lines by means of two different chemistries (SYBR ® Green I dye and TaqMan ® probes).

Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.

PubMed

Depuydt, C E; Boulet, G A V; Horvath, C A J; Benoy, I H; Vereecken, A J; Bogers, J J

2007-01-01

The causal relationship between persistent infection with high-risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type-specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type-specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow-up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type-specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type-specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type-specific PCRs could be used in a clinical setting as a reliable screening tool.

A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

PubMed

Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

2017-11-15

A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

Use of internally controlled real-time genome amplification for detection of variola virus and other orthopoxviruses infecting humans.

PubMed

Fedele, C G; Negredo, A; Molero, F; Sánchez-Seco, M P; Tenorio, A

2006-12-01

Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3'-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.

Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.

PubMed

Zarrinfar, Hossein; Makimura, Koichi; Satoh, Kazuo; Khodadadi, Hossein; Mirhendi, Hossein

2013-05-01

Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group. © 2013 Wiley Periodicals, Inc.

Novel and highly sensitive sybr® green real-time pcr for poxvirus detection in odontocete cetaceans.

PubMed

Sacristán, Carlos; Luiz Catão-Dias, José; Ewbank, Ana Carolina; Machado, Eduardo Ferreira; Neves, Elena; Santos-Neto, Elitieri Batista; Azevedo, Alexandre; Laison-Brito, José; De Castilho, Pedro Volkmer; Daura-Jorge, Fábio Gonçalves; Simões-Lopes, Paulo César; Carballo, Matilde; García-Párraga, Daniel; Manuel Sánchez-Vizcaíno, José; Esperón, Fernando

2018-06-08

Poxviruses are emerging pathogens in cetaceans, temporarily named 'Cetaceanpoxvirus' (CePV, family Poxviridae), classified into two main lineages: CePV-1 in odontocetes and CePV-2 in mysticetes. Only a few studies performed the molecular detection of CePVs, based on DNA-polymerase gene and/or DNA-topoisomerase I gene amplification. Herein we describe a new real-time PCR assay based on SYBR ® Green and a new primer set to detect a 150 bp fragment of CePV DNA-polymerase gene, also effective for conventional PCR detection. The novel real-time PCR was able to detect 5 up to 5 × 10 6 copies per reaction of a cloned positive control. Both novel PCR methods were 1000 to 100,000-fold more sensitive than those previously described in the literature. Samples of characteristic poxvirus skin lesions ('tattoo') from one Risso's dolphin (Grampus griseus), two striped dolphins (Stenella coeruleoalba) and two Guiana dolphins (Sotalia guianensis) were all positive to both our novel real time- and conventional PCR methods, even though three of these animals (a Risso's dolphin, a striped dolphin, and a Guiana dolphin) were previously negative to the conventional PCRs previously available. To our knowledge, this is the first real-time PCR detection method for Cetaceanpoxvirus, a much more sensitive tool for the detection of CePV-1 infections. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

PubMed Central

Schaeffer, Anke; Henrich, Birgit

2008-01-01

Background Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men. Results The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1) -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H) -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male. Conclusion This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies. PMID:18447917

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

PubMed Central

Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

2006-01-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages. PMID:16957227

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.

PubMed

Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

2006-09-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

Detection of adulterated murine components in meat products by TaqMan© real-time PCR.

PubMed

Fang, Xin; Zhang, Chi

2016-02-01

Using murine meat to substitute mutton has been identified as a new type of meat fraud in China, yet no detection method for murine species has been reported. Here, three kinds of rodent were used as target species to establish a murine-specific real-time PCR method of detection. The mitochondrial cytochrome b gene (cytb) of each target was sequenced and a TaqMan probe was designed based on the cytb. Simultaneously, an internal positive control (IPC) plasmid along with its respective probe were designed to monitor the PCR reaction. As a result, the duplex real-time PCR system was verified to be specific. The limit of detection (LOD) was lower than 1 pg of DNA per reaction and 0.1% murine contamination in meat mixtures. Standard curves were generated for a quantitative analysis. Thus, this study provided a new tool to control the quality of meat products for official and third-party laboratories. Copyright © 2015. Published by Elsevier Ltd.

Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

PubMed Central

Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

2014-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

A handheld real time thermal cycler for bacterial pathogen detection.

PubMed

Higgins, James A; Nasarabadi, Shanavaz; Karns, Jeffrey S; Shelton, Daniel R; Cooper, Mary; Gbakima, Aiah; Koopman, Ronald P

2003-08-15

The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.

Development of duplex real-time RT-PCR based on Taqman technology for detecting simultaneously the genome of pan-enterovirus and enterovirus 71.

PubMed

Hwang, Seoyeon; Kang, Byunghak; Hong, Jiyoung; Kim, Ahyoun; Kim, Hyejin; Kim, Kisang; Cheon, Doo-Sung

2013-07-01

Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks. Copyright © 2013 Wiley Periodicals, Inc.

[Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].

PubMed

Zhang, Qi-wei; You, Shang-you; Sun, Ji-min; Wu, Qi; Yu, Chun-hua; Zhang, Chu-yu

2005-07-01

To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

Development of a real-time quantitative PCR assay to enumerate Yersinia pestis in fleas.

PubMed

Gabitzsch, Elizabeth S; Vera-Tudela, Rommelle; Eisen, Rebecca J; Bearden, Scott W; Gage, Kenneth L; Zeidner, Nordin S

2008-07-01

A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.

Real-time PCR detection of Vibrio vulnificus in oysters: comparison of oligonucleotide primers and probes targeting vvhA.

PubMed

Panicker, Gitika; Bej, Asim K

2005-10-01

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C(T)) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10(3) V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 x 10(3) CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

PubMed Central

LOPES, Estela Gallucci; GERALDO, Carlos Alberto; MARCILI, Arlei; SILVA, Ricardo Duarte; KEID, Lara Borges; OLIVEIRA, Trícia Maria Ferreira da Silva; SOARES, Rodrigo Martins

2016-01-01

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs. PMID:27253743

Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans▿

PubMed Central

Fedele, C. G.; Negredo, A.; Molero, F.; Sánchez-Seco, M. P.; Tenorio, A.

2006-01-01

Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3′-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube. PMID:17065259

The tracing of mycobacteria in drinking water supply systems by culture, conventional, and real time PCRs.

PubMed

Klanicova, Barbora; Seda, Jaromir; Slana, Iva; Slany, Michal; Pavlik, Ivo

2013-12-01

Mycobacteria are widely present in diverse aquatic habitats, where they can survive for months or years while some species can even proliferate. The resistance of different mycobacterial species to disinfection methods like chlorination or ozonation could result in their presence in the final tap water of consumers. In this study, the culture method, Mycobacterium tuberculosis complex conventional duplex PCR for detection of non-tuberculous mycobacteria (NTM) and quantitative real-time PCR (qPCR) to detect three subspecies of M. avium species (M. a. avium, M. a. hominissuis, and M. a. paratuberculosis) were used to trace their possible path of transmission from the watershed through the reservoir and drinking water plant to raw drinking water and finally to households. A total of 124 samples from four drinking water supply systems in the Czech Republic, 52 dam sediments, 34 water treatment plant sludge samples, and 38 tap water household sediments, were analyzed. NTM of 11 different species were isolated by culture from 42 (33.9 %) samples; the most prevalent were M. gordonae (16.7 %), M. triplex (14.3 %), M. lentiflavum (9.5 %), M. a. avium (7.1 %), M. montefiorenase (7.1 %), and M. nonchromogenicum (7.1 %). NTM DNA was detected in 92 (76.7 %) samples. By qPCR analysis a statistically significant decrease (P < 0.01) was observed along the route from the reservoir (dam sediments), through water treatment sludge and finally to household sediments. The concentrations ranged from 10(0) to 10(4) DNA cells/g. It was confirmed that drinking water supply systems (watershed-reservoir-drinking water treatment plant-household) might be a potential transmission route for mycobacteria.

Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus.

PubMed

Letellier, C; Kerkhofs, P

2003-12-01

Since two genotypes of bovine viral diarrhea viruses (BVDV) occur in Belgian herds, their differentiation is important for disease surveillance. A quantitative real-time PCR assay was developed to detect and classify bovine viral diarrhea viruses in genotype I and II. A pair of primers specific for highly conserved regions of the 5'UTR and two TaqMan probes were designed. The FAM and VIC-labeled probe sequences differed by three nucleotides, allowing the differentiation between genotype I and II. The assay detectability of genotype I and II real-time PCR assay was 1000 and 100 copies, respectively. Highly reproducible data were obtained as the coefficients of variation of threshold cycle values in inter-runs were less than 2.2%. The correct classification of genotype I and II viruses was assessed by using reference strains and characterized field isolates of both genotypes. The application to clinical diagnosis was evaluated on pooled blood samples by post run measurement of the FAM- and VIC-associated fluorescence. The 100% agreement with the conventional RT-PCR method confirmed that this new technique could be used for routine detection of persistently infected immunotolerant animals.

Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

PubMed

Li, Baoguang; Liu, Huanli; Wang, Weimin

2017-11-09

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency. The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella

Detection of sesame seed DNA in foods using real-time PCR.

PubMed

Brzezinski, Jennifer L

2007-04-01

The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.

Smallpox and pan-orthopox virus detection by real-time 3'-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms.

PubMed

Kulesh, David A; Baker, Robert O; Loveless, Bonnie M; Norwood, David; Zwiers, Susan H; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P; Huggins, John; Jahrling, Peter B

2004-02-01

We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs

The PCRS (Parent-Child Reading System) Specialist's Guide; The Des Moines Family Learning Project.

ERIC Educational Resources Information Center

Miller, Martin, Ed.

The Parent-Child Reading System (PCRS), a way of organizing instructional materials for reading so that parents can become continuously involved in helping to improve their children's reading abilities, may be used in connection with family learning center (FLC) workshops, in schools, or in institutions maintaining contact with schools. This…

The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations.

PubMed

Teh, L-K; Lee, T-Y; Tan, J A M A; Lai, M-I; George, E

2015-02-01

In Malaysia, β-thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR-incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β-thalassaemia mutation detection. ARMS allows single-mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β-thalassaemia mutations can be identified in samples with low DNA concentrations. Genotype identification of common β-thalassaemia mutations in Malays was carried out using Taqman genotyping assays. Results show that the Taqman assays allow mutation detection with DNA template concentrations as low as 2-100 ng. In addition, consistent reproducibility was observed in the Taqman assays when repeated eight times and at different time intervals. The developed sensitive Taqman assays allow molecular characterization of β-thalassaemia mutations in samples with low DNA concentrations. The Taqman genotyping assays have potential as a diagnostic tool for foetal blood, chorionic villi or pre-implantation genetic diagnosis where DNA is limited and precious. © 2014 John Wiley & Sons Ltd.

MethPrimer: designing primers for methylation PCRs.

PubMed

Li, Long-Cheng; Dahiya, Rajvir

2002-11-01

DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.

Development of real-time PCR methods to quantify patulin-producing molds in food products.

PubMed

Rodríguez, Alicia; Luque, M Isabel; Andrade, María J; Rodríguez, Mar; Asensio, Miguel A; Córdoba, Juan J

2011-09-01

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g(-1) per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Cobas AmpliPrep/Cobas TaqMan HCV test, version 2.0, real-time PCR assay accurately quantifies hepatitis C virus genotype 4 RNA.

PubMed

Chevaliez, Stéphane; Bouvier-Alias, Magali; Rodriguez, Christophe; Soulier, Alexandre; Poveda, Jean-Dominique; Pawlotsky, Jean-Michel

2013-04-01

Accurate hepatitis C virus (HCV) RNA quantification is mandatory for the management of chronic hepatitis C therapy. The first-generation Cobas AmpliPrep/Cobas TaqMan HCV test (CAP/CTM HCV) underestimated HCV RNA levels by >1-log10 international units/ml in a number of patients infected with HCV genotype 4 and occasionally failed to detect it. The aim of this study was to evaluate the ability of the Cobas AmpliPrep/Cobas TaqMan HCV test, version 2.0 (CAP/CTM HCV v2.0), to accurately quantify HCV RNA in a large series of patients infected with different subtypes of HCV genotype 4. Group A comprised 122 patients with chronic HCV genotype 4 infection, and group B comprised 4 patients with HCV genotype 4 in whom HCV RNA was undetectable using the CAP/CTM HCV. Each specimen was tested with the third-generation branched DNA (bDNA) assay, CAP/CTM HCV, and CAP/CTM HCV v2.0. The HCV RNA level was lower in CAP/CTM HCV than in bDNA in 76.2% of cases, regardless of the HCV genotype 4 subtype. In contrast, the correlation between bDNA and CAP/CTM HCV v2.0 values was excellent. CAP/CTM HCV v2.0 accurately quantified HCV RNA levels in the presence of an A-to-T substitution at position 165 alone or combined with a G-to-A substitution at position 145 of the 5' untranslated region of HCV genome. In conclusion, CAP/CTM HCV v2.0 accurately quantifies HCV RNA in genotype 4 clinical specimens, regardless of the subtype, and can be confidently used in clinical trials and clinical practice with this genotype.

Real-Time Systems

DTIC Science & Technology

1992-02-01

Postgraduate School Autonomous Under Vehicle (AUV) are then examined. Autonomous underwater vehicle (AUV), hard real-time system, real - time operating system , real-time programming language, real-time system, soft real-time system.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

PubMed Central

Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M.; Zhao, Hui; Ma, Xiaoyue; Ellison, James A.; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian

2017-01-01

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

PubMed

Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu

2017-01-01

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay.

PubMed

Maksyutov, Rinat A; Gavrilova, Elena V; Shchelkunov, Sergei N

2016-10-01

A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species. Copyright © 2016 Elsevier B.V. All rights reserved.

Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform.

PubMed

Opota, Onya; Brouillet, René; Greub, Gilbert; Jaton, Katia

2017-01-01

The advances in molecular biology of the last decades have dramatically improved the field of diagnostic bacteriology. In particular, PCR-based technologies have impacted the diagnosis of infections caused by obligate intracellular bacteria such as pathogens from the Chlamydiacae family. Here, we describe a real-time PCR-based method using the Taqman technology for the diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus infection. The method presented here can be applied to various clinical samples and can be adapted on opened molecular diagnostic platforms.

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

PubMed

Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

2008-12-01

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

PubMed Central

Kulesh, David A.; Baker, Robert O.; Loveless, Bonnie M.; Norwood, David; Zwiers, Susan H.; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P.; Huggins, John; Jahrling, Peter B.

2004-01-01

We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However

Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay.

PubMed

Iijima, Yoshio; Asako, Nahoko T; Aihara, Masanori; Hayashi, Kozaburo

2004-07-01

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including Salmonella enterica, Vibrio parahaemolyticus, Campylobacter jejuni and Shiga toxin-producing Escherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40%) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70%) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18%) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6%) patients were found to be positive by PCR analysis.

Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

PubMed

Cassler, M; Peterson, C L; Ledger, A; Pomponi, S A; Wright, A E; Winegar, R; McCarthy, P J; Lopez, J V

2008-04-01

In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

Characterization of Phytophthora nicotianae isolates in southeast Spain and their detection and quantification through a real-time TaqMan PCR.

PubMed

Blaya, Josefa; Lacasa, Carmen; Lacasa, Alfredo; Martínez, Victoriano; Santísima-Trinidad, Ana B; Pascual, Jose A; Ros, Margarita

2015-04-01

The soil-borne pathogens Phytophthora nicotianae and P. capsici are the causal agents of root and stem rot of many plant species. Although P. capsici was considered the causal agent in one of the main pepper production areas of Spain to date, evidence of the presence of P. nicotianae was found. We aimed to survey the presence of P. nicotianae and study the variability in its populations in this area in order to improve the management of Tristeza disease. A new specific primer and a TaqMan probe were designed based on the internal transcribed spacer regions of ribosomal DNA to detect and quantify P. nicotianae. Both morphological and molecular analysis showed its presence and confirmed it to be the causal agent of the Phytophthora disease symptoms in the studied area. The genetic characterization among P. nicotianae populations showed a low variability of genetic diversity among the isolates. Only isolates of the A2 mating type were detected. Not only is a specific and early detection of P. nicotianae essential but also the study of genetic variability among isolates for the appropriate management of the disease, above all, in producing areas with favorable conditions for the advance of the disease. © 2014 Society of Chemical Industry.

Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus.

PubMed

Liu, Jie; Ochieng, Caroline; Wiersma, Steve; Ströher, Ute; Towner, Jonathan S; Whitmer, Shannon; Nichol, Stuart T; Moore, Christopher C; Kersh, Gilbert J; Kato, Cecilia; Sexton, Christopher; Petersen, Jeannine; Massung, Robert; Hercik, Christine; Crump, John A; Kibiki, Gibson; Maro, Athanasia; Mujaga, Buliga; Gratz, Jean; Jacob, Shevin T; Banura, Patrick; Scheld, W Michael; Juma, Bonventure; Onyango, Clayton O; Montgomery, Joel M; Houpt, Eric; Fields, Barry

2016-01-01

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DNA barcoding and real-time PCR detection of Bactrocera xanthodes (Tephritidae: Diptera) complex.

PubMed

Li, D; Waite, D W; Gunawardana, D N; McCarthy, B; Anderson, D; Flynn, A; George, S

2018-05-06

Immature fruit fly stages of the family Tephritidae are commonly intercepted on breadfruit from Pacific countries at the New Zealand border but are unable to be identified to the species level using morphological characters. Subsequent molecular identification showed that they belong to Bactrocera xanthodes, which is part of a species complex that includes Bactrocera paraxanthodes, Bactrocera neoxanthodes and an undescribed species. To establish a more reliable molecular identification system for B. xanthodes, a reference database of DNA barcode sequences for the 5'-fragment of COI gene region was constructed for B. xanthodes from Fiji, Samoa and Tonga. To better understand the species complex, B. neoxanthodes from Vanuatu and B. paraxanthodes from New Caledonia were also barcoded. Using the results of this analysis, real-time TaqMan polymerase chain reaction (PCR) assays for the detection of B. xanthodes complex and for the three individual species of the complex were developed and validated. The assay showed high specificity for the target species, with no cross-reaction observed for closely related organisms. Each of the real-time PCR assays is sensitive, detecting the target sequences at concentrations as low as ten copies µl-1 and can be used as either singleplex or multiplex formats. This real-time PCR assay for B. xanthodes has been successfully applied at the borders in New Zealand, leading to the rapid identification of intercepted Tephritidae eggs and larvae. The developed assays will be useful biosecurity tools for rapid detection of species in the B. xanthodes complex worldwide.

Comparison of Two Commercial Automated Nucleic Acid Extraction and Integrated Quantitation Real-Time PCR Platforms for the Detection of Cytomegalovirus in Plasma

PubMed Central

Tsai, Huey-Pin; Tsai, You-Yuan; Lin, I-Ting; Kuo, Pin-Hwa; Chen, Tsai-Yun; Chang, Kung-Chao; Wang, Jen-Ren

2016-01-01

Quantitation of cytomegalovirus (CMV) viral load in the transplant patients has become a standard practice for monitoring the response to antiviral therapy. The cut-off values of CMV viral load assays for preemptive therapy are different due to the various assay designs employed. To establish a sensitive and reliable diagnostic assay for preemptive therapy of CMV infection, two commercial automated platforms including m2000sp extraction system integrated the Abbott RealTime (m2000rt) and the Roche COBAS AmpliPrep for extraction integrated COBAS Taqman (CAP/CTM) were evaluated using WHO international CMV standards and 110 plasma specimens from transplant patients. The performance characteristics, correlation, and workflow of the two platforms were investigated. The Abbott RealTime assay correlated well with the Roche CAP/CTM assay (R2 = 0.9379, P<0.01). The Abbott RealTime assay exhibited higher sensitivity for the detection of CMV viral load, and viral load values measured with Abbott RealTime assay were on average 0.76 log10 IU/mL higher than those measured with the Roche CAP/CTM assay (P<0.0001). Workflow analysis on a small batch size at one time, using the Roche CAP/CTM platform had a shorter hands-on time than the Abbott RealTime platform. In conclusion, these two assays can provide reliable data for different purpose in a clinical virology laboratory setting. PMID:27494707

Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 RNA 3.0 assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes.

PubMed

Gomes, Perpétua; Palma, Ana Carolina; Cabanas, Joaquim; Abecasis, Ana; Carvalho, Ana Patrícia; Ziermann, Rainer; Diogo, Isabel; Gonçalves, Fátima; Lobo, Céu Sousa; Camacho, Ricardo

2006-08-01

Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland-Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples.

Real-Time CORBA

DTIC Science & Technology

2000-10-01

control systems and prototyped the approach by porting the ILU ORB from Xerox to the Lynx real - time operating system . They then provided a distributed...compliant real - time operating system , a real-time ORB, and an ODMG-compliant real-time ODBMS [12]. The MITRE system is an infrastructure for...the server’s local operating system can handle. For instance, on a node controlled by the VXWorks real - time operating system with 256 local

Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.

PubMed

Peng, Xiujuan; Nguyen, Alex; Ghosh, Debadyuti

2018-02-01

TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*10 1 -2.75*10 8 genome copies(gc)/μL and 2.66*10 1 -2.66*10 8 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (E s ) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75-2.75*10 7 gc/μL and 2.66*10 1 -2.66*10 7 gc/μL of M13 and T7 phage DNA, with an amplification efficiency E s of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS). Copyright © 2017 Elsevier B.V. All rights reserved.

Real-Time PCR Detection of Dogwood Anthracnose Fungus in Historical Herbarium Specimens from Asia.

PubMed

Miller, Stephen; Masuya, Hayato; Zhang, Jian; Walsh, Emily; Zhang, Ning

2016-01-01

Cornus species (dogwoods) are popular ornamental trees and important understory plants in natural forests of northern hemisphere. Dogwood anthracnose, one of the major diseases affecting the native North American Cornus species, such as C. florida, is caused by the fungal pathogen Discula destructiva. The origin of this fungus is not known, but it is hypothesized that it was imported to North America with its host plants from Asia. In this study, a TaqMan real-time PCR assay was used to detect D. destructiva in dried herbarium and fresh Cornus samples. Several herbarium specimens from Japan and China were detected positive for D. destructiva, some of which were collected before the first report of the dogwood anthracnose in North America. Our findings further support that D. destructiva was introduced to North America from Asia where the fungus likely does not cause severe disease.

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus.

PubMed

Tantibhedhyangkul, Wiwit; Wongsawat, Ekkarat; Silpasakorn, Saowaluk; Waywa, Duangdao; Saenyasiri, Nuttawut; Suesuay, Jintapa; Thipmontree, Wilawan; Suputtamongkol, Yupin

2017-05-01

Scrub typhus, caused by Orientia tsutsugamushi , is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targeting O. tsutsugamushi 47-kDa, groEL , and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness. Copyright © 2017 American Society for Microbiology.

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

PubMed Central

Li, Jiandong; Qu, Jing; He, Chengcheng; Zhang, Shuo; Li, Chuan; Zhang, Quanfu; Liang, Mifang; Li, Dexin

2014-01-01

Background Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Results Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Conclusions Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive

Real-time PCR quantification of six periodontal pathogens in saliva samples from healthy young adults.

PubMed

Zhou, Xiaodong; Liu, Xiaoli; Li, Jing; Aprecio, Raydolfo M; Zhang, Wu; Li, Yiming

2015-05-01

The use of saliva as a diagnostic fluid for the evaluation of periodontal health has gained attention recently. Most published real-time PCR assays focused on quantification of bacteria in subgingival plaque, not in saliva. The aims of this study were to develop a real-time PCR assay for quantification of six periodontal pathogens in saliva and to establish a relationship between the amount of DNA (fg) and colony-forming unit (CFU). TaqMan primers/probe sets were used for the detection of Aggregatibacter actinomycetemcomitans (Aa), Eikenella corrodens (Ec), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf), and total bacteria. Six periodontal pathogens and total bacteria in saliva from 24 periodontally healthy individuals were determined. The relationship between the amount of DNA (fg) and CFU was established by measuring the concentrations of extracted bacterial DNA and CFU per milliliter of bacteria on agar plates. Fn, Ec, and Pi were detected in all saliva samples, while 58.5, 45.8, and 33.3% were detected for Tf, Pg, and Aa, respectively. Numbers of Ec and Fn in saliva were highly correlated (R(2) = 0.93, P < 0.01). The values of DNA (fg) per CFU ranged from 64 for Ec to 121 for Pg. The real-time PCR assay in combination with the relationship between DNA (fg) and CFU can be used to quantitate periodontal pathogens in saliva and estimate the number of live bacteria (CFU). This real-time PCR assay in combination with the relationship between DNA (fg) and CFU has the potential to be an adjunct in evaluation of periodontal health status.

Market analysis of food products for detection of allergenic walnut (Juglans regia) and pecan (Carya illinoinensis) by real-time PCR.

PubMed

López-Calleja, Inés María; de la Cruz, Silvia; González, Isabel; García, Teresa; Martín, Rosario

2015-06-15

Two real-time polymerase chain reaction (PCR)-based assays for detection of walnut (Juglans regia) and pecan (Carya illinoinensis) traces in a wide range of processed foods are described here. The method consists on a real-time PCR assay targeting the ITS1 region, using a nuclease (TaqMan) probe labeled with FAM and BBQ. The method was positive for walnut and pecan respectively, and negative for all other heterologous plants and animals tested. Using a series of model samples with defined raw walnut in wheat flour and heat-treated walnut in wheat flour with a range of concentrations of 0.1-100,000 mg kg(-1), a practical detection limit of 0.1 mg kg(-1) of walnut content was estimated. Identical binary mixtures were done for pecan, reaching the same limit of detection of 0.1 mg kg(-1). The assay was successfully trialed on a total of 232 commercial foodstuffs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Real Time Revisited

NASA Astrophysics Data System (ADS)

Allen, Phillip G.

1985-12-01

The call for abolishing photo reconnaissance in favor of real time is once more being heard. Ten years ago the same cries were being heard with the introduction of the Charge Coupled Device (CCD). The real time system problems that existed then and stopped real time proliferation have not been solved. The lack of an organized program by either DoD or industry has hampered any efforts to solve the problems, and as such, very little has happened in real time in the last ten years. Real time is not a replacement for photo, just as photo is not a replacement for infra-red or radar. Operational real time sensors can be designed only after their role has been defined and improvements made to the weak links in the system. Plodding ahead on a real time reconnaissance suite without benefit of evaluation of utility will allow this same paper to be used ten years from now.

Real-time PCR array as a universal platform for the detection of genetically modified crops and its application in identifying unapproved genetically modified crops in Japan.

PubMed

Mano, Junichi; Shigemitsu, Natsuki; Futo, Satoshi; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Furui, Satoshi; Kitta, Kazumi

2009-01-14

We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination. The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html .

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.

Development of real-time PCR assay for genetic identification of the mottled skate, Beringraja pulchra.

PubMed

Hwang, In Kwan; Lee, Hae Young; Kim, Min-Hee; Jo, Hyun-Su; Choi, Dong-Ho; Kang, Pil-Won; Lee, Yang-Han; Cho, Nam-Soo; Park, Ki-Won; Chae, Ho Zoon

2015-10-01

The mottled skate, Beringraja pulchra is one of the commercially important fishes in the market today. However, B. pulchra identification methods have not been well developed. The current study reports a novel real-time PCR method based on TaqMan technology developed for the genetic identification of B. pulchra. The mitochondrial cytochrome oxidase subunit 1 (COI) nucleotide sequences of 29 B. pulchra, 157 skates and rays reported in GenBank DNA database were comparatively analyzed and the COI sequences specific to B. pulchra was identified. Based on this information, a system of specific primers and Minor Groove Binding (MGB) TaqMan probe were designed. The assay successfully discriminated in 29 specimens of B. pulchra and 27 commercial samples with unknown species identity. For B. pulchra DNA, an average Threshold Cycle (Ct) value of 19.1±0.1 was obtained. Among 27 commercial samples, two samples showed average Ct values 19.1±0.0 and 26.7±0.1, respectively and were confirmed to be B. pulchra based on sequencing. The other samples tested showed undetectable or extremely weak signals for the target fragment, which was also consistent with the sequencing results. These results reveal that the method developed is a rapid and efficient tool to identify B. pulchra and might prevent fraud or mislabeling during the distribution of B. pulchra products. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

USGS Publications Warehouse

Chase, D.M.; Elliott, D.G.; Pascho, R.J.

2006-01-01

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

PubMed

Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

2016-11-01

Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

Detection of cashew nut DNA in spiked baked goods using a real-time polymerase chain reaction method.

PubMed

Brzezinski, Jennifer L

2006-01-01

The detection of potentially allergenic foods, such as tree nuts, in food products is a major concern for the food processing industry. A real-time polymerase chain reaction (PCR) method was designed to determine the presence of cashew DNA in food products. The PCR amplifies a 67 bp fragment of the cashew 2S albumin gene, which is detected with a cashew-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other tree nut species, such as almond, Brazil nut, hazelnut, and walnut, as well as 4 varieties of peanut. This assay was sensitive enough to detect 5 pg purified cashew DNA as well as cashew DNA in a spiked chocolate cookie sample containing 0.01% (100 mg/kg) cashew.

"Fast" Is Not "Real-Time": Designing Effective Real-Time AI Systems

NASA Astrophysics Data System (ADS)

O'Reilly, Cindy A.; Cromarty, Andrew S.

1985-04-01

Realistic practical problem domains (such as robotics, process control, and certain kinds of signal processing) stand to benefit greatly from the application of artificial intelligence techniques. These problem domains are of special interest because they are typified by complex dynamic environments in which the ability to select and initiate a proper response to environmental events in real time is a strict prerequisite to effective environmental interaction. Artificial intelligence systems developed to date have been sheltered from this real-time requirement, however, largely by virtue of their use of simplified problem domains or problem representations. The plethora of colloquial and (in general) mutually inconsistent interpretations of the term "real-time" employed by workers in each of these domains further exacerbates the difficul-ties in effectively applying state-of-the-art problem solving tech-niques to time-critical problems. Indeed, the intellectual waters are by now sufficiently muddied that the pursuit of a rigorous treatment of intelligent real-time performance mandates the redevelopment of proper problem perspective on what "real-time" means, starting from first principles. We present a simple but nonetheless formal definition of real-time performance. We then undertake an analysis of both conventional techniques and AI technology with respect to their ability to meet substantive real-time performance criteria. This analysis provides a basis for specification of problem-independent design requirements for systems that would claim real-time performance. Finally, we discuss the application of these design principles to a pragmatic problem in real-time signal understanding.

Improving clinical laboratory efficiency: a time-motion evaluation of the Abbott m2000 RealTime and Roche COBAS AmpliPrep/COBAS TaqMan PCR systems for the simultaneous quantitation of HIV-1 RNA and HCV RNA.

PubMed

Amendola, Alessandra; Coen, Sabrina; Belladonna, Stefano; Pulvirenti, F Renato; Clemens, John M; Capobianchi, M Rosaria

2011-08-01

Diagnostic laboratories need automation that facilitates efficient processing and workflow management to meet today's challenges for expanding services and reducing cost, yet maintaining the highest levels of quality. Processing efficiency of two commercially available automated systems for quantifying HIV-1 and HCV RNA, Abbott m2000 system and Roche COBAS Ampliprep/COBAS TaqMan 96 (docked) systems (CAP/CTM), was evaluated in a mid/high throughput workflow laboratory using a representative daily workload of 24 HCV and 72 HIV samples. Three test scenarios were evaluated: A) one run with four batches on the CAP/CTM system, B) two runs on the Abbott m2000 and C) one run using the Abbott m2000 maxCycle feature (maxCycle) for co-processing these assays. Cycle times for processing, throughput and hands-on time were evaluated. Overall processing cycle time was 10.3, 9.1 and 7.6 h for Scenarios A), B) and C), respectively. Total hands-on time for each scenario was, in order, 100.0 (A), 90.3 (B) and 61.4 min (C). The interface of an automated analyzer to the laboratory workflow, notably system set up for samples and reagents and clean up functions, are as important as the automation capability of the analyzer for the overall impact to processing efficiency and operator hands-on time.

Real-time PCR method applied to seafood products for authentication of European sole (Solea solea) and differentiation of common substitute species.

PubMed

Herrero, Beatriz; Lago, Fátima C; Vieites, Juan M; Espiñeira, Montserrat

2012-01-01

Judged by quality and taste, the European sole (Solea solea) is considered one of the finest flatfish and is, thus, of considerable commercial value. In the present work, a specific fast real-time PCR was developed for the authentication of S. solea, i.e. to distinguish it from other related species and avoid substitution of this species, either deliberately or unintentionally. The method is based on a species-specific set of primers and MGB Taqman probe which amplifies a 116-bp fragment of the internal transcribed spacer 1 (ITS 1) ribosomal DNA region. This assay combines the high specificity and sensitivity of real-time PCR with the rapidity of the fast mode, allowing the detection of S. solea in a short period of time. The present methodology was validated for application to all types of manufactured products for the presence of S. solea, with successful results. Subsequently, the method was applied to 40 commercial samples to determine whether correct labeling had been employed in the market. It was demonstrated that the assay is a useful tool in monitoring and verifying food labeling regulations.

Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil.

PubMed

Savazzini, Federica; Longa, Claudia Maria Oliveira; Pertot, Ilaria; Gessler, Cesare

2008-05-01

Trichoderma (Hypocreales, Ascomycota) is a widespread genus in nature and several Trichoderma species are used in industrial processes and as biocontrol agents against crop diseases. It is very important that the persistence and spread of microorganisms released on purpose into the environment are accurately monitored. Real-time PCR methods for genus/species/strain identification of microorganisms are currently being developed to overcome the difficulties of classical microbiological and enzymatic methods for monitoring these populations. The aim of the present study was to develop and validate a specific real-time PCR-based method for detecting Trichoderma atroviride SC1 in soil. We developed a primer and TaqMan probe set constructed on base mutations in an endochitinase gene. This tool is highly specific for the detection and quantification of the SC1 strain. The limits of detection and quantification calculated from the relative standard deviation were 6000 and 20,000 haploid genome copies per gram of soil. Together with the low throughput time associated with this procedure, which allows the evaluation of many soil samples within a short time period, these results suggest that this method could be successfully used to trace the fate of T. atroviride SC1 applied as an open-field biocontrol agent.

Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials.

PubMed

Soejima, Mikiko; Tsuchiya, Yuji; Egashira, Kouichi; Kawano, Hiroyuki; Sagawa, Kimitaka; Koda, Yoshiro

2010-06-01

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

Development of a taqman-based real-time PCR assay for the rapid and specific detection of novel duck- origin goose parvovirus.

PubMed

Wang, Jianchang; Wang, Jinfeng; Cui, Yuan; Nan, Huizhu; Yuan, Wanzhe

2017-08-01

A real-time PCR assay was developed for specific detection of novel duck-origin goose parvovirus (N-GPV), the etiological agent of duck beak atrophy and dwarfism syndrome (BADS). The detection limit of the assay was 10 2 copies. The assay was useful in the prevention and control of BADS. Copyright © 2017 Elsevier Ltd. All rights reserved.

Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification.

PubMed

Montalvo, A M; Fraga, J; Maes, I; Dujardin, J-C; Van der Auwera, G

2012-07-01

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the New and Old World, using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Current PCR presents limitations in terms of sensitivity, which hampers its use for analyzing clinical and biological samples, and specificity, which makes it inappropriate to discriminate between Leishmania and other trypanosomatids. The aim of the study was to improve the sensitivity and specificity of a previously reported hsp70 PCR using alternative PCR primers and RFLPs. Following in silico analysis of available sequences, three new PCR primer sets and restriction digest schemes were tested on a globally representative panel of 114 Leishmania strains, various other infectious agents, and clinical samples. The largest new PCR fragment retained the discriminatory power from RFLP, while two smaller fragments discriminated less species. The detection limit of the new PCRs was between 0.05 and 0.5 parasite genomes, they amplified clinical samples more efficiently, and were Leishmania specific. We succeeded in significantly improving the specificity and sensitivity of the PCRs for hsp70 Leishmania species typing. The improved PCR-RFLP assays can impact diagnosis, treatment, and epidemiological studies of leishmaniasis in any setting worldwide.

Detection of minute virus of mice using real time quantitative PCR in assessment of virus clearance during the purification of Mammalian cell substrate derived biotherapeutics.

PubMed

Zhan, Dejin; Roy, Margaret R; Valera, Christine; Cardenas, Jesse; Vennari, Joann C; Chen, Janice W; Liu, Shengjiang

2002-12-01

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

Efficiency of semi-automated fluorescent multiplex PCRs with 11 microsatellite markers for genetic studies of deer populations.

PubMed

Bonnet, A; Thévenon, S; Maudet, F; Maillard, J C

2002-10-01

Thirty bovine and eight ovine microsatellite primer pairs were tested on four tropical deer species: Eld's and Swamp deer (highly threatened) and Rusa and Vietnamese Sika deer (economically important). Thirty markers gave an amplified product in all four species (78.9%). The number of polymorphic microsatellite markers varied among the species from 14 in Eld's deer (47%) to 20 in Swamp deer (67%). Among them, 11 microsatellite loci were multiplexed in three polymerase chain reactions (PCRs) and labelled with three different fluorochromes that can be loaded in one gel-lane. To test the efficiency of the multiplex, primary genetic studies (mean number of alleles, expected heterozygosities and Fis values) were carried out on four deer populations. Parentage exclusion probability and probability of identity were computed and discussed on a Swamp deer population. These multiplexes PCRs were also tested on several other deer species and subspecies. The aim of this study is to establish a tool useful for genetic studies of population structure and diversity in four tropical deer species which with few modifications can be applied to other species of the genus Cervus.

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.).

PubMed

Lee-Montero, I; Navarro, A; Borrell, Y; García-Celdrán, M; Martín, N; Negrín-Báez, D; Blanco, G; Armero, E; Berbel, C; Zamorano, M J; Sánchez, J J; Estévez, A; Ramis, G; Manchado, M; Afonso, J M

2013-08-01

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

PubMed

Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J

2012-08-01

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products. Copyright © 2012 Elsevier Ltd. All rights reserved.

Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

PubMed

Schutten, M; Peters, D; Back, N K T; Beld, M; Beuselinck, K; Foulongne, V; Geretti, A-M; Pandiani, L; Tiemann, C; Niesters, H G M

2007-06-01

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.

Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam.

PubMed

Vital, Pierangeli G; Van Ha, Nguyen Thi; Tuyet, Le Thi Hong; Widmer, Kenneth W

2017-02-01

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

PubMed

Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PubMed Central

Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8�

Simplex and duplex event-specific analytical methods for functional biotech maize.

PubMed

Lee, Seong-Hun; Kim, Su-Jeong; Yi, Bu-Young

2009-08-26

Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens.

PubMed

Okamoto, Kiyoko; Mori, Yoshio; Komagome, Rika; Nagano, Hideki; Miyoshi, Masahiro; Okano, Motohiko; Aoki, Yoko; Ogura, Atsushi; Hotta, Chiemi; Ogawa, Tomoko; Saikusa, Miwako; Kodama, Hiroe; Yasui, Yoshihiro; Minagawa, Hiroko; Kurata, Takako; Kanbayashi, Daiki; Kase, Tetsuo; Murata, Sachiko; Shirabe, Komei; Hamasaki, Mitsuhiro; Kato, Takashi; Otsuki, Noriyuki; Sakata, Masafumi; Komase, Katsuhiro; Takeda, Makoto

2016-07-01

An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination. Copyright © 2016 Elsevier B.V. All rights reserved.

Dependable Real-Time Systems

DTIC Science & Technology

1991-09-30

0196 or 413 545-0720 PI E-mail Address: krithi@nirvan.cs.umass.edu, stankovic(ocs.umass.edu Grant or Contract Title: Dependable Real - Time Systems Grant...Dependable Real - Time Systems " Grant or Contract Number: N00014-85-k-0398 L " Reporting Period: 1 Oct 87 - 30 Sep 91 , 2. Summary of Accomplishments ’ 2.1 Our...in developing a sound approach to scheduling tasks in complex real - time systems , (2) developed a real-time operating system kernel, a preliminary

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients.

PubMed

Kumar, Jyoti S; Saxena, Divyasha; Parida, Manmohan

2014-01-01

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009-10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid real-time PCR methods to distinguish Salmonella Enteritidis wildtype field isolates from vaccine strains Salmovac SE/Gallivac SE and AviPro SALMONELLA VAC E.

PubMed

Maurischat, Sven; Szabo, Istvan; Baumann, Beatrice; Malorny, Burkhard

2015-05-01

Salmonella enterica serovar Enteritidis is a major non-typhoid Salmonella serovar causing human salmonellosis mainly associated with the consumption of poultry and products thereof. To reduce infections in poultry, S. Enteritidis live vaccine strains AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE have been licensed and used in several countries worldwide. To definitively diagnose a S. Enteritidis contamination in vaccinated herds a reliable and fast method for the differentiation between vaccine and wildtype field isolates is required. In this study, we developed and validated real-time PCR (qPCR) assays to distinguish those variants genetically. Suitable target sequences were identified by whole genome sequencing (WGS) using the Illumina MiSeq system. SNP regions in kdpA and nhaA proved to be most useful for differentiation of AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE, respectively, from wildtype strains. For each vaccine strain one TaqMan-qPCR assay and one alternative approach using High Resolution Melting (HRM) analysis was designed. All 30 Salmovac SE and 7 AviPro SALMONELLA VAC E vaccine strain reisolates tested were correctly identified by both approaches (100% inclusivity). Furthermore, all 137 (TaqMan) and 97 (HRM) Salmonella non-vaccine and related Enterobacteriaceae strains tested were excluded (100% exclusivity). The analytical detection limits were determined to be approx. 10(2) genome copies/reaction for the TaqMan and 10(4) genome copies/reaction for the HRM approach. The real-time PCR assays proved to be a reliable and fast alternative to the cultural vaccine strain identification tests helping decision makers in control measurements to take action within a shorter period of time. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and validation of a novel hydrolysis probe real-time polymerase chain reaction for agamid adenovirus 1 in the central bearded dragon (Pogona vitticeps).

PubMed

Fredholm, Daniel V; Coleman, James K; Childress, April L; Wellehan, James F X

2015-03-01

Agamid adenovirus 1 (AgAdv-1) is a significant cause of disease in bearded dragons (Pogona sp.). Clinical manifestations of AgAdv-1 infection are variable and often nonspecific; the manifestations range from lethargy, weight loss, and inappetence, to severe enteritis, hepatitis, and sudden death. Currently, diagnosis of AgAdv-1 infection is achieved through a single published method: standard nested polymerase chain reaction (nPCR) and sequencing. Standard nPCR with sequencing provides reliable sensitivity, specificity, and validation of PCR products. However, this process is comparatively expensive, laborious, and slow. Probe hybridization, as used in a TaqMan assay, represents the best option for validating PCR products aside from the time-consuming process of sequencing. This study developed a real-time PCR (qPCR) assay using a TaqMan probe-based assay, targeting a highly conserved region of the AgAdv-1 genome. Standard curves were generated, detection results were compared with the gold standard conventional PCR and sequencing assay, and limits of detection were determined. Additionally, the qPCR assay was run on samples known to be positive for AgAdv-1 and samples known to be positive for other adenoviruses. Based on the results of these evaluations, this assay allows for a less expensive, rapid, quantitative detection of AgAdv-1 in bearded dragons. © 2015 The Author(s).

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

PubMed Central

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

2013-01-01

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

Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia.

PubMed

Abdeldaim, G; Herrmann, B; Mölling, P; Holmberg, H; Blomberg, J; Olcén, P; Strålin, K

2010-08-01

In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.

Detection of Mycobacterium tuberculosis Complex in Paraffin-Embedded Tissues by the New Automated Abbott RealTime MTB Assay.

PubMed

Fu, Yung-Chieh; Liao, I-Chuang; Chen, Hung-Mo; Yan, Jing-Jou

2016-07-01

The Abbott RealTime MTB assay, launched in June 2014, has been shown to have a competitive performance in the detection of the Mycobacterium tuberculosis (MTB) complex in respiratory specimens. The present study was conducted to investigate the usefulness of the Abbott MTB Realtime assay in the detection of MTB in formalin-fixed paraffin-embedded (FFPE) tissues. A total of 96 FFPE specimens obtained from microbiologically proven MTB cases (N=60) and nontuberculous Mycobacterium cases (N=36) were analyzed. The performance of the Abbott MTB Realtime assay was compared with that of the Roche Cobas TaqMan MTB assay. The overall sensitivity and specificity of the Abbott assay were 63.3% and 97.2%, respectively, compared with 11.7% and 100% for the Cobas assay. The detection rate of the Abbott assay was much higher among 37 acid-fast-positive specimens than among 23 acid-fast-negative specimens (89.3% versus 21.7%, respectively). The detection rate of the assay was higher among 29 resection specimens than among 31 small biopsy specimens (86.2% versus 41.9%, respectively). Our results suggest that the Abbott RealTime MTB assay can be used to differentiate MTB from nontuberculous mycobacterial infections in acid-fast-positive FFPE tissues. © 2016 by the Association of Clinical Scientists, Inc.

Real-time Kp predictions from ACE real time solar wind

NASA Astrophysics Data System (ADS)

Detman, Thomas; Joselyn, Joann

1999-06-01

The Advanced Composition Explorer (ACE) spacecraft provides nearly continuous monitoring of solar wind plasma, magnetic fields, and energetic particles from the Sun-Earth L1 Lagrange point upstream of Earth in the solar wind. The Space Environment Center (SEC) in Boulder receives ACE telemetry from a group of international network of tracking stations. One-minute, and 1-hour averages of solar wind speed, density, temperature, and magnetic field components are posted on SEC's World Wide Web page within 3 to 5 minutes after they are measured. The ACE Real Time Solar Wind (RTSW) can be used to provide real-time warnings and short term forecasts of geomagnetic storms based on the (traditional) Kp index. Here, we use historical data to evaluate the performance of the first real-time Kp prediction algorithm to become operational.

Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues

PubMed Central

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R.; Mrug, Michal

2009-01-01

Background Endogenous internal controls (‘reference’ or ‘housekeeping’ genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. Methods To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used ‘reference genes’ in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan® RT-PCR analyses and Affymetrix GeneChip® arrays, were normalized and tested for overall variance and equivalence of the means. Results Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. Conclusion A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. PMID:19729889

Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens▿

PubMed Central

Boddicker, Jennifer D.; Rota, Paul A.; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B.; Thompson, Robert; Bellini, William J.; Pentella, Michael; DesJardin, Lucy E.

2007-01-01

The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques. PMID:17652480

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

USGS Publications Warehouse

Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

2015-01-01

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

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

PubMed

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

2013-05-01

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

Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections

PubMed Central

Gade, Lalitha; Grgurich, Dale E.; Kerkering, Thomas M.; Brandt, Mary E.

2014-01-01

Exserohilum rostratum was the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range and E. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection of E. rostratum in body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive for E. rostratum to 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data. PMID:25520443

Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system.

PubMed

Volle, Romain; Nourrisson, Céline; Mirand, Audrey; Regagnon, Christel; Chambon, Martine; Henquell, Cécile; Bailly, Jean-Luc; Peigue-Lafeuille, Hélène; Archimbaud, Christine

2012-10-01

Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections. Copyright © 2012 Elsevier B.V. All rights reserved.

SOFTWARE DESIGN FOR REAL-TIME SYSTEMS.

DTIC Science & Technology

Real-time computer systems and real-time computations are defined for the purposes of this report. The design of software for real - time systems is...discussed, employing the concept that all real - time systems belong to one of two types. The types are classified according to the type of control...program used; namely: Pre-assigned Iterative Cycle and Real-time Queueing. The two types of real - time systems are described in general, with supplemental

Development and validation of a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines.

PubMed

Petrov, Anja; Beer, Martin; Blome, Sandra

2014-01-01

Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene expression

Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR

USGS Publications Warehouse

Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.

2002-01-01

A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.

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

PubMed

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

2015-09-01

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

Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

PubMed

Rodríguez, Alicia; Werning, María L; Rodríguez, Mar; Bermúdez, Elena; Córdoba, Juan J

2012-12-01

A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain. Copyright © 2012 Elsevier Ltd. All rights reserved.

Fault Tolerant Real-Time Systems

DTIC Science & Technology

1993-09-30

The ART (Advanced Real-Time Technology) Project of Carnegie Mellon University is engaged in wide ranging research on hard real - time systems . The...including hardware and software fault tolerance using temporal redundancy and analytic redundancy to permit the construction of real - time systems whose

Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus.

PubMed

Lakshmi, I Karthika; Putty, Kalyani; Raut, Satya Samparna; Patil, Sunil R; Rao, P P; Bhagyalakshmi, B; Jyothi, Y Krishna; Susmitha, B; Reddy, Y Vishnuvardhan; Kasulanati, Sowmya; Jyothi, J Shiva; Reddy, Y N

2018-04-01

The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India. A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 10 5 ml and RNA was isolated by the Trizol method. Both reverse transcription-PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV. The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269E×10 3 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 10 3 TCID 50/ml and 10 4 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 10 2 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative. Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples

An immunomagnetic separation-real-time PCR system for the detection of Alicyclobacillus acidoterrestris in fruit products.

PubMed

Wang, Zhouli; Cai, Rui; Yuan, Yahong; Niu, Chen; Hu, Zhongqiu; Yue, Tianli

2014-04-03

Alicyclobacillus acidoterrestris is the most important spoilage species within the Alicyclobacillus genus and has become a major issue in the pasteurized fruit juice industry. The aim of this study was to develop a method combining immunomagnetic separation (IMS) with real-time PCR system (IMS-PCR) for rapid and specific detection of A. acidoterrestris in fruit products. A real-time PCR with the TaqMan system was designed to target the 16S rDNA genes with specific primer and probe set. The specificity of the assay was confirmed using 9 A. acidoterrestris strains and 21 non-A. acidoterrestris strains. The results indicated that no combination of the designed primers and probe was found in any Alicyclobacillus genus except A. acidoterrestris. The detection limit of the established IMS-PCR was less than 10CFU/mL and the testing process was accomplished in 2-3h. For the three types of samples (sterile water, apple juice and kiwi juice), the correlation coefficient of standard curves was greater than 0.991, and the calculated PCR efficiencies were from 108% to 109%. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS-PCR for 196 naturally contaminated fruit products were 90.0%, 98.3% and 97.5%, respectively. The results exhibited that the proposed IMS-PCR method was effective for the rapid detection of A. acidoterrestris in fruit products. Copyright © 2014. Published by Elsevier B.V.

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting.

PubMed

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile; Bamford, Colleen

2017-01-01

Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation.

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting

PubMed Central

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile

2017-01-01

Introduction Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. Methods We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. Results From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. Discussion In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation. PMID:28346504

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

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

Jothikumar, N., E-mail: jin2@cdc.gov; Hill, Vincent R.

Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forwardmore » or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource

VERSE - Virtual Equivalent Real-time Simulation

NASA Technical Reports Server (NTRS)

Zheng, Yang; Martin, Bryan J.; Villaume, Nathaniel

2005-01-01

Distributed real-time simulations provide important timing validation and hardware in the- loop results for the spacecraft flight software development cycle. Occasionally, the need for higher fidelity modeling and more comprehensive debugging capabilities - combined with a limited amount of computational resources - calls for a non real-time simulation environment that mimics the real-time environment. By creating a non real-time environment that accommodates simulations and flight software designed for a multi-CPU real-time system, we can save development time, cut mission costs, and reduce the likelihood of errors. This paper presents such a solution: Virtual Equivalent Real-time Simulation Environment (VERSE). VERSE turns the real-time operating system RTAI (Real-time Application Interface) into an event driven simulator that runs in virtual real time. Designed to keep the original RTAI architecture as intact as possible, and therefore inheriting RTAI's many capabilities, VERSE was implemented with remarkably little change to the RTAI source code. This small footprint together with use of the same API allows users to easily run the same application in both real-time and virtual time environments. VERSE has been used to build a workstation testbed for NASA's Space Interferometry Mission (SIM PlanetQuest) instrument flight software. With its flexible simulation controls and inexpensive setup and replication costs, VERSE will become an invaluable tool in future mission development.

Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora.

PubMed

Ott, Stephan J; Musfeldt, Meike; Ullmann, Uwe; Hampe, Jochen; Schreiber, Stefan

2004-06-01

The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10(1) to 10(3) bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.

Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs.

PubMed

Conrad, Cheyenne C; Gilroyed, Brandon H; McAllister, Tim A; Reuter, Tim

2012-10-01

Non-O157 Shiga toxin producing Escherichia coli (STEC) are gaining recognition as human pathogens, but no standardized method exists to identify them. Sequence analysis revealed that STEC can be classified on the base of variable O antigen regions into different O serotypes. Polymerase chain reaction is a powerful technique for thorough screening and complex diagnosis for these pathogens, but requires a positive control to verify qualitative and/or quantitative DNA-fragment amplification. Due to the pathogenic nature of STEC, controls are not readily available and cell culturing of STEC reference strains requires biosafety conditions of level 2 or higher. In order to bypass this limitation, controls of stacked O-type specific DNA-fragments coding for primer recognition sites were designed to screen for nine STEC serotypes frequently associated with human infection. The synthetic controls were amplified by PCR, cloned into a plasmid vector and transferred into bacteria host cells. Plasmids amplified by bacterial expression were purified, serially diluted and tested as standards for real-time PCR using SYBR Green and TaqMan assays. Utility of synthetic DNA controls was demonstrated in conventional and real-time PCR assays and validated with DNA from natural STEC strains. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of Simplexa HSV 1 & 2 PCR with culture, immunofluorescence, and laboratory-developed TaqMan PCR for detection of herpes simplex virus in swab specimens.

PubMed

Gitman, Melissa R; Ferguson, David; Landry, Marie L

2013-11-01

The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type.

NDBC - NDBC Real-Time Data

Science.gov Websites

Subtropical Storm Alberto. NDBC Real-Time Data NDBC moored buoy, C-MAN, and drifting buoy data are available in real-time through selecting either: NDBC Station locator map: a series of regional maps which show : a tabular list of station identifiers. Real-time data are available for the last 45 days (at least

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.

PubMed

Weighardt, Florian; Barbati, Cristina; Paoletti, Claudia; Querci, Maddalena; Kay, Simon; De Beuckeleer, Marc; Van den Eede, Guy

2004-01-01

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement

Evaluation and utilization of preassembled frozen commercial fast real-time qPCR master mixes for detection of cytomegalovirus and BK virus.

PubMed

Glover, William A; Atienza, Ederlyn E; Nesbitt, Shannon; Kim, Woo J; Castor, Jared; Cook, Linda; Jerome, Keith R

2016-01-01

Quantitative DNA detection of cytomegalovirus (CMV) and BK virus (BKV) is critical in the management of transplant patients. Quantitative laboratory-developed procedures for CMV and BKV have been described in which much of the processing is automated, resulting in rapid, reproducible, and high-throughput testing of transplant patients. To increase the efficiency of such assays, the performance and stability of four commercial preassembled frozen fast qPCR master mixes (Roche FastStart Universal Probe Master Mix with Rox, Bio-Rad SsoFast Probes Supermix with Rox, Life Technologies TaqMan FastAdvanced Master Mix, and Life Technologies Fast Universal PCR Master Mix), in combination with in-house designed primers and probes, was evaluated using controls and standards from standard CMV and BK assays. A subsequent parallel evaluation using patient samples was performed comparing the performance of freshly prepared assay mixes versus aliquoted frozen master mixes made with two of the fast qPCR mixes (Life Technologies TaqMan FastAdvanced Master Mix, and Bio-Rad SsoFast Probes Supermix with Rox), chosen based on their performance and compatibility with existing PCR cycling conditions. The data demonstrate that the frozen master mixes retain excellent performance over a period of at least 10 weeks. During the parallel testing using clinical specimens, no difference in quantitative results was observed between the preassembled frozen master mixes and freshly prepared master mixes. Preassembled fast real-time qPCR frozen master mixes perform well and represent an additional strategy laboratories can implement to reduce assay preparation times, and to minimize technical errors and effort necessary to perform clinical PCR. © 2015 Wiley Periodicals, Inc.

Comparison of Versant HBV DNA 3.0 and COBAS AmpliPrep-COBAS TaqMan assays for hepatitis B DNA quantitation: Possible clinical implications.

PubMed

Garbuglia, A R; Angeletti, C; Lauria, F N; Zaccaro, P; Cocca, A M; Pisciotta, M; Solmone, M; Capobianchi, M R

2007-12-01

We compared two commercial assays for HBV DNA quantitation, Versant HBV 3.0, System 340 (bDNA; Bayer Diagnostics) and COBAS AmpliPrep-COBAS TaqMan HBV Test (TaqMan; Roche Diagnostics). Analytical sensitivity, calculated on WHO International Standard, predicted 95% detection rate at 11.4 and 520.2IU/ml for TaqMan and bDNA, respectively. Specificity, established on 50 blood donor samples, was 100% and 84% for TaqMan and bDNA, respectively. When using clinical samples, HBV DNA was detected by TaqMan in 21/55 samples negative to bDNA. Mean values of HBV DNA obtained with bDNA were higher than those obtained with TaqMan (4.09log(10)+/-1.90 versus 3.39log(10)+/-2.41, p<0.001), and 24.4% of samples showed differences in viral load values >0.5log(10), without association with HBV genotype. There was a good correlation for HBV DNA concentrations measured by the two assays (r=0.94; p<0.001) within the overlapping range, and the distribution of results with respect to relevant clinical threshold recently confirmed (20,000 and 2000IU/ml) was similar. Approximately 50% of samples with low HBV DNA, appreciated by TaqMan but not by bDNA, were successfully sequenced in pol region, where drug resistance mutations are located.

Ring trial 2016 for Bluetongue virus detection by real-time RT-PCR in France.

PubMed

Sailleau, Corinne; Viarouge, Cyril; Breard, Emmanuel; Vitour, Damien; Zientara, Stephan

2017-05-01

Since the unexpected emergence of BTV-8 in Northern Europe and the incursion of BTV-8 and 1 in France in 2006-2007, molecular diagnosis has considerably evolved. Several real-time RT-PCR (rtRT-PCR) methods have been developed and published, and are currently being used in many countries across Europe for BTV detection and typing. In France, the national reference laboratory (NRL) for orbiviruses develops and validates 'ready-to-use' kits with private companies for viral RNA detection. The regional laboratories network that was set up to deal with a heavy demand for analyses has used these available kits. From 2007, ring tests were organized to monitor the performance of the French laboratories. This study presents the results of 63 regional laboratories in the ring trial organized in 2016. Blood samples were sent to the laboratories. Participants were asked to use the rtRT-PCR methods in place in their laboratory, for detection of all BTV serotypes and specifically BTV-8. The French regional laboratories are able to detect and genotype BTV in affected animals. Despite the use of several methods (i.e. RNA extraction and different commercial rtRT-PCRs), the network is homogeneous. The ring trial demonstrated that the French regional veterinary laboratories have reliable and robust BTV diagnostic tools for BTV genome detection.

Real-Time Simulation

NASA Technical Reports Server (NTRS)

1997-01-01

Coryphaeus Software, founded in 1989 by former NASA electronic engineer Steve Lakowske, creates real-time 3D software. Designer's Workbench, the company flagship product, is a modeling and simulation tool for the development of both static and dynamic 3D databases. Other products soon followed. Activation, specifically designed for game developers, allows developers to play and test the 3D games before they commit to a target platform. Game publishers can shorten development time and prove the "playability" of the title, maximizing their chances of introducing a smash hit. Another product, EasyT, lets users create massive, realistic representation of Earth terrains that can be viewed and traversed in real time. Finally, EasyScene software control the actions among interactive objects within a virtual world. Coryphaeus products are used on Silican Graphics workstation and supercomputers to simulate real-world performance in synthetic environments. Customers include aerospace, aviation, architectural and engineering firms, game developers, and the entertainment industry.

Characterization of real-time computers

NASA Technical Reports Server (NTRS)

Shin, K. G.; Krishna, C. M.

1984-01-01

A real-time system consists of a computer controller and controlled processes. Despite the synergistic relationship between these two components, they have been traditionally designed and analyzed independently of and separately from each other; namely, computer controllers by computer scientists/engineers and controlled processes by control scientists. As a remedy for this problem, in this report real-time computers are characterized by performance measures based on computer controller response time that are: (1) congruent to the real-time applications, (2) able to offer an objective comparison of rival computer systems, and (3) experimentally measurable/determinable. These measures, unlike others, provide the real-time computer controller with a natural link to controlled processes. In order to demonstrate their utility and power, these measures are first determined for example controlled processes on the basis of control performance functionals. They are then used for two important real-time multiprocessor design applications - the number-power tradeoff and fault-masking and synchronization.

Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches.

PubMed

Fernandes, Telmo J R; Costa, Joana; Oliveira, M Beatriz P P; Mafra, Isabel

2018-04-15

Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. In this work, two real-time PCR systems based on the EvaGreen dye and a TaqMan probe are proposed and compared. New primers were designed to target the 16S rRNA gene, as a universal maker for fish detection, with fully demonstrated specificity for a wide range of fish species. Both systems showed similar absolute sensitivities, down to 0.01 pg of fish DNA, and adequate real-time PCR performance parameters. The probe system showed higher relative sensitivity and dynamic range (0.0001-50%) than the EvaGreen (0.05-50%). They were both precise, but trueness was compromised at the highest tested level with the EvaGreen assay. Therefore, both systems were successful, although the probe one exhibited the best performance. Its application to verify labelling compliance of foodstuffs suggested a high level of mislabelling and/or fraudulent practices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Acting to gain information: Real-time reasoning meets real-time perception

NASA Technical Reports Server (NTRS)

Rosenschein, Stan

1994-01-01

Recent advances in intelligent reactive systems suggest new approaches to the problem of deriving task-relevant information from perceptual systems in real time. The author will describe work in progress aimed at coupling intelligent control mechanisms to real-time perception systems, with special emphasis on frame rate visual measurement systems. A model for integrated reasoning and perception will be discussed, and recent progress in applying these ideas to problems of sensor utilization for efficient recognition and tracking will be described.

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl.

PubMed

Smith, Matthew M; Schmutz, Joel; Apelgren, Chloe; Ramey, Andrew M

2015-04-01

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n=105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R(2)=0.694, P=0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species. Published by Elsevier B.V.

A real-time PCR approach to detect predation on anchovy and sardine early life stages

NASA Astrophysics Data System (ADS)

Cuende, Elsa; Mendibil, Iñaki; Bachiller, Eneko; Álvarez, Paula; Cotano, Unai; Rodriguez-Ezpeleta, Naiara

2017-12-01

Recruitment of sardine (Sardina pilchardus Walbaum, 1792) and anchovy (Engraulis encrasicolus Linnaeus, 1758) is thought to be regulated by predation of their eggs and larvae. Predators of sardine and anchovy can be identified by visual taxonomic identification of stomach contents, but this method is time consuming, tedious and may underestimate predation, especially in small predators such as fish larvae. Alternatively, genetic tools may offer a more cost-effective and accurate alternative. Here, we have developed a multiplex real-time polymerase chain reaction (RT-PCR) assay based on TaqMan probes to simultaneously detect sardine and anchovy remains in gut contents of potential predators. The assay combines previously described and newly generated species-specific primers and probes for anchovy and sardine detection respectively, and allows the detection of 0,001 ng of target DNA (which corresponds to about one hundredth of the total DNA present in a single egg). We applied the method to candidate anchovy and sardine egg predators in the Bay of Biscay, Atlantic Mackerel (Scomber scombrus) larvae. Egg predation observed was limited primarily to those stations where sardine and/or anchovy eggs were present. Our developed assay offers a suitable tool to understand the effects of predation on the survival of anchovy and sardine early life stages.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

PubMed

Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

2017-07-01

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

An OPTIMIZE study retrospective analysis for management of telaprevir-treated hepatitis C virus (HCV)-infected patients by use of the Abbott RealTime HCV RNA assay.

PubMed

Sarrazin, Christoph; Dierynck, Inge; Cloherty, Gavin; Ghys, Anne; Janssen, Katrien; Luo, Donghan; Witek, James; Buti, Maria; Picchio, Gaston; De Meyer, Sandra

2015-04-01

Protease inhibitor (PI)-based response-guided triple therapies for hepatitis C virus (HCV) infection are still widely used. Noncirrhotic treatment-naive and prior relapser patients receiving telaprevir-based treatment are eligible for shorter, 24-week total therapy if HCV RNA is undetectable at both weeks 4 and 12. In this study, the concordance in HCV RNA assessments between the Roche High Pure System/Cobas TaqMan and Abbott RealTime HCV RNA assays and the impacts of different HCV RNA cutoffs on treatment outcome were evaluated. A total of 2,629 samples from 663 HCV genotype 1 patients receiving telaprevir/pegylated interferon/ribavirin in OPTIMIZE were analyzed using the High Pure System and reanalyzed using Abbott RealTime (limits of detection, 15.1 IU/ml versus 8.3 IU/ml; limits of quantification, 25 IU/ml versus 12 IU/ml, respectively). Overall, good concordance was observed between the assays. Using undetectable HCV RNA at week 4, 34% of the patients would be eligible for shorter treatment duration with Abbott RealTime versus 72% with the High Pure System. However, using <12 IU/ml for Abbott RealTime, a similar proportion (74%) would be eligible. Of the patients receiving 24-week total therapy, 87% achieved a sustained virologic response with undetectable HCV RNA by the High Pure System or <12 IU/ml by Abbott RealTime; however, 92% of the patients with undetectable HCV RNA by Abbott RealTime achieved a sustained virologic response. Using undetectable HCV RNA as the cutoff, the more sensitive Abbott RealTime assay would identify fewer patients eligible for shorter treatment than the High Pure System. Our data confirm the <12-IU/ml cutoff, as previously established in other studies of the Abbott RealTime assay, to determine eligibility for shortened PI-based HCV treatment. (The study was registered with ClinicalTrials.gov under registration no. NCT01241760.). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

PubMed Central

Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

2017-01-01

Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates. PMID:28120891

Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

PubMed Central

Ranieri, Matthew L.; Ivy, Reid A.; Mitchell, W. Robert; Call, Emma; Masiello, Stephanie N.; Wiedmann, Martin

2012-01-01

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (CT) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (CT > 40), 3 Bacillus isolates showed very weak positive signals (CT = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 101 CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product. PMID:22685148

Diagnosis of bacteremia in pediatric oncologic patients by in-house real-time PCR.

PubMed

Quiles, Milene Gonçalves; Menezes, Liana Carballo; Bauab, Karen de Castro; Gumpl, Elke Kreuscher; Rocchetti, Talita Trevizani; Palomo, Flavia Silva; Carlesse, Fabianne; Pignatari, Antonio Carlos Campos

2015-07-23

Infections are the major cause of morbidity and mortality in children with cancer. Gaining a favorable prognosis for these patients depends on selecting the appropriate therapy, which in turn depends on rapid and accurate microbiological diagnosis. This study employed real-time PCR (qPCR) to identify the main pathogens causing bloodstream infection (BSI) in patients treated at the Pediatric Oncology Institute IOP-GRAACC-UNIFESP-Brazil. Antimicrobial resistance genes were also investigated using this methodology. A total of 248 samples from BACTEC® blood culture bottles and 99 whole-blood samples collected in tubes containing EDTA K2 Gel were isolated from 137 patients. All samples were screened by specific Gram probes for multiplex qPCR. Seventeen sequences were evaluated using gender-specific TaqMan probes and the resistance genes bla SHV, bla TEM, bla CTX, bla KPC, bla IMP, bla SPM, bla VIM, vanA, vanB and mecA were detected using the SYBR Green method. Positive qPCR results were obtained in 112 of the blood culture bottles (112/124), and 90 % agreement was observed between phenotypic and molecular microbial detection methods. For bacterial and fungal identification, the performance test showed: sensitivity 87 %; specificity 91 %; NPV 90 %; PPV 89 % and accuracy of 89 % when compared with the phenotypic method. The mecA gene was detected in 37 samples, extended-spectrum β-lactamases were detected in six samples and metallo-β-lactamase coding genes in four samples, with 60 % concordance between the two methods. The qPCR on whole blood detected eight samples possessing the mecA gene and one sample harboring the vanB gene. The bla KPC, bla VIM, bla IMP and bla SHV genes were not detected in this study. Real-time PCR is a useful tool in the early identification of pathogens and antimicrobial resistance genes from bloodstream infections of pediatric oncologic patients.

Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR

PubMed Central

Mull, Bonnie J.; Narayanan, Jothikumar; Hill, Vincent R.

2013-01-01

Primary amebic meningoencephalitis (PAM) is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri. In 2010, the first confirmed case of PAM acquired in Minnesota highlighted the need for improved detection and quantification methods in order to study the changing ecology of N. fowleri and to evaluate potential risk factors for increased exposure. An immunomagnetic separation (IMS) procedure and real-time PCR TaqMan assay were developed to recover and quantify N. fowleri in water and sediment samples. When one liter of lake water was seeded with N. fowleri strain CDC:V212, the method had an average recovery of 46% and detection limit of 14 amebas per liter of water. The method was then applied to sediment and water samples with unknown N. fowleri concentrations, resulting in positive direct detections by real-time PCR in 3 out of 16 samples and confirmation of N. fowleri culture in 6 of 16 samples. This study has resulted in a new method for detection and quantification of N. fowleri in water and sediment that should be a useful tool to facilitate studies of the physical, chemical, and biological factors associated with the presence and dynamics of N. fowleri in environmental systems. PMID:24228172

A real-time architecture for time-aware agents.

PubMed

Prouskas, Konstantinos-Vassileios; Pitt, Jeremy V

2004-06-01

This paper describes the specification and implementation of a new three-layer time-aware agent architecture. This architecture is designed for applications and environments where societies of humans and agents play equally active roles, but interact and operate in completely different time frames. The architecture consists of three layers: the April real-time run-time (ART) layer, the time aware layer (TAL), and the application agents layer (AAL). The ART layer forms the underlying real-time agent platform. An original online, real-time, dynamic priority-based scheduling algorithm is described for scheduling the computation time of agent processes, and it is shown that the algorithm's O(n) complexity and scalable performance are sufficient for application in real-time domains. The TAL layer forms an abstraction layer through which human and agent interactions are temporally unified, that is, handled in a common way irrespective of their temporal representation and scale. A novel O(n2) interaction scheduling algorithm is described for predicting and guaranteeing interactions' initiation and completion times. The time-aware predicting component of a workflow management system is also presented as an instance of the AAL layer. The described time-aware architecture addresses two key challenges in enabling agents to be effectively configured and applied in environments where humans and agents play equally active roles. It provides flexibility and adaptability in its real-time mechanisms while placing them under direct agent control, and it temporally unifies human and agent interactions.

Real Time Conference 2014 Overview

NASA Astrophysics Data System (ADS)

Nomachi, Masaharu

2015-06-01

This article presents an overview of the 19th Real Time Conference held last May 26-30, 2014, at the Nara Prefectural New Public Hall, Nara, Japan, organized by the Research Center for Nuclear Physics of the Osaka University. The program included many invited talks and oral sessions offering an extensive overview on the following topics: real-time system architectures, intelligent signal processing, fast data transfer links and networks, trigger systems, data acquisition, processing-farms, control, monitoring and test systems, emerging real-time technologies, new standards, real-time safety and security, and some feedback on experiences. In parallel to the oral and poster presentations, industrial exhibits by companies, workshops and short courses also ran through the week.

Real-time flutter identification

NASA Technical Reports Server (NTRS)

Roy, R.; Walker, R.

1985-01-01

The techniques and a FORTRAN 77 MOdal Parameter IDentification (MOPID) computer program developed for identification of the frequencies and damping ratios of multiple flutter modes in real time are documented. Physically meaningful model parameterization was combined with state of the art recursive identification techniques and applied to the problem of real time flutter mode monitoring. The performance of the algorithm in terms of convergence speed and parameter estimation error is demonstrated for several simulated data cases, and the results of actual flight data analysis from two different vehicles are presented. It is indicated that the algorithm is capable of real time monitoring of aircraft flutter characteristics with a high degree of reliability.

HEVC real-time decoding

NASA Astrophysics Data System (ADS)

Bross, Benjamin; Alvarez-Mesa, Mauricio; George, Valeri; Chi, Chi Ching; Mayer, Tobias; Juurlink, Ben; Schierl, Thomas

2013-09-01

The new High Efficiency Video Coding Standard (HEVC) was finalized in January 2013. Compared to its predecessor H.264 / MPEG4-AVC, this new international standard is able to reduce the bitrate by 50% for the same subjective video quality. This paper investigates decoder optimizations that are needed to achieve HEVC real-time software decoding on a mobile processor. It is shown that HEVC real-time decoding up to high definition video is feasible using instruction extensions of the processor while decoding 4K ultra high definition video in real-time requires additional parallel processing. For parallel processing, a picture-level parallel approach has been chosen because it is generic and does not require bitstreams with special indication.

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

PubMed Central

2010-01-01

Background The hepatitis C virus (HCV) genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM), at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection. PMID:20529244

Real-time PCR in virology.

PubMed

Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

2002-03-15

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials

PubMed Central

Ahmed, Ahmed; Engelberts, Mirjam F. M.; Boer, Kimberly R.; Ahmed, Niyaz; Hartskeerl, Rudy A.

2009-01-01

Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the

MARTe: A Multiplatform Real-Time Framework

NASA Astrophysics Data System (ADS)

Neto, André C.; Sartori, Filippo; Piccolo, Fabio; Vitelli, Riccardo; De Tommasi, Gianmaria; Zabeo, Luca; Barbalace, Antonio; Fernandes, Horacio; Valcarcel, Daniel F.; Batista, Antonio J. N.

2010-04-01

Development of real-time applications is usually associated with nonportable code targeted at specific real-time operating systems. The boundary between hardware drivers, system services, and user code is commonly not well defined, making the development in the target host significantly difficult. The Multithreaded Application Real-Time executor (MARTe) is a framework built over a multiplatform library that allows the execution of the same code in different operating systems. The framework provides the high-level interfaces with hardware, external configuration programs, and user interfaces, assuring at the same time hard real-time performances. End-users of the framework are required to define and implement algorithms inside a well-defined block of software, named Generic Application Module (GAM), that is executed by the real-time scheduler. Each GAM is reconfigurable with a set of predefined configuration meta-parameters and interchanges information using a set of data pipes that are provided as inputs and required as output. Using these connections, different GAMs can be chained either in series or parallel. GAMs can be developed and debugged in a non-real-time system and, only once the robustness of the code and correctness of the algorithm are verified, deployed to the real-time system. The software also supplies a large set of utilities that greatly ease the interaction and debugging of a running system. Among the most useful are a highly efficient real-time logger, HTTP introspection of real-time objects, and HTTP remote configuration. MARTe is currently being used to successfully drive the plasma vertical stabilization controller on the largest magnetic confinement fusion device in the world, with a control loop cycle of 50 ?s and a jitter under 1 ?s. In this particular project, MARTe is used with the Real-Time Application Interface (RTAI)/Linux operating system exploiting the new ?86 multicore processors technology.

GNSS global real-time augmentation positioning: Real-time precise satellite clock estimation, prototype system construction and performance analysis

NASA Astrophysics Data System (ADS)

Chen, Liang; Zhao, Qile; Hu, Zhigang; Jiang, Xinyuan; Geng, Changjiang; Ge, Maorong; Shi, Chuang

2018-01-01

Lots of ambiguities in un-differenced (UD) model lead to lower calculation efficiency, which isn't appropriate for the high-frequency real-time GNSS clock estimation, like 1 Hz. Mixed differenced model fusing UD pseudo-range and epoch-differenced (ED) phase observations has been introduced into real-time clock estimation. In this contribution, we extend the mixed differenced model for realizing multi-GNSS real-time clock high-frequency updating and a rigorous comparison and analysis on same conditions are performed to achieve the best real-time clock estimation performance taking the efficiency, accuracy, consistency and reliability into consideration. Based on the multi-GNSS real-time data streams provided by multi-GNSS Experiment (MGEX) and Wuhan University, GPS + BeiDou + Galileo global real-time augmentation positioning prototype system is designed and constructed, including real-time precise orbit determination, real-time precise clock estimation, real-time Precise Point Positioning (RT-PPP) and real-time Standard Point Positioning (RT-SPP). The statistical analysis of the 6 h-predicted real-time orbits shows that the root mean square (RMS) in radial direction is about 1-5 cm for GPS, Beidou MEO and Galileo satellites and about 10 cm for Beidou GEO and IGSO satellites. Using the mixed differenced estimation model, the prototype system can realize high-efficient real-time satellite absolute clock estimation with no constant clock-bias and can be used for high-frequency augmentation message updating (such as 1 Hz). The real-time augmentation message signal-in-space ranging error (SISRE), a comprehensive accuracy of orbit and clock and effecting the users' actual positioning performance, is introduced to evaluate and analyze the performance of GPS + BeiDou + Galileo global real-time augmentation positioning system. The statistical analysis of real-time augmentation message SISRE is about 4-7 cm for GPS, whlile 10 cm for Beidou IGSO/MEO, Galileo and about 30 cm

Determining miRNA Expression Levels in Degraded RNA Samples Using Real-Time RT-qPCR and Microarray Technologies

PubMed Central

Tighe, S.; Holbrook, J.; Nadella, V.; Carmical, R.; Sol-Church, K.; Yueng, A.T.; Chittur, S.

2011-01-01

The Nucleic Acid Research Group (NARG) has previously conducted studies evaluating the impact of RNA integrity and priming strategies on cDNA synthesis and real-time RT-qPCR. The results of last year's field study as it relates to degraded RNA will be presented. In continuation of the RNA integrity theme, this year's study was designed to evaluate the impact of RNA integrity on the analysis of miRNA expression using real-time RT-qPCR. Target section was based on data obtained by the Microarray Research Group (MARG) and other published data from next gen sequencing. These 9 miRNAs represent three groups of miRNA that are expressed at low, medium or high levels in the First Choice human brain reference RNA sample. Two popular RT priming strategies tested in this study include the Megaplex miRNA TaqMan assay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences). The basis for the ABI assay design is a target-specific stem-loop structure and reverse-transcription primer, while the Qiagen design combines poly(A) tailing and a universal reverse transcription in one cDNA synthesis reaction. For this study, the human brain reference RNA was subject to controlled degradation using RNase A to RIN (RNA Integrity Number) values of 7 (good), 4 (moderately degraded), and 2 (severely degraded).These templates were then used to assess both RT methods. In addition to this real-time RT-qPCR data, the same RNA templates were further analyzed using universal poly(A) tailing and hybridization to Affymetrix miRNA GeneChips. This talk will provide insights into RT priming strategies for miRNA and contrast the qPCR results obtained using different technologies.

Comparison of Simplexa HSV 1 & 2 PCR with Culture, Immunofluorescence, and Laboratory-Developed TaqMan PCR for Detection of Herpes Simplex Virus in Swab Specimens

PubMed Central

Gitman, Melissa R.; Ferguson, David

2013-01-01

The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type. PMID:24006008

Scheduling Dependent Real-Time Activities

DTIC Science & Technology

1990-08-01

dependency relationships in a way that is suitable for all real - time systems . This thesis provides an algorithm, called DASA, that is effective for...scheduling the class of real - time systems known as supervisory control systems. Simulation experiments that account for the time required to make scheduling

Real-Time Nonlinear Optical Information Processing.

DTIC Science & Technology

1979-06-01

operations aree presented. One approach realizes the halftone method of nonlinear optical processing in real time by replacing the conventional...photographic recording medium with a real-time image transducer. In the second approach halftoning is eliminated and the real-time device is used directly

Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR.

PubMed

Espiñeira, Montserrat; Vieites, Juan M

2012-12-15

The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay. The method can be applied to all kinds of products; fresh, frozen and processed, including those undergoing intensive processes of transformation. This methodology was validated to check how the degree of food processing affects the method and the detection of each species. Moreover, it was applied to 34 commercial samples to evaluate the labeling of products made from them. The methodology herein developed is useful to check the fulfillment of labeling regulations for seafood products and to verify traceability in commercial trade and for fisheries control. Copyright © 2012 Elsevier Ltd. All rights reserved.

Real-Time and Near Real-Time Data for Space Weather Applications and Services

NASA Astrophysics Data System (ADS)

Singer, H. J.; Balch, C. C.; Biesecker, D. A.; Matsuo, T.; Onsager, T. G.

2015-12-01

Space weather can be defined as conditions in the vicinity of Earth and in the interplanetary environment that are caused primarily by solar processes and influenced by conditions on Earth and its atmosphere. Examples of space weather are the conditions that result from geomagnetic storms, solar particle events, and bursts of intense solar flare radiation. These conditions can have impacts on modern-day technologies such as GPS or electric power grids and on human activities such as astronauts living on the International Space Station or explorers traveling to the moon or Mars. While the ultimate space weather goal is accurate prediction of future space weather conditions, for many applications and services, we rely on real-time and near-real time observations and model results for the specification of current conditions. In this presentation, we will describe the space weather system and the need for real-time and near-real time data that drive the system, characterize conditions in the space environment, and are used by models for assimilation and validation. Currently available data will be assessed and a vision for future needs will be given. The challenges for establishing real-time data requirements, as well as acquiring, processing, and disseminating the data will be described, including national and international collaborations. In addition to describing how the data are used for official government products, we will also give examples of how these data are used by both the public and private sector for new applications that serve the public.

Rapid and Reliable Genotyping of HLA-B*57:01 in Four Chinese Populations Using a Single-Tube Duplex Real-Time Polymerase Chain Reaction Assay.

PubMed

Han, Min; Kang, Xing; Liu, Zhengbin; Zhang, Tingting; Li, Yanwei; Chen, Chao; Wang, Huijuan

2017-07-01

HLA-B*57:01 is strongly associated with severe adverse drug reaction induced by the anti-HIV drug abacavir (ABC) and antibiotic flucloxacillin. This study was dedicated to establishing a new method for HLA-B*57:01 genotyping and investigating the HLA-B*57:01 distribution pattern in four Chinese populations. A single-tube duplex real-time polymerase chain reaction (PCR) system was established by combining the amplification refractory mutation system and TaqMan probe. The reliability of this assay was validated by comparing the genotyping results with those by sequence-based typing. With this assay, the distribution of HLA-B*57:01 in 354 blood samples from four ethnic groups, namely, Han, Tibetan, Uighur, and Buyei, was determined. A 100% concordance was observed between the results of real-time PCR and sequence-based typing in 50 Uighur samples. As low as 0.016 ng DNA that carried HLA-B*57:01 could be detected with this assay. HLA-B*57:01 carriers identified in 100 Northern Han Chinese, 104 Buyeis, 100 Tibetans, and 50 Uighurs were 0, 1 (0.96%), 3 (3%), and 6 (12%), respectively. The carrier rate of HLA-B*57:01 in Uighur was significantly higher than those in Northern Han (p = .001) and Buyei (p = .005). The newly established real-time PCR assay provides a rapid and reliable tool for HLA-B*57:01 allele screening before the prescription of ABC and flucloxacillin in clinical practice.

Development of a Rickettsia bellii-Specific TaqMan Assay Targeting the Citrate Synthase Gene.

PubMed

Hecht, Joy A; Allerdice, Michelle E J; Krawczak, Felipe S; Labruna, Marcelo B; Paddock, Christopher D; Karpathy, Sandor E

2016-11-01

Rickettsia bellii is a rickettsial species of unknown pathogenicity that infects argasid and ixodid ticks throughout the Americas. Many molecular assays used to detect spotted fever group (SFG) Rickettsia species do not detect R. bellii, so that infection with this bacterium may be concealed in tick populations when assays are used that screen specifically for SFG rickettsiae. We describe the development and validation of a R. bellii-specific, quantitative, real-time PCR TaqMan assay that targets a segment of the citrate synthase (gltA) gene. The specificity of this assay was validated against a panel of DNA samples that included 26 species of Rickettsia, Orientia, Ehrlichia, Anaplasma, and Bartonella, five samples of tick and human DNA, and DNA from 20 isolates of R. bellii, including 11 from North America and nine from South America. A R. bellii control plasmid was constructed, and serial dilutions of the plasmid were used to determine the limit of detection of the assay to be one copy per 4 µl of template DNA. This assay can be used to better determine the role of R. bellii in the epidemiology of tick-borne rickettsioses in the Western Hemisphere. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

Software Design Methods for Real-Time Systems

DTIC Science & Technology

1989-12-01

This module describes the concepts and methods used in the software design of real time systems . It outlines the characteristics of real time systems , describes...the role of software design in real time system development, surveys and compares some software design methods for real - time systems , and

T.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory

PubMed Central

Monnier, Stéphanie; Cox, David G; Albion, Tim; Canzian, Federico

2005-01-01

Background Single Nucleotide Polymorphism (SNP) genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS) need tools to organize the data flow. Results We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements. Conclusion We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories whithout a more sophisticated and expensive system, such as a LIMS. PMID:16221298

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

PubMed

Harvey, John J; Chester, Stephanie; Burke, Stephen A; Ansbro, Marisela; Aden, Tricia; Gose, Remedios; Sciulli, Rebecca; Bai, Jing; DesJardin, Lucy; Benfer, Jeffrey L; Hall, Joshua; Smole, Sandra; Doan, Kimberly; Popowich, Michael D; St George, Kirsten; Quinlan, Tammy; Halse, Tanya A; Li, Zhen; Pérez-Osorio, Ailyn C; Glover, William A; Russell, Denny; Reisdorf, Erik; Whyte, Thomas; Whitaker, Brett; Hatcher, Cynthia; Srinivasan, Velusamy; Tatti, Kathleen; Tondella, Maria Lucia; Wang, Xin; Winchell, Jonas M; Mayer, Leonard W; Jernigan, Daniel; Mawle, Alison C

2016-02-01

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators. Copyright © 2015 Elsevier B.V. All rights reserved.

Research of real-time communication software

NASA Astrophysics Data System (ADS)

Li, Maotang; Guo, Jingbo; Liu, Yuzhong; Li, Jiahong

2003-11-01

Real-time communication has been playing an increasingly important role in our work, life and ocean monitor. With the rapid progress of computer and communication technique as well as the miniaturization of communication system, it is needed to develop the adaptable and reliable real-time communication software in the ocean monitor system. This paper involves the real-time communication software research based on the point-to-point satellite intercommunication system. The object-oriented design method is adopted, which can transmit and receive video data and audio data as well as engineering data by satellite channel. In the real-time communication software, some software modules are developed, which can realize the point-to-point satellite intercommunication in the ocean monitor system. There are three advantages for the real-time communication software. One is that the real-time communication software increases the reliability of the point-to-point satellite intercommunication system working. Second is that some optional parameters are intercalated, which greatly increases the flexibility of the system working. Third is that some hardware is substituted by the real-time communication software, which not only decrease the expense of the system and promotes the miniaturization of communication system, but also aggrandizes the agility of the system.

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

PubMed Central

2012-01-01

Background Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. Methods A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly. Results The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. Conclusions The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either

Hard real-time closed-loop electrophysiology with the Real-Time eXperiment Interface (RTXI)

PubMed Central

George, Ansel; Dorval, Alan D.; Christini, David J.

2017-01-01

The ability to experimentally perturb biological systems has traditionally been limited to static pre-programmed or operator-controlled protocols. In contrast, real-time control allows dynamic probing of biological systems with perturbations that are computed on-the-fly during experimentation. Real-time control applications for biological research are available; however, these systems are costly and often restrict the flexibility and customization of experimental protocols. The Real-Time eXperiment Interface (RTXI) is an open source software platform for achieving hard real-time data acquisition and closed-loop control in biological experiments while retaining the flexibility needed for experimental settings. RTXI has enabled users to implement complex custom closed-loop protocols in single cell, cell network, animal, and human electrophysiology studies. RTXI is also used as a free and open source, customizable electrophysiology platform in open-loop studies requiring online data acquisition, processing, and visualization. RTXI is easy to install, can be used with an extensive range of external experimentation and data acquisition hardware, and includes standard modules for implementing common electrophysiology protocols. PMID:28557998

ControlShell - A real-time software framework

NASA Technical Reports Server (NTRS)

Schneider, Stanley A.; Ullman, Marc A.; Chen, Vincent W.

1991-01-01

ControlShell is designed to enable modular design and impplementation of real-time software. It is an object-oriented tool-set for real-time software system programming. It provides a series of execution and data interchange mechansims that form a framework for building real-time applications. These mechanisms allow a component-based approach to real-time software generation and mangement. By defining a set of interface specifications for intermodule interaction, ControlShell provides a common platform that is the basis for real-time code development and exchange.

OPAD-EDIFIS Real-Time Processing

NASA Technical Reports Server (NTRS)

Katsinis, Constantine

1997-01-01

The Optical Plume Anomaly Detection (OPAD) detects engine hardware degradation of flight vehicles through identification and quantification of elemental species found in the plume by analyzing the plume emission spectra in a real-time mode. Real-time performance of OPAD relies on extensive software which must report metal amounts in the plume faster than once every 0.5 sec. OPAD software previously written by NASA scientists performed most necessary functions at speeds which were far below what is needed for real-time operation. The research presented in this report improved the execution speed of the software by optimizing the code without changing the algorithms and converting it into a parallelized form which is executed in a shared-memory multiprocessor system. The resulting code was subjected to extensive timing analysis. The report also provides suggestions for further performance improvement by (1) identifying areas of algorithm optimization, (2) recommending commercially available multiprocessor architectures and operating systems to support real-time execution and (3) presenting an initial study of fault-tolerance requirements.

Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.

PubMed

Bièche, I; Olivi, M; Champème, M H; Vidaud, D; Lidereau, R; Vidaud, M

1998-11-23

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccndl and erbB2) in breast tumors. Extra copies of myc, ccndl and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings.

Research in Distributed Real-Time Systems

NASA Technical Reports Server (NTRS)

Mukkamala, R.

1997-01-01

This document summarizes the progress we have made on our study of issues concerning the schedulability of real-time systems. Our study has produced several results in the scalability issues of distributed real-time systems. In particular, we have used our techniques to resolve schedulability issues in distributed systems with end-to-end requirements. During the next year (1997-98), we propose to extend the current work to address the modeling and workload characterization issues in distributed real-time systems. In particular, we propose to investigate the effect of different workload models and component models on the design and the subsequent performance of distributed real-time systems.

Real-time Space-time Integration in GIScience and Geography

PubMed Central

Richardson, Douglas B.

2013-01-01

Space-time integration has long been the topic of study and speculation in geography. However, in recent years an entirely new form of space-time integration has become possible in GIS and GIScience: real-time space-time integration and interaction. While real-time spatiotemporal data is now being generated almost ubiquitously, and its applications in research and commerce are widespread and rapidly accelerating, the ability to continuously create and interact with fused space-time data in geography and GIScience is a recent phenomenon, made possible by the invention and development of real-time interactive (RTI) GPS/GIS technology and functionality in the late 1980s and early 1990s. This innovation has since functioned as a core change agent in geography, cartography, GIScience and many related fields, profoundly realigning traditional relationships and structures, expanding research horizons, and transforming the ways geographic data is now collected, mapped, modeled, and used, both in geography and in science and society more broadly. Real-time space-time interactive functionality remains today the underlying process generating the current explosion of fused spatiotemporal data, new geographic research initiatives, and myriad geospatial applications in governments, businesses, and society. This essay addresses briefly the development of these real-time space-time functions and capabilities; their impact on geography, cartography, and GIScience; and some implications for how discovery and change can occur in geography and GIScience, and how we might foster continued innovation in these fields. PMID:24587490

Real-time Space-time Integration in GIScience and Geography.

PubMed

Richardson, Douglas B

2013-01-01

Space-time integration has long been the topic of study and speculation in geography. However, in recent years an entirely new form of space-time integration has become possible in GIS and GIScience: real-time space-time integration and interaction. While real-time spatiotemporal data is now being generated almost ubiquitously, and its applications in research and commerce are widespread and rapidly accelerating, the ability to continuously create and interact with fused space-time data in geography and GIScience is a recent phenomenon, made possible by the invention and development of real-time interactive (RTI) GPS/GIS technology and functionality in the late 1980s and early 1990s. This innovation has since functioned as a core change agent in geography, cartography, GIScience and many related fields, profoundly realigning traditional relationships and structures, expanding research horizons, and transforming the ways geographic data is now collected, mapped, modeled, and used, both in geography and in science and society more broadly. Real-time space-time interactive functionality remains today the underlying process generating the current explosion of fused spatiotemporal data, new geographic research initiatives, and myriad geospatial applications in governments, businesses, and society. This essay addresses briefly the development of these real-time space-time functions and capabilities; their impact on geography, cartography, and GIScience; and some implications for how discovery and change can occur in geography and GIScience, and how we might foster continued innovation in these fields.

A Real-Time Systems Symposium Preprint.

DTIC Science & Technology

1983-09-01

Real - Time Systems Symposium Preprint Interim Tech...estimate of the occurence of the error. Unclassii ledSECUqITY CLASSIF’ICA T" NO MI*IA If’ inDI /’rrd erter for~~ble. ’Corrputnqg A REAL - TIME SYSTEMS SYMPOSIUM...ABSTRACT This technical report contains a preprint of a paper accepted for presentation at the REAL - TIME SYSTEMS SYMPOSIUM, Arlington,

Analysis of real-time vibration data

USGS Publications Warehouse

Safak, E.

2005-01-01

In recent years, a few structures have been instrumented to provide continuous vibration data in real time, recording not only large-amplitude motions generated by extreme loads, but also small-amplitude motions generated by ambient loads. The main objective in continuous recording is to track any changes in structural characteristics, and to detect damage after an extreme event, such as an earthquake or explosion. The Fourier-based spectral analysis methods have been the primary tool to analyze vibration data from structures. In general, such methods do not work well for real-time data, because real-time data are mainly composed of ambient vibrations with very low amplitudes and signal-to-noise ratios. The long duration, linearity, and the stationarity of ambient data, however, allow us to utilize statistical signal processing tools, which can compensate for the adverse effects of low amplitudes and high noise. The analysis of real-time data requires tools and techniques that can be applied in real-time; i.e., data are processed and analyzed while being acquired. This paper presents some of the basic tools and techniques for processing and analyzing real-time vibration data. The topics discussed include utilization of running time windows, tracking mean and mean-square values, filtering, system identification, and damage detection.

Flexible real-time magnetic resonance imaging framework.

PubMed

Santos, Juan M; Wright, Graham A; Pauly, John M

2004-01-01

The extension of MR imaging to new applications has demonstrated the limitations of the architecture of current real-time systems. Traditional real-time implementations provide continuous acquisition of data and modification of basic sequence parameters on the fly. We have extended the concept of real-time MRI by designing a system that drives the examinations from a real-time localizer and then gets reconfigured for different imaging modes. Upon operator request or automatic feedback the system can immediately generate a new pulse sequence or change fundamental aspects of the acquisition such as gradient waveforms excitation pulses and scan planes. This framework has been implemented by connecting a data processing and control workstation to a conventional clinical scanner. Key components on the design of this framework are the data communication and control mechanisms, reconstruction algorithms optimized for real-time and adaptability, flexible user interface and extensible user interaction. In this paper we describe the various components that comprise this system. Some of the applications implemented in this framework include real-time catheter tracking embedded in high frame rate real-time imaging and immediate switching between real-time localizer and high-resolution volume imaging for coronary angiography applications.

Real-time photo-magnetic imaging.

PubMed

Nouizi, Farouk; Erkol, Hakan; Luk, Alex; Unlu, Mehmet B; Gulsen, Gultekin

2016-10-01

We previously introduced a new high resolution diffuse optical imaging modality termed, photo-magnetic imaging (PMI). PMI irradiates the object under investigation with near-infrared light and monitors the variations of temperature using magnetic resonance thermometry (MRT). In this paper, we present a real-time PMI image reconstruction algorithm that uses analytic methods to solve the forward problem and assemble the Jacobian matrix much faster. The new algorithm is validated using real MRT measured temperature maps. In fact, it accelerates the reconstruction process by more than 250 times compared to a single iteration of the FEM-based algorithm, which opens the possibility for the real-time PMI.

Making real-time reactive systems reliable

NASA Technical Reports Server (NTRS)

Marzullo, Keith; Wood, Mark

1990-01-01

A reactive system is characterized by a control program that interacts with an environment (or controlled program). The control program monitors the environment and reacts to significant events by sending commands to the environment. This structure is quite general. Not only are most embedded real time systems reactive systems, but so are monitoring and debugging systems and distributed application management systems. Since reactive systems are usually long running and may control physical equipment, fault tolerance is vital. The research tries to understand the principal issues of fault tolerance in real time reactive systems and to build tools that allow a programmer to design reliable, real time reactive systems. In order to make real time reactive systems reliable, several issues must be addressed: (1) How can a control program be built to tolerate failures of sensors and actuators. To achieve this, a methodology was developed for transforming a control program that references physical value into one that tolerates sensors that can fail and can return inaccurate values; (2) How can the real time reactive system be built to tolerate failures of the control program. Towards this goal, whether the techniques presented can be extended to real time reactive systems is investigated; and (3) How can the environment be specified in a way that is useful for writing a control program. Towards this goal, whether a system with real time constraints can be expressed as an equivalent system without such constraints is also investigated.

Visualization of Real-Time Data

NASA Technical Reports Server (NTRS)

Stansifer, Ryan; Engrand, Peter

1996-01-01

In this project we explored various approaches to presenting real-time data from the numerous systems monitored on the space shuttle to computer users. We examined the approach that several projects at the Kennedy Space Center (KSC) used to accomplish this. We undertook to build a prototype system to demonstrate that the Internet and the Java programming language could be used to present the real-time data conveniently. Several Java programs were developed that presented real-time data in different forms including one form that emulated the display screens of the PC GOAL system which is familiar to many at KSC. Also, we developed several communications programs to supply the data continuously. Furthermore, a framework was created using the World Wide Web (WWW) to organize the collection and presentation of the real-time data. We believe our demonstration project shows the great flexibility of the approach. We had no particular use of the data in mind, instead we wanted the most general and the least complex framework possible. People who wish to view data need only know how to use a WWW browser and the address (the URL). People wanting to build WWW documents containing real-time data need only know the values of a few parameters, they do not need to program in Java or any other language. These are stunning advantages over more monolithic systems.

Real-time Enhanced Vision System

NASA Technical Reports Server (NTRS)

Hines, Glenn D.; Rahman, Zia-Ur; Jobson, Daniel J.; Woodell, Glenn A.; Harrah, Steven D.

2005-01-01

Flying in poor visibility conditions, such as rain, snow, fog or haze, is inherently dangerous. However these conditions can occur at nearly any location, so inevitably pilots must successfully navigate through them. At NASA Langley Research Center (LaRC), under support of the Aviation Safety and Security Program Office and the Systems Engineering Directorate, we are developing an Enhanced Vision System (EVS) that combines image enhancement and synthetic vision elements to assist pilots flying through adverse weather conditions. This system uses a combination of forward-looking infrared and visible sensors for data acquisition. A core function of the system is to enhance and fuse the sensor data in order to increase the information content and quality of the captured imagery. These operations must be performed in real-time for the pilot to use while flying. For image enhancement, we are using the LaRC patented Retinex algorithm since it performs exceptionally well for improving low-contrast range imagery typically seen during poor visibility conditions. In general, real-time operation of the Retinex requires specialized hardware. To date, we have successfully implemented a single-sensor real-time version of the Retinex on several different Digital Signal Processor (DSP) platforms. In this paper we give an overview of the EVS and its performance requirements for real-time enhancement and fusion and we discuss our current real-time Retinex implementations on DSPs.

Real-time enhanced vision system

NASA Astrophysics Data System (ADS)

Hines, Glenn D.; Rahman, Zia-ur; Jobson, Daniel J.; Woodell, Glenn A.; Harrah, Steven D.

2005-05-01

Flying in poor visibility conditions, such as rain, snow, fog or haze, is inherently dangerous. However these conditions can occur at nearly any location, so inevitably pilots must successfully navigate through them. At NASA Langley Research Center (LaRC), under support of the Aviation Safety and Security Program Office and the Systems Engineering Directorate, we are developing an Enhanced Vision System (EVS) that combines image enhancement and synthetic vision elements to assist pilots flying through adverse weather conditions. This system uses a combination of forward-looking infrared and visible sensors for data acquisition. A core function of the system is to enhance and fuse the sensor data in order to increase the information content and quality of the captured imagery. These operations must be performed in real-time for the pilot to use while flying. For image enhancement, we are using the LaRC patented Retinex algorithm since it performs exceptionally well for improving low-contrast range imagery typically seen during poor visibility poor visibility conditions. In general, real-time operation of the Retinex requires specialized hardware. To date, we have successfully implemented a single-sensor real-time version of the Retinex on several different Digital Signal Processor (DSP) platforms. In this paper we give an overview of the EVS and its performance requirements for real-time enhancement and fusion and we discuss our current real-time Retinex implementations on DSPs.

Quantitative analysis of the cutaneous Malassezia microbiota in 770 healthy Japanese by age and gender using a real-time PCR assay.

PubMed

Sugita, Takashi; Suzuki, Miho; Goto, Seiko; Nishikawa, Akemi; Hiruma, Masataro; Yamazaki, Takashi; Makimura, Koichi

2010-03-01

Although the lipophilic yeasts of the genus Malassezia are part of the cutaneous microbiota in healthy individuals, they are also associated with several skin diseases, such as seborrheic dermatitis. However, the effects of age and gender on the Malassezia microbiota have not been completely elucidated. We analyzed the cutaneous Malassezia microbiota of 770 healthy Japanese using the highly accurate real-time PCR with a TaqMan probe to investigate the effects of age and gender on the Malassezia population. The numbers of Malassezia cells increased in males up to 16-18 years of age and in females to 10-12 years old, and subsequently decreased gradually in both genders until senescence. Malassezia restricta overwhelmingly predominated at ages over 16-18 years in males and 23-29 years in females. M. globosa and M. restricta together accounted for more than 70% of Malassezia spp. recovered regardless of gender. The total colonization of Malassezia and the ratio of the two major species change with age and gender in humans.

Hard Real-Time: C++ Versus RTSJ

NASA Technical Reports Server (NTRS)

Dvorak, Daniel L.; Reinholtz, William K.

2004-01-01

In the domain of hard real-time systems, which language is better: C++ or the Real-Time Specification for Java (RTSJ)? Although ordinary Java provides a more productive programming environment than C++ due to its automatic memory management, that benefit does not apply to RTSJ when using NoHeapRealtimeThread and non-heap memory areas. As a result, RTSJ programmers must manage non-heap memory explicitly. While that's not a deterrent for veteran real-time programmers-where explicit memory management is common-the lack of certain language features in RTSJ (and Java) makes that manual memory management harder to accomplish safely than in C++. This paper illustrates the problem for practitioners in the context of moving data and managing memory in a real-time producer/consumer pattern. The relative ease of implementation and safety of the C++ programming model suggests that RTSJ has a struggle ahead in the domain of hard real-time applications, despite its other attractive features.

Research Directions in Real-Time Systems.

DTIC Science & Technology

1996-09-01

This report summarizes a survey of published research in real time systems . Material is presented that provides an overview of the topic, focusing on...communications protocols and scheduling techniques. It is noted that real - time systems deserve special attention separate from other areas because of...formal tools for design and analysis of real - time systems . The early work on applications as well as notable theoretical advances are summarized

Real-time dynamic simulation of the Cassini spacecraft using DARTS. Part 2: Parallel/vectorized real-time implementation

NASA Technical Reports Server (NTRS)

Fijany, A.; Roberts, J. A.; Jain, A.; Man, G. K.

1993-01-01

Part 1 of this paper presented the requirements for the real-time simulation of Cassini spacecraft along with some discussion of the DARTS algorithm. Here, in Part 2 we discuss the development and implementation of parallel/vectorized DARTS algorithm and architecture for real-time simulation. Development of the fast algorithms and architecture for real-time hardware-in-the-loop simulation of spacecraft dynamics is motivated by the fact that it represents a hard real-time problem, in the sense that the correctness of the simulation depends on both the numerical accuracy and the exact timing of the computation. For a given model fidelity, the computation should be computed within a predefined time period. Further reduction in computation time allows increasing the fidelity of the model (i.e., inclusion of more flexible modes) and the integration routine.

Real-Time MENTAT programming language and architecture

NASA Technical Reports Server (NTRS)

Grimshaw, Andrew S.; Silberman, Ami; Liu, Jane W. S.

1989-01-01

Real-time MENTAT, a programming environment designed to simplify the task of programming real-time applications in distributed and parallel environments, is described. It is based on the same data-driven computation model and object-oriented programming paradigm as MENTAT. It provides an easy-to-use mechanism to exploit parallelism, language constructs for the expression and enforcement of timing constraints, and run-time support for scheduling and exciting real-time programs. The real-time MENTAT programming language is an extended C++. The extensions are added to facilitate automatic detection of data flow and generation of data flow graphs, to express the timing constraints of individual granules of computation, and to provide scheduling directives for the runtime system. A high-level view of the real-time MENTAT system architecture and programming language constructs is provided.

[Application of transcription mediated amplification and real-time reverse transcription polymerase chain reaction in detection of human immunodeficiency virus RNA].

PubMed

Wu, Daxian; Tao, Shuhui; Liu, Shuiping; Zhou, Jiebin; Tan, Deming; Hou, Zhouhua

2017-07-28

To observe the sensitivity of transcription mediated amplification (TMA), and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA).
 Methods: TMA system was established with TaqMan probes, specific primers, moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase, and reaction substrates. The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro. A total of 60 plasma of HIV infected patients were measured by TMA and Cobas Amplicor HIV-1 Monitor test to observe the positive rate. The correlation and concordance of the above two technologies were investigated by linear regression and Bland-Altman analysis.
 Results: TMA system was established successfully and HIV RNA transcribed standards at concentration of equal or more than 10 copies/mL could be detected by TMA technology. Among 60 samples of plasma from HIV infected patients, 46 were positively detected and 12 were negatively amplified by both TMA and Cobas reagents; 2 samples were positively tested by Cobas reagent but negatively tested by TMA system. The concordance rate of the two methods was 97.1% and the difference of positive detection rate between the two methods was not statistically significant (P>0.05). Linear regression was used for 46 samples which were positively detected by both TMA and Cobas reagents and showed an excellent correlation between the two reagents (r=0.997, P<0.001). Bland-Altma analysis revealed that the mean different value of HIV RNA levels for denary logarithm was 0.02. Forty-four samples were included in 95% of credibility interval of concordance.
 Conclusion: TMA system has the potential of high sensitivity. TMA and real-time RT-PCR keep an excellent correlation and consistency in detecting HIV RNA.

Real Time Conference 2016 Overview

NASA Astrophysics Data System (ADS)

Luchetta, Adriano

2017-06-01

This is a special issue of the IEEE Transactions on Nuclear Science containing papers from the invited, oral, and poster presentation of the 20th Real Time Conference (RT2016). The conference was held June 6-10, 2016, at Centro Congressi Padova “A. Luciani,” Padova, Italy, and was organized by Consorzio RFX (CNR, ENEA, INFN, Università di Padova, Acciaierie Venete SpA) and the Istituto Nazionale di Fisica Nucleare. The Real Time Conference is multidisciplinary and focuses on the latest developments in real-time techniques in high-energy physics, nuclear physics, astrophysics and astroparticle physics, nuclear fusion, medical physics, space instrumentation, nuclear power instrumentation, general radiation instrumentation, and real-time security and safety. Taking place every second year, it is sponsored by the Computer Application in Nuclear and Plasma Sciences technical committee of the IEEE Nuclear and Plasma Sciences Society. RT2016 attracted more than 240 registrants, with a large proportion of young researchers and engineers. It had an attendance of 67 students from many countries.

Real-Time Network Management

DTIC Science & Technology

1998-07-01

Report No. WH97JR00-A002 Sponsored by REAL-TIME NETWORK MANAGEMENT FINAL TECHNICAL REPORT K CD July 1998 CO CO O W O Defense Advanced...Approved for public release; distribution unlimited. t^GquALmmsPEami Report No. WH97JR00-A002 REAL-TIME NETWORK MANAGEMENT Synectics Corporation...2.1.2.1 WAN-class Networks 12 2.1.2.2 IEEE 802.3-class Networks 13 2.2 Task 2 - Object Modeling for Architecture 14 2.2.1 Managed Objects 14 2.2.2

Real-Time Embedded High Performance Computing: Communications Scheduling.

DTIC Science & Technology

1995-06-01

real - time operating system must explicitly limit the degradation of the timing performance of all processes as the number of processes...adequately supported by a real - time operating system , could compound the development problems encountered in the past. Many experts feel that the... real - time operating system support for an MPP, although they all provide some support for distributed real-time applications. A distributed real

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

PubMed

Carvalho, Maria da Gloria S; Tondella, Maria Lucia; McCaustland, Karen; Weidlich, Luciana; McGee, Lesley; Mayer, Leonard W; Steigerwalt, Arnold; Whaley, Melissa; Facklam, Richard R; Fields, Barry; Carlone, George; Ades, Edwin W; Dagan, Ron; Sampson, Jacquelyn S

2007-08-01

The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA

Designing Real-Time Systems in Ada (Trademark).

DTIC Science & Technology

1986-01-01

e a. T * .K Ada .e 6 4J (FINAL REPORT) Real - Time Systems in Ada* Abstract Real-time software differs from other kinds of software in the sense that it...1-2 1.2.2 Functional Focus ...... ................ 1-2 1.3 ROLE OF ADA IN REAL - TIME SYSTEMS DESIGN. ..... 1-3 1.4 SCOPE OF THIS...MODELS OF REAL TIME SYSTEMS 8.1 REQUIREMENTS FOR TEMPORAL BEHAVIOR ANALYSIS . 8-1 8.2 METHODS OF TEMPORAL BEHAVIOR ANALYSIS.... ....... 8-4 8.3

Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

PubMed

Wang, Li-Ping; Lei, Kun

2016-12-01

Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

Detection of Diarrhea Etiology Among U.S. Military Personnel During Exercise Balikatan 2014, Philippines, Using TaqMan Array Cards.

PubMed

Lertsethtakarn, Paphavee; Nakjarung, Kaewkanya; Silapong, Sasikorn; Neesanant, Pimmnapar; Sakpaisal, Pimmada; Bodhidatta, Ladaporn; Liu, Jie; Houpt, Eric; Velasco, John Mark; Macareo, Louis R; Swierczewski, Brett E; Mason, Carl J

2016-11-01

Military personnel are vulnerable to diarrhea. Diarrheal disease is common when deployed for operations or exercise in developing countries. Although diarrheal disease is transient, cumulative time lost and medical asset can have a significant impact on military operations. Currently, diagnostics of diarrheal etiology typically relies on a mixture of conventional bacteriology, enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR)-based methods including real-time PCR. These methods, however, can be time and labor intensive, although the identification of diarrheal etiology needs to be informative and rapid for treatment and prevention. Real-time PCR has been increasingly used to identify pathogens. Real-time PCR panels of common diarrheal pathogens have been developed, but several diarrheal pathogens are not included in the panel. An expanded and customizable panel to detect diarrhea etiology has been developed employing TaqMan Array Card (TAC) technology. TAC performs 384 real-time PCR reactions simultaneously. As currently configured for diarrheal disease by the University of Virginia, a maximum of 8 samples can be tested simultaneously with approximately 48 target pathogens per sample including bacteria, fungi, helminths, protozoan parasites, and viruses. TAC diarrheal disease panels have been successfully applied to detect pathogens in acute diarrheal stool samples from young children in several international multicenter diarrhea studies. In this study, TAC was applied to stool samples collected under an approved human use protocol from military personnel with acute diarrhea participating in the annual joint military exercise, Balikatan, between the Republic of the Philippines and the United States in 2014. Several established pathogen-specific real-time PCR detection assays were also performed in parallel for comparative purposes. TAC was applied to 7 stool samples. Campylobacter spp. was the most common diarrheal disease pathogen detected

A tool for modeling concurrent real-time computation

NASA Technical Reports Server (NTRS)

Sharma, D. D.; Huang, Shie-Rei; Bhatt, Rahul; Sridharan, N. S.

1990-01-01

Real-time computation is a significant area of research in general, and in AI in particular. The complexity of practical real-time problems demands use of knowledge-based problem solving techniques while satisfying real-time performance constraints. Since the demands of a complex real-time problem cannot be predicted (owing to the dynamic nature of the environment) powerful dynamic resource control techniques are needed to monitor and control the performance. A real-time computation model for a real-time tool, an implementation of the QP-Net simulator on a Symbolics machine, and an implementation on a Butterfly multiprocessor machine are briefly described.

Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae)

PubMed Central

Li, Dongmei; Fan, Qing-Hai; Waite, David W.; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith

2015-01-01

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide. PMID:26147599

Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

PubMed

Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

2005-09-01

1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

Real-time video quality monitoring

NASA Astrophysics Data System (ADS)

Liu, Tao; Narvekar, Niranjan; Wang, Beibei; Ding, Ran; Zou, Dekun; Cash, Glenn; Bhagavathy, Sitaram; Bloom, Jeffrey

2011-12-01

The ITU-T Recommendation G.1070 is a standardized opinion model for video telephony applications that uses video bitrate, frame rate, and packet-loss rate to measure the video quality. However, this model was original designed as an offline quality planning tool. It cannot be directly used for quality monitoring since the above three input parameters are not readily available within a network or at the decoder. And there is a great room for the performance improvement of this quality metric. In this article, we present a real-time video quality monitoring solution based on this Recommendation. We first propose a scheme to efficiently estimate the three parameters from video bitstreams, so that it can be used as a real-time video quality monitoring tool. Furthermore, an enhanced algorithm based on the G.1070 model that provides more accurate quality prediction is proposed. Finally, to use this metric in real-world applications, we present an example emerging application of real-time quality measurement to the management of transmitted videos, especially those delivered to mobile devices.

17 CFR 23.205 - Real-time public reporting.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 17 Commodity and Securities Exchanges 1 2013-04-01 2013-04-01 false Real-time public reporting. 23... Swap Dealers and Major Swap Participants § 23.205 Real-time public reporting. (a) Real-time public... information and swap transaction and pricing data required to be reported in accordance with the real-time...

17 CFR 23.205 - Real-time public reporting.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 1 2014-04-01 2014-04-01 false Real-time public reporting. 23... Swap Dealers and Major Swap Participants § 23.205 Real-time public reporting. (a) Real-time public... information and swap transaction and pricing data required to be reported in accordance with the real-time...

Heterogeneous Embedded Real-Time Systems Environment

DTIC Science & Technology

2003-12-01

AFRL-IF-RS-TR-2003-290 Final Technical Report December 2003 HETEROGENEOUS EMBEDDED REAL - TIME SYSTEMS ENVIRONMENT Integrated...HETEROGENEOUS EMBEDDED REAL - TIME SYSTEMS ENVIRONMENT 6. AUTHOR(S) Cosmo Castellano and James Graham 5. FUNDING NUMBERS C - F30602-97-C-0259

Satellite clock corrections estimation to accomplish real time ppp: experiments for brazilian real time network

NASA Astrophysics Data System (ADS)

Marques, Haroldo; Monico, João; Aquino, Marcio; Melo, Weyller

2014-05-01

The real time PPP method requires the availability of real time precise orbits and satellites clocks corrections. Currently, it is possible to apply the solutions of clocks and orbits available by BKG within the context of IGS Pilot project or by using the operational predicted IGU ephemeris. The accuracy of the satellite position available in the IGU is enough for several applications requiring good quality. However, the satellites clocks corrections do not provide enough accuracy (3 ns ~ 0.9 m) to accomplish real time PPP with the same level of accuracy. Therefore, for real time PPP application it is necessary to further research and develop appropriated methodologies for estimating the satellite clock corrections in real time with better accuracy. Currently, it is possible to apply the real time solutions of clocks and orbits available by Federal Agency for Cartography and Geodesy (BKG) within the context of IGS Pilot project. The BKG corrections are disseminated by a new proposed format of the RTCM 3.x and can be applied in the broadcasted orbits and clocks. Some investigations have been proposed for the estimation of the satellite clock corrections using GNSS code and phase observable at the double difference level between satellites and epochs (MERVAT, DOUSA, 2007). Another possibility consists of applying a Kalman Filter in the PPP network mode (HAUSCHILD, 2010) and it is also possible the integration of both methods, using network PPP and observables at double difference level in specific time intervals (ZHANG; LI; GUO, 2010). For this work the methodology adopted consists in the estimation of the satellite clock corrections based on the data adjustment in the PPP mode, but for a network of GNSS stations. The clock solution can be solved by using two types of observables: code smoothed by carrier phase or undifferenced code together with carrier phase. In the former, we estimate receiver clock error; satellite clock correction and troposphere, considering

Temporal Proof Methodologies for Real-Time Systems,

DTIC Science & Technology

1990-09-01

real time systems that communicate either through shared variables or by message passing and real time issues such as time-outs, process priorities (interrupts) and process scheduling. The authors exhibit two styles for the specification of real - time systems . While the first approach uses bounded versions of temporal operators the second approach allows explicit references to time through a special clock variable. Corresponding to two styles of specification the authors present and compare two fundamentally different proof

Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus.

PubMed

Raue, R; Hess, M

1998-08-01

Three different polymerase chain reactions (PCRs), two of them combined with restriction enzyme analysis (REA), were developed for detection and differentiation of all 12 fowl adenovirus (FAV) serotypes and the egg drop syndrome (EDS) virus. For primer construction FAV1, FAV10 and EDS virus hexon proteins were aligned and conserved and variable regions were determined. Two primer sets (H1/H2 and H3/H4) for single use were constructed which hybridize in three conserved regions of hexon genes. Each primer pair amplifies approximately half of the hexon gene including two loop regions. An amplification product was detected with both primer sets using purified DNA from all FAV1-12 reference strains. Viral EDS DNA was negative using the H1/H2 or H3/H4 primer pair. HaeII digestion of the H1/H2 amplification products differentiates between all viruses except FAV4 and FAV5. In comparison, much more clustering among genomic closely related FAV serotypes was seen after HpaII digestion of the H3/H4 PCR products. Oligonucleotides H5/H6 located in the variable regions of EDS virus hexon gene do not detect any of the FAV serotypes. The PCRs and REA described are suitable to detect all avian adenoviruses infecting chickens, to distinguish all 12 FAV reference strains and to differentiate FAVs from the EDS virus.

Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems.

PubMed

Fischer, Melina; Schirrmeier, Horst; Wernike, Kerstin; Wegelt, Anne; Beer, Martin; Hoffmann, Bernd

2013-11-05

Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable

Real Time Data System (RTDS)

NASA Technical Reports Server (NTRS)

Muratore, John F.

1991-01-01

Lessons learned from operational real time expert systems are examined. The basic system architecture is discussed. An expert system is any software that performs tasks to a standard that would normally require a human expert. An expert system implies knowledge contained in data rather than code. And an expert system implies the use of heuristics as well as algorithms. The 15 top lessons learned by the operation of a real time data system are presented.

PERTS: A Prototyping Environment for Real-Time Systems

NASA Technical Reports Server (NTRS)

Liu, Jane W. S.; Lin, Kwei-Jay; Liu, C. L.

1991-01-01

We discuss an ongoing project to build a Prototyping Environment for Real-Time Systems, called PERTS. PERTS is a unique prototyping environment in that it has (1) tools and performance models for the analysis and evaluation of real-time prototype systems, (2) building blocks for flexible real-time programs and the support system software, (3) basic building blocks of distributed and intelligent real time applications, and (4) an execution environment. PERTS will make the recent and future theoretical advances in real-time system design and engineering readily usable to practitioners. In particular, it will provide an environment for the use and evaluation of new design approaches, for experimentation with alternative system building blocks and for the analysis and performance profiling of prototype real-time systems.

Comparison of allelic discrimination by dHPLC, HRM, and TaqMan in the detection of BRAF mutation V600E.

PubMed

Carbonell, Pablo; Turpin, María C; Torres-Moreno, Daniel; Molina-Martínez, Irene; García-Solano, José; Perez-Guillermo, Miguel; Conesa-Zamora, Pablo

2011-09-01

The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffin-embedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent (κ = 0.9411 and κ = 0.8988, respectively); for HRM, the concordances were good (κ = 0.7973 and κ = 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in archival paraffin-embedded tissues. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

StarBase: A Firm Real-Time Database Manager for Time-Critical Applications

DTIC Science & Technology

1995-01-01

Mellon University [IO]. StarBase differs from previous RT-DBMS work [l, 2, 31 in that a) it relies on a real - time operating system which provides...simulation studies, StarBase uses a real - time operating system to provide basic real-time functionality and deals with issues beyond transaction...re- source scheduling provided by the underlying real - time operating system . Issues of data contention are dealt with by use of a priority

On Real-Time Systems Using Local Area Networks.

DTIC Science & Technology

1987-07-01

87-35 July, 1987 CS-TR-1892 On Real - Time Systems Using Local Area Networks*I VShem-Tov Levi Department of Computer Science Satish K. Tripathit...1892 On Real - Time Systems Using Local Area Networks* Shem-Tov Levi Department of Computer Science Satish K. Tripathit Department of Computer Science...constraints and the clock systems that feed the time to real - time systems . A model for real-time system based on LAN communication is presented in

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

PubMed Central

Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

2005-01-01

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated. PMID:16145106

Approaching near real-time biosensing: microfluidic microsphere based biosensor for real-time analyte detection.

PubMed

Cohen, Noa; Sabhachandani, Pooja; Golberg, Alexander; Konry, Tania

2015-04-15

In this study we describe a simple lab-on-a-chip (LOC) biosensor approach utilizing well mixed microfluidic device and a microsphere-based assay capable of performing near real-time diagnostics of clinically relevant analytes such cytokines and antibodies. We were able to overcome the adsorption kinetics reaction rate-limiting mechanism, which is diffusion-controlled in standard immunoassays, by introducing the microsphere-based assay into well-mixed yet simple microfluidic device with turbulent flow profiles in the reaction regions. The integrated microsphere-based LOC device performs dynamic detection of the analyte in minimal amount of biological specimen by continuously sampling micro-liter volumes of sample per minute to detect dynamic changes in target analyte concentration. Furthermore we developed a mathematical model for the well-mixed reaction to describe the near real time detection mechanism observed in the developed LOC method. To demonstrate the specificity and sensitivity of the developed real time monitoring LOC approach, we applied the device for clinically relevant analytes: Tumor Necrosis Factor (TNF)-α cytokine and its clinically used inhibitor, anti-TNF-α antibody. Based on the reported results herein, the developed LOC device provides continuous sensitive and specific near real-time monitoring method for analytes such as cytokines and antibodies, reduces reagent volumes by nearly three orders of magnitude as well as eliminates the washing steps required by standard immunoassays. Copyright © 2014 Elsevier B.V. All rights reserved.

Static Schedulers for Embedded Real-Time Systems

DTIC Science & Technology

1989-12-01

Because of the need for having efficient scheduling algorithms in large scale real time systems , software engineers put a lot of effort on developing...provide static schedulers for he Embedded Real Time Systems with single processor using Ada programming language. The independent nonpreemptable...support the Computer Aided Rapid Prototyping for Embedded Real Time Systems so that we determine whether the system, as designed, meets the required

A Real-Time Linux for Multicore Platforms

DTIC Science & Technology

2013-12-20

under ARO support) to obtain a fully-functional OS for supporting real-time workloads on multicore platforms. This system, called LITMUS -RT...to be specified as plugin components. LITMUS -RT is open-source software (available at The views, opinions and/or findings contained in this report... LITMUS -RT (LInux Testbed for MUltiprocessor Scheduling in Real-Time systems), allows different multiprocessor real-time scheduling and

ALMA Correlator Real-Time Data Processor

NASA Astrophysics Data System (ADS)

Pisano, J.; Amestica, R.; Perez, J.

2005-10-01

The design of a real-time Linux application utilizing Real-Time Application Interface (RTAI) to process real-time data from the radio astronomy correlator for the Atacama Large Millimeter Array (ALMA) is described. The correlator is a custom-built digital signal processor which computes the cross-correlation function of two digitized signal streams. ALMA will have 64 antennas with 2080 signal streams each with a sample rate of 4 giga-samples per second. The correlator's aggregate data output will be 1 gigabyte per second. The software is defined by hard deadlines with high input and processing data rates, while requiring interfaces to non real-time external computers. The designed computer system - the Correlator Data Processor or CDP, consists of a cluster of 17 SMP computers, 16 of which are compute nodes plus a master controller node all running real-time Linux kernels. Each compute node uses an RTAI kernel module to interface to a 32-bit parallel interface which accepts raw data at 64 megabytes per second in 1 megabyte chunks every 16 milliseconds. These data are transferred to tasks running on multiple CPUs in hard real-time using RTAI's LXRT facility to perform quantization corrections, data windowing, FFTs, and phase corrections for a processing rate of approximately 1 GFLOPS. Highly accurate timing signals are distributed to all seventeen computer nodes in order to synchronize them to other time-dependent devices in the observatory array. RTAI kernel tasks interface to the timing signals providing sub-millisecond timing resolution. The CDP interfaces, via the master node, to other computer systems on an external intra-net for command and control, data storage, and further data (image) processing. The master node accesses these external systems utilizing ALMA Common Software (ACS), a CORBA-based client-server software infrastructure providing logging, monitoring, data delivery, and intra-computer function invocation. The software is being developed in tandem

Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

PubMed

Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

2016-08-01

Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect

Software-safety and software quality assurance in real-time applications Part 2: Real-time structures and languages

NASA Astrophysics Data System (ADS)

Schoitsch, Erwin

1988-07-01

Our society is depending more and more on the reliability of embedded (real-time) computer systems even in every-day life. Considering the complexity of the real world, this might become a severe threat. Real-time programming is a discipline important not only in process control and data acquisition systems, but also in fields like communication, office automation, interactive databases, interactive graphics and operating systems development. General concepts of concurrent programming and constructs for process-synchronization are discussed in detail. Tasking and synchronization concepts, methods of process communication, interrupt- and timeout handling in systems based on semaphores, signals, conditional critical regions or on real-time languages like Concurrent PASCAL, MODULA, CHILL and ADA are explained and compared with each other and with respect to their potential to quality and safety.

Real-time data flow and product generating for GNSS

NASA Technical Reports Server (NTRS)

Muellerschoen, Ronald J.; Caissy, Mark

2004-01-01

The last IGS workshop with the theme 'Towards Real-Time' resulted in the design of a prototype for real-time data and sharing within the IGS. A prototype real-time network is being established that will serve as a test bed for real-time activities within the IGS. We review the developments of the prototype and discuss some of the existing methods and related products of real-time GNSS systems. Recommendations are made concerning real-time data distribution and product generation.

Distributed Issues for Ada Real-Time Systems

DTIC Science & Technology

1990-07-23

NUMBERS Distributed Issues for Ada Real - Time Systems MDA 903-87- C- 0056 S. AUTHOR(S) Thomas E. Griest 7. PERFORMING ORGANiZATION NAME(S) AND ADORESS(ES) 8...considerations. I Adding to the problem of distributed real - time systems is the issue of maintaining a common sense of time among all of the processors...because -omeone is waiting for the final output of a very large set of computations. However in real - time systems , consistent meeting of short-term

Design Specifications for Adaptive Real-Time Systems

DTIC Science & Technology

1991-12-01

TICfl \\ E CT E Design Specifications for JAN’\\ 1992 Adaptive Real - Time Systems fl Randall W. Lichota U, Alice H. Muntz - December 1991 \\ \\\\/ 0 / r...268-2056 Technical Report CMU/SEI-91-TR-20 ESD-91-TR-20 December 1991 Design Specifications for Adaptive Real - Time Systems Randall W. Lichota Hughes...Design Specifications for Adaptive Real - Time Systems Abstract: The design specification method described in this report treats a software

Design Recovery Technology for Real-Time Systems.

DTIC Science & Technology

1995-10-01

RL-TR-95-208 Final Technical Report October 1995 DESIGN RECOVERY TECHNOLOGY FOR REAL TIME SYSTEMS The MITRE Corporation Lester J. Holtzblatt...92 - Jan 95 4. TTTLE AND SUBTITLE DESIGN RECOVERY TECHNOLOGY FOR REAL - TIME SYSTEMS 6. AUTHOR(S) Lester J. Holtzblatt, Richard Piazza, and Susan...behavior of real - time systems in general, our initial efforts have centered on recovering this information from one system in particular, the Modular

Real Time Large Memory Optical Pattern Recognition.

DTIC Science & Technology

1984-06-01

AD-Ri58 023 REAL TIME LARGE MEMORY OPTICAL PATTERN RECOGNITION(U) - h ARMY MISSILE COMMAND REDSTONE ARSENAL AL RESEARCH DIRECTORATE D A GREGORY JUN...TECHNICAL REPORT RR-84-9 Ln REAL TIME LARGE MEMORY OPTICAL PATTERN RECOGNITION Don A. Gregory Research Directorate US Army Missile Laboratory JUNE 1984 L...RR-84-9 , ___/_ _ __ _ __ _ __ _ __"__ _ 4. TITLE (and Subtitle) S. TYPE OF REPORT & PERIOD COVERED Real Time Large Memory Optical Pattern Technical

[Real time 3D echocardiography

NASA Technical Reports Server (NTRS)

Bauer, F.; Shiota, T.; Thomas, J. D.

2001-01-01

Three-dimensional representation of the heart is an old concern. Usually, 3D reconstruction of the cardiac mass is made by successive acquisition of 2D sections, the spatial localisation and orientation of which require complex guiding systems. More recently, the concept of volumetric acquisition has been introduced. A matricial emitter-receiver probe complex with parallel data processing provides instantaneous of a pyramidal 64 degrees x 64 degrees volume. The image is restituted in real time and is composed of 3 planes (planes B and C) which can be displaced in all spatial directions at any time during acquisition. The flexibility of this system of acquisition allows volume and mass measurement with greater accuracy and reproducibility, limiting inter-observer variability. Free navigation of the planes of investigation allows reconstruction for qualitative and quantitative analysis of valvular heart disease and other pathologies. Although real time 3D echocardiography is ready for clinical usage, some improvements are still necessary to improve its conviviality. Then real time 3D echocardiography could be the essential tool for understanding, diagnosis and management of patients.

Real-Time Precise Point Positioning (RTPPP) with raw observations and its application in real-time regional ionospheric VTEC modeling

NASA Astrophysics Data System (ADS)

Liu, Teng; Zhang, Baocheng; Yuan, Yunbin; Li, Min

2018-01-01

Precise Point Positioning (PPP) is an absolute positioning technology mainly used in post data processing. With the continuously increasing demand for real-time high-precision applications in positioning, timing, retrieval of atmospheric parameters, etc., Real-Time PPP (RTPPP) and its applications have drawn more and more research attention in recent years. This study focuses on the models, algorithms and ionospheric applications of RTPPP on the basis of raw observations, in which high-precision slant ionospheric delays are estimated among others in real time. For this purpose, a robust processing strategy for multi-station RTPPP with raw observations has been proposed and realized, in which real-time data streams and State-Space-Representative (SSR) satellite orbit and clock corrections are used. With the RTPPP-derived slant ionospheric delays from a regional network, a real-time regional ionospheric Vertical Total Electron Content (VTEC) modeling method is proposed based on Adjusted Spherical Harmonic Functions and a Moving-Window Filter. SSR satellite orbit and clock corrections from different IGS analysis centers are evaluated. Ten globally distributed real-time stations are used to evaluate the positioning performances of the proposed RTPPP algorithms in both static and kinematic modes. RMS values of positioning errors in static/kinematic mode are 5.2/15.5, 4.7/17.4 and 12.8/46.6 mm, for north, east and up components, respectively. Real-time slant ionospheric delays from RTPPP are compared with those from the traditional Carrier-to-Code Leveling (CCL) method, in terms of function model, formal precision and between-receiver differences of short baseline. Results show that slant ionospheric delays from RTPPP are more precise and have a much better convergence performance than those from the CCL method in real-time processing. 30 real-time stations from the Asia-Pacific Reference Frame network are used to model the ionospheric VTECs over Australia in real time

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

PubMed Central

Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

2016-01-01

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS® TAQMAN® HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.

PubMed

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Marcos, MaAngeles; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

2017-10-01

Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories STUDY DESIGN: Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS ® TaqMan ® HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT ® HBV Assay (Versant), and 74 specimens tested with Veris and artus ® HBV RG PCR kit (artus). Bland-Altman analysis showed average bias of -0.46 log 10 IU/mL between Veris and Cobas, -0.46 log 10 IU/mL between Veris and RealTime, -0.36 log 10 IU/mL between Veris and Versant, and -0.12 log 10 IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris. Copyright © 2017 Elsevier B.V. All rights reserved.

Time-gated real-time pump-probe imaging spectroscopy

NASA Astrophysics Data System (ADS)

Ferrari, Raffaele; D'Andrea, Cosimo; Bassi, Andrea; Valentini, Gianluca; Cubeddu, Rinaldo

2007-07-01

An experimental technique which allows one to perform pump-probe transient absorption spectroscopy in real-time is an important tool to study irreversible processes. This is particularly interesting in the case of biological samples which easily deteriorate upon exposure to light pulses, with the formation of permanent photoproducts and structural changes. In particular pump-probe spectroscopy can provide fundamental information for the design of optical chromophores. In this work a real-time pump-probe imaging spectroscopy system has been realized and we have explored the possibility to further reduce the number of laser pulses by using a time-gated camera. We believe that the use of a time-gated camera can provide an important step towards the final goal of pump-probe single shot spectroscopy.

Real-time quasi-3D tomographic reconstruction

NASA Astrophysics Data System (ADS)

Buurlage, Jan-Willem; Kohr, Holger; Palenstijn, Willem Jan; Joost Batenburg, K.

2018-06-01

Developments in acquisition technology and a growing need for time-resolved experiments pose great computational challenges in tomography. In addition, access to reconstructions in real time is a highly demanded feature but has so far been out of reach. We show that by exploiting the mathematical properties of filtered backprojection-type methods, having access to real-time reconstructions of arbitrarily oriented slices becomes feasible. Furthermore, we present , software for visualization and on-demand reconstruction of slices. A user of can interactively shift and rotate slices in a GUI, while the software updates the slice in real time. For certain use cases, the possibility to study arbitrarily oriented slices in real time directly from the measured data provides sufficient visual and quantitative insight. Two such applications are discussed in this article.

Real-time logic modelling on SpaceWire

NASA Astrophysics Data System (ADS)

Zhou, Qiang; Ma, Yunpeng; Fei, Haidong; Wang, Xingyou

2017-04-01

A SpaceWire is a standard for on-board satellite networks as the basis for future data-handling architectures. However, it cannot meet the deterministic requirement for safety/time critical application in spacecraft, where the delay of real-time (RT) message streams must be guaranteed. Therefore, SpaceWire-D is developed that provides deterministic delivery over a SpaceWire network. Formal analysis and verification of real-time systems is critical to their development and safe implementation, and is a prerequisite for obtaining their safety certification. Failure to meet specified timing constraints such as deadlines in hard real-time systems may lead to catastrophic results. In this paper, a formal verification method, Real-Time Logic (RTL), has been proposed to specify and verify timing properties of SpaceWire-D network. Based on the principal of SpaceWire-D protocol, we firstly analyze the timing properties of fundamental transactions, such as RMAP WRITE, and RMAP READ. After that, the RMAP WRITE transaction structure is modeled in Real-Time Logic (RTL) and Presburger Arithmetic representations. And then, the associated constraint graph and safety analysis is provided. Finally, it is suggested that RTL method can be useful for the protocol evaluation and provision of recommendation for further protocol evolutions.

The Real Time Display Builder (RTDB)

NASA Technical Reports Server (NTRS)

Kindred, Erick D.; Bailey, Samuel A., Jr.

1989-01-01

The Real Time Display Builder (RTDB) is a prototype interactive graphics tool that builds logic-driven displays. These displays reflect current system status, implement fault detection algorithms in real time, and incorporate the operational knowledge of experienced flight controllers. RTDB utilizes an object-oriented approach that integrates the display symbols with the underlying operational logic. This approach allows the user to specify the screen layout and the driving logic as the display is being built. RTDB is being developed under UNIX in C utilizing the MASSCOMP graphics environment with appropriate functional separation to ease portability to other graphics environments. RTDB grew from the need to develop customized real-time data-driven Space Shuttle systems displays. One display, using initial functionality of the tool, was operational during the orbit phase of STS-26 Discovery. RTDB is being used to produce subsequent displays for the Real Time Data System project currently under development within the Mission Operations Directorate at NASA/JSC. The features of the tool, its current state of development, and its applications are discussed.

Geographically distributed real-time digital simulations using linear prediction

DOE PAGES

Liu, Ren; Mohanpurkar, Manish; Panwar, Mayank; ...

2016-07-04

Real time simulation is a powerful tool for analyzing, planning, and operating modern power systems. For analyzing the ever evolving power systems and understanding complex dynamic and transient interactions larger real time computation capabilities are essential. These facilities are interspersed all over the globe and to leverage unique facilities geographically-distributed real-time co-simulation in analyzing the power systems is pursued and presented. However, the communication latency between different simulator locations may lead to inaccuracy in geographically distributed real-time co-simulations. In this paper, the effect of communication latency on geographically distributed real-time co-simulation is introduced and discussed. In order to reduce themore » effect of the communication latency, a real-time data predictor, based on linear curve fitting is developed and integrated into the distributed real-time co-simulation. Two digital real time simulators are used to perform dynamic and transient co-simulations with communication latency and predictor. Results demonstrate the effect of the communication latency and the performance of the real-time data predictor to compensate it.« less

Geographically distributed real-time digital simulations using linear prediction

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

Liu, Ren; Mohanpurkar, Manish; Panwar, Mayank

Real time simulation is a powerful tool for analyzing, planning, and operating modern power systems. For analyzing the ever evolving power systems and understanding complex dynamic and transient interactions larger real time computation capabilities are essential. These facilities are interspersed all over the globe and to leverage unique facilities geographically-distributed real-time co-simulation in analyzing the power systems is pursued and presented. However, the communication latency between different simulator locations may lead to inaccuracy in geographically distributed real-time co-simulations. In this paper, the effect of communication latency on geographically distributed real-time co-simulation is introduced and discussed. In order to reduce themore » effect of the communication latency, a real-time data predictor, based on linear curve fitting is developed and integrated into the distributed real-time co-simulation. Two digital real time simulators are used to perform dynamic and transient co-simulations with communication latency and predictor. Results demonstrate the effect of the communication latency and the performance of the real-time data predictor to compensate it.« less

Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

PubMed

Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

2016-07-01

Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

Real Time Linux - The RTOS for Astronomy?

NASA Astrophysics Data System (ADS)

Daly, P. N.

The BoF was attended by about 30 participants and a free CD of real time Linux-based upon RedHat 5.2-was available. There was a detailed presentation on the nature of real time Linux and the variants for hard real time: New Mexico Tech's RTL and DIAPM's RTAI. Comparison tables between standard Linux and real time Linux responses to time interval generation and interrupt response latency were presented (see elsewhere in these proceedings). The present recommendations are to use RTL for UP machines running the 2.0.x kernels and RTAI for SMP machines running the 2.2.x kernel. Support, both academically and commercially, is available. Some known limitations were presented and the solutions reported e.g., debugging and hardware support. The features of RTAI (scheduler, fifos, shared memory, semaphores, message queues and RPCs) were described. Typical performance statistics were presented: Pentium-based oneshot tasks running > 30kHz, 486-based oneshot tasks running at ~ 10 kHz, periodic timer tasks running in excess of 90 kHz with average zero jitter peaking to ~ 13 mus (UP) and ~ 30 mus (SMP). Some detail on kernel module programming, including coding examples, were presented showing a typical data acquisition system generating simulated (random) data writing to a shared memory buffer and a fifo buffer to communicate between real time Linux and user space. All coding examples were complete and tested under RTAI v0.6 and the 2.2.12 kernel. Finally, arguments were raised in support of real time Linux: it's open source, free under GPL, enables rapid prototyping, has good support and the ability to have a fully functioning workstation capable of co-existing hard real time performance. The counter weight-the negatives-of lack of platforms (x86 and PowerPC only at present), lack of board support, promiscuous root access and the danger of ignorance of real time programming issues were also discussed. See ftp://orion.tuc.noao.edu/pub/pnd/rtlbof.tgz for the StarOffice overheads

Network protocols for real-time applications

NASA Technical Reports Server (NTRS)

Johnson, Marjory J.

1987-01-01

The Fiber Distributed Data Interface (FDDI) and the SAE AE-9B High Speed Ring Bus (HSRB) are emerging standards for high-performance token ring local area networks. FDDI was designed to be a general-purpose high-performance network. HSRB was designed specifically for military real-time applications. A workshop was conducted at NASA Ames Research Center in January, 1987 to compare and contrast these protocols with respect to their ability to support real-time applications. This report summarizes workshop presentations and includes an independent comparison of the two protocols. A conclusion reached at the workshop was that current protocols for the upper layers of the Open Systems Interconnection (OSI) network model are inadequate for real-time applications.

Critical (of) Issues in Real-Time Systems.

DTIC Science & Technology

1988-05-03

IACCESON 11 T IT LE (Include Security Classification) Cr it ic, (of) issues in real .- time systems A Position Paper 12 PERPSONAL AU THOR(S) Fr ed B...are obsolete %0%’ 4.’.C %" Critical (of) issues in real - time systems ’ • "A Position Paper Acc, son For NTIS R& DTrC TAB May 3, 1988 Uyjannotuce~d...m . 2C:. r Ithaca, New York 14853 Dist 1 -.1 g- It is time to place the development of real - time systems on a firm scientific basis. Unlike other

Managing Contention and Timing Constraints in a Real-Time Database System

DTIC Science & Technology

1995-01-01

In order to realize many of these goals, StarBase is constructed on top of RT-Mach, a real - time operating system developed at Carnegie Mellon...University [ll]. StarBase differs from previous RT-DBMS work [l, 2, 31 in that a) it relies on a real - time operating system which provides priority...CPU and resource scheduling pro- vided by tlhe underlying real - time operating system . Issues of data contention are dealt with by use of a priority

A Programmable Microkernel for Real-Time Systems

DTIC Science & Technology

2003-06-01

A Programmable Microkernel for Real - Time Systems Christoph M. Kirsch Thomas A. Henzinger Marco A.A. Sanvido Report No. UCB/CSD-3-1250 June 2003...TITLE AND SUBTITLE A Programmable Microkernel for Real - Time Systems 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 A Programmable Microkernel for Real - Time Systems ∗ Christoph M

Predictable and Adaptable Complex Real-Time Systems

DTIC Science & Technology

1993-09-30

Predictable and Adaptable Complex Real - Time Systems Grant or Contract Number: N00014-92-J-1048 Reporting Period: 1 Oct 91 - 30 Sep 93 1... Real - Time Systems Grant or Contract Number: N00014-92-J-1048 Reporting Period: 1 Oct 91 - 30 Sep 93 2. Summary of Technical Progress Our...cs.umass.edu Grant or Contract Title: Predictable and Adaptable Complex Real - Time Systems Grant or Contract Number: N00014-92-J-1048 Reporting Period: 1 Oct 91

Tactical AI in Real Time Strategy Games

DTIC Science & Technology

2015-03-26

TACTICAL AI IN REAL TIME STRATEGY GAMES THESIS Donald A. Gruber, Capt, USAF AFIT-ENG-MS-15-M-021 DEPARTMENT OF THE AIR FORCE AIR UNIVERSITY AIR FORCE...protection in the United States. AFIT-ENG-MS-15-M-021 TACTICAL AI IN REAL TIME STRATEGY GAMES THESIS Presented to the Faculty Department of Electrical...DISTRIBUTION STATEMENT A APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED. AFIT-ENG-MS-15-M-021 TACTICAL AI IN REAL TIME STRATEGY GAMES THESIS Donald A

Real-time simulation of large-scale floods

NASA Astrophysics Data System (ADS)

Liu, Q.; Qin, Y.; Li, G. D.; Liu, Z.; Cheng, D. J.; Zhao, Y. H.

2016-08-01

According to the complex real-time water situation, the real-time simulation of large-scale floods is very important for flood prevention practice. Model robustness and running efficiency are two critical factors in successful real-time flood simulation. This paper proposed a robust, two-dimensional, shallow water model based on the unstructured Godunov- type finite volume method. A robust wet/dry front method is used to enhance the numerical stability. An adaptive method is proposed to improve the running efficiency. The proposed model is used for large-scale flood simulation on real topography. Results compared to those of MIKE21 show the strong performance of the proposed model.

23 CFR 511.311 - Real-time information program establishment.

Code of Federal Regulations, 2011 CFR

2011-04-01

... INFRASTRUCTURE MANAGEMENT REAL-TIME SYSTEM MANAGEMENT INFORMATION PROGRAM Real-Time System Management Information... operated by the State. In addition, the real-time information program shall complement current... 23 Highways 1 2011-04-01 2011-04-01 false Real-time information program establishment. 511.311...

23 CFR 511.311 - Real-time information program establishment.

Code of Federal Regulations, 2014 CFR

2014-04-01

... INFRASTRUCTURE MANAGEMENT REAL-TIME SYSTEM MANAGEMENT INFORMATION PROGRAM Real-Time System Management Information... operated by the State. In addition, the real-time information program shall complement current... 23 Highways 1 2014-04-01 2014-04-01 false Real-time information program establishment. 511.311...

23 CFR 511.311 - Real-time information program establishment.

Code of Federal Regulations, 2013 CFR

2013-04-01

... INFRASTRUCTURE MANAGEMENT REAL-TIME SYSTEM MANAGEMENT INFORMATION PROGRAM Real-Time System Management Information... operated by the State. In addition, the real-time information program shall complement current... 23 Highways 1 2013-04-01 2013-04-01 false Real-time information program establishment. 511.311...

23 CFR 511.311 - Real-time information program establishment.

Code of Federal Regulations, 2012 CFR

2012-04-01

... INFRASTRUCTURE MANAGEMENT REAL-TIME SYSTEM MANAGEMENT INFORMATION PROGRAM Real-Time System Management Information... operated by the State. In addition, the real-time information program shall complement current... 23 Highways 1 2012-04-01 2012-04-01 false Real-time information program establishment. 511.311...

Automated real-time software development

NASA Technical Reports Server (NTRS)

Jones, Denise R.; Walker, Carrie K.; Turkovich, John J.

1993-01-01

A Computer-Aided Software Engineering (CASE) system has been developed at the Charles Stark Draper Laboratory (CSDL) under the direction of the NASA Langley Research Center. The CSDL CASE tool provides an automated method of generating source code and hard copy documentation from functional application engineering specifications. The goal is to significantly reduce the cost of developing and maintaining real-time scientific and engineering software while increasing system reliability. This paper describes CSDL CASE and discusses demonstrations that used the tool to automatically generate real-time application code.

Real-Time Occupancy Change Analyzer

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

2005-03-30

The Real-Time Occupancy Change Analyzer (ROCA) produces an occupancy grid map of an environment around the robot, scans the environment to generate a current obstacle map relative to a current robot position, and converts the current obstacle map to a current occupancy grid map. Changes in the occupancy grid can be reported in real time to support a number of tracking capabilities. The benefit of ROCA is that rather than only providing a vector to the detected change, it provides the actual x,y position of the change.

Development and Application of a Multiplex Real-Time PCR Assay as an Indicator of Potential Allergenicity in Citrus Fruits.

PubMed

Wu, Jinlong; Chen, Lin; Lin, Dingbo; Ma, Zhaocheng; Deng, Xiuxin

2016-11-30

The effects of tissue type, harvest maturity, and genetic factors on the expression of genes that related to citrus fruit allergies remain poorly understood. In the present study, a multiplex real-time PCR assay was developed to monitor the expression of citrus allergen genes individually with the advantages of much fewer sample requirements and simultaneously multiple target genes detection. Gene specific primer pairs and Taqman probes of three citrus allergen genes Cit s 1.01, Cit s 2.01, and Cit s 3.01 and the house-keeping gene β-actin were designed based on gene sequence differences. The PCR results showed that differential expression patterns were found during the ripening process. The expression levels of Cit s 3.01 were much higher than those of Cit s 1.01 and Cit s 2.01 in both peel and pulp tissues among 10 citrus cultivars. Data suggested that Kao Phuang Pummelo could be safely consumed with a potential low risk in allergenicity. Considering that assessing allergenicity is one of the tests in food safety, this assay might also facilitate the breeding and production of "allergy-friendly" citrus fruits.

17 CFR 38.157 - Real-time market monitoring.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 1 2014-04-01 2014-04-01 false Real-time market monitoring... DESIGNATED CONTRACT MARKETS Compliance With Rules § 38.157 Real-time market monitoring. A designated contract market must conduct real-time market monitoring of all trading activity on its electronic trading...

17 CFR 38.157 - Real-time market monitoring.

Code of Federal Regulations, 2013 CFR

2013-04-01

... DESIGNATED CONTRACT MARKETS Compliance With Rules § 38.157 Real-time market monitoring. A designated contract market must conduct real-time market monitoring of all trading activity on its electronic trading... 17 Commodity and Securities Exchanges 1 2013-04-01 2013-04-01 false Real-time market monitoring...

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid.

PubMed

Wisselink, Henk J; Cornelissen, Jan B W J; van der Wal, Fimme J; Kooi, Engbert A; Koene, Miriam G; Bossers, Alex; Smid, Bregtje; de Bree, Freddy M; Antonis, Adriaan F G

2017-07-13

Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10 -1 to 2 × 10 0  CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

A real-time simulator of a turbofan engine

NASA Technical Reports Server (NTRS)

Litt, Jonathan S.; Delaat, John C.; Merrill, Walter C.

1989-01-01

A real-time digital simulator of a Pratt and Whitney F100 engine has been developed for real-time code verification and for actuator diagnosis during full-scale engine testing. This self-contained unit can operate in an open-loop stand-alone mode or as part of closed-loop control system. It can also be used for control system design and development. Tests conducted in conjunction with the NASA Advanced Detection, Isolation, and Accommodation program show that the simulator is a valuable tool for real-time code verification and as a real-time actuator simulator for actuator fault diagnosis. Although currently a small perturbation model, advances in microprocessor hardware should allow the simulator to evolve into a real-time, full-envelope, full engine simulation.

Geomagnetic Observatory Data for Real-Time Applications

NASA Astrophysics Data System (ADS)

Love, J. J.; Finn, C. A.; Rigler, E. J.; Kelbert, A.; Bedrosian, P.

2015-12-01

The global network of magnetic observatories represents a unique collective asset for the scientific community. Historically, magnetic observatories have supported global magnetic-field mapping projects and fundamental research of the Earth's interior and surrounding space environment. More recently, real-time data streams from magnetic observatories have become an important contributor to multi-sensor, operational monitoring of evolving space weather conditions, especially during magnetic storms. In this context, the U.S. Geological Survey (1) provides real-time observatory data to allied space weather monitoring projects, including those of NOAA, the U.S. Air Force, NASA, several international agencies, and private industry, (2) collaborates with Schlumberger to provide real-time geomagnetic data needed for directional drilling for oil and gas in Alaska, (3) develops products for real-time evaluation of hazards for the electric-power grid industry that are associated with the storm-time induction of geoelectric fields in the Earth's conducting lithosphere. In order to implement strategic priorities established by the USGS Natural Hazards Mission Area and the National Science and Technology Council, and with a focus on developing new real-time products, the USGS is (1) leveraging data management protocols already developed by the USGS Earthquake Program, (2) developing algorithms for mapping geomagnetic activity, a collaboration with NASA and NOAA, (3) supporting magnetotelluric surveys and developing Earth conductivity models, a collaboration with Oregon State University and the NSF's EarthScope Program, (4) studying the use of geomagnetic activity maps and Earth conductivity models for real-time estimation of geoelectric fields, (5) initiating geoelectric monitoring at several observatories, (6) validating real-time estimation algorithms against historical geomagnetic and geoelectric data. The success of these long-term projects is subject to funding constraints

Real-time earthquake data feasible

NASA Astrophysics Data System (ADS)

Bush, Susan

Scientists agree that early warning devices and monitoring of both Hurricane Hugo and the Mt. Pinatubo volcanic eruption saved thousands of lives. What would it take to develop this sort of early warning and monitoring system for earthquake activity?Not all that much, claims a panel assigned to study the feasibility, costs, and technology needed to establish a real-time earthquake monitoring (RTEM) system. The panel, drafted by the National Academy of Science's Committee on Seismology, has presented its findings in Real-Time Earthquake Monitoring. The recently released report states that “present technology is entirely capable of recording and processing data so as to provide real-time information, enabling people to mitigate somewhat the earthquake disaster.” RTEM systems would consist of two parts—an early warning system that would give a few seconds warning before severe shaking, and immediate postquake information within minutes of the quake that would give actual measurements of the magnitude. At this time, however, this type of warning system has not been addressed at the national level for the United States and is not included in the National Earthquake Hazard Reduction Program, according to the report.

Real-time interferometric diagnostics of rubidium plasma

NASA Astrophysics Data System (ADS)

Djotyan, G. P.; Bakos, J. S.; Kedves, M. Á.; Ráczkevi, B.; Dzsotjan, D.; Varga-Umbrich, K.; Sörlei, Zs.; Szigeti, J.; Ignácz, P.; Lévai, P.; Czitrovszky, A.; Nagy, A.; Dombi, P.; Rácz, P.

2018-03-01

A method of interferometric real-time diagnostics is developed and applied to rubidium plasma created by strong laser pulses in the femtosecond duration range at different initial rubidium vapor densities using a Michelson-type interferometer. A cosine fit with an exponentially decaying relative phase is applied to the obtained time-dependent interferometry signals to measure the density-length product of the created plasma and its recombination time constant. The presented technique may be applicable for real-time measurements of rubidium plasma dynamics in the AWAKE experiment at CERN, as well as for real-time diagnostics of plasmas created in different gaseous media and on surfaces of solid targets.

Real-time UNIX in HEP data acquisition

NASA Astrophysics Data System (ADS)

Buono, S.; Gaponenko, I.; Jones, R.; Mapelli, L.; Mornacchi, G.; Prigent, D.; Sanchez-Corral, E.; Skiadelli, M.; Toppers, A.; Duval, P. Y.; Ferrato, D.; Le Van Suu, A.; Qian, Z.; Rondot, C.; Ambrosini, G.; Fumagalli, G.; Aguer, M.; Huet, M.

1994-12-01

Today's experimentation in high energy physics is characterized by an increasing need for sensitivity to rare phenomena and complex physics signatures, which require the use of huge and sophisticated detectors and consequently a high performance readout and data acquisition. Multi-level triggering, hierarchical data collection and an always increasing amount of processing power, distributed throughout the data acquisition layers, will impose a number of features on the software environment, especially the need for a high level of standardization. Real-time UNIX seems, today, the best solution for the platform independence, operating system interface standards and real-time features necessary for data acquisition in HEP experiments. We present the results of the evaluation, in a realistic application environment, of a Real-Time UNIX operating system: the EP/LX real-time UNIX system.

Temporal Specification and Verification of Real-Time Systems.

DTIC Science & Technology

1991-08-30

of concrete real - time systems can be modeled adequately. Specification: We present two conservative extensions of temporal logic that allow for the...logic. We present both model-checking algorithms for the automatic verification of finite-state real - time systems and proof methods for the deductive verification of real - time systems .

The New Aptima HCV Quant Dx Real-time TMA Assay Accurately Quantifies Hepatitis C Virus Genotype 1-6 RNA.

PubMed

Chevaliez, Stéphane; Dubernet, Fabienne; Dauvillier, Claude; Hézode, Christophe; Pawlotsky, Jean-Michel

2017-06-01

Sensitive and accurate hepatitis C virus (HCV) RNA detection and quantification is essential for the management of chronic hepatitis C therapy. Currently available platforms and assays are usually batched and require at least 5hours of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed Aptima HCV Quant Dx assay that eliminates the need for batch processing and automates all aspects of nucleic acid testing in a single step, to accurately detect and quantify HCV RNA in a large series of patients infected with different HCV genotypes. The limit of detection was estimated to be 2.3 IU/mL. The specificity of the assay was 98.6% (95% confidence interval: 96.1%-99.5%). Intra-assay and inter-assay coefficients of variation ranged from 0.09% to 5.61%, and 1.05% to 3.65%, respectively. The study of serum specimens from patients infected with HCV genotypes 1 to 6 showed a satisfactory relationship between HCV RNA levels measured by the Aptima HCV Quant Dx assay, and both real-time PCR comparators (Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays). the new Aptima HCV Quant Dx assay is rapid, sensitive, reasonably specific and reproducible and accurately quantifies HCV RNA in serum samples from patients with chronic HCV infection, including patients on antiviral treatment. The Aptima HCV Quant Dx assay can thus be confidently used to detect and quantify HCV RNA in both clinical trials with new anti-HCV drugs and clinical practice in Europe and the US. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-time scheduling using minimum search

NASA Technical Reports Server (NTRS)

Tadepalli, Prasad; Joshi, Varad

1992-01-01

In this paper we consider a simple model of real-time scheduling. We present a real-time scheduling system called RTS which is based on Korf's Minimin algorithm. Experimental results show that the schedule quality initially improves with the amount of look-ahead search and tapers off quickly. So it sppears that reasonably good schedules can be produced with a relatively shallow search.

Global Real-Time Ocean Forecast System

Science.gov Websites

services. Marine Modeling and Analysis Branch Logo Click here to go to the MMAB home page Global Real-Time 17 Oct 2017 at 0Z, the Global RTOFS model has been upgraded to version 1.1.2. Changes include: The ). The global operational Real-Time Ocean Forecast System (Global RTOFS) at the National Centers for

Real-Time Visualization of Tissue Ischemia

NASA Technical Reports Server (NTRS)

Bearman, Gregory H. (Inventor); Chrien, Thomas D. (Inventor); Eastwood, Michael L. (Inventor)

2000-01-01

A real-time display of tissue ischemia which comprises three CCD video cameras, each with a narrow bandwidth filter at the correct wavelength is discussed. The cameras simultaneously view an area of tissue suspected of having ischemic areas through beamsplitters. The output from each camera is adjusted to give the correct signal intensity for combining with, the others into an image for display. If necessary a digital signal processor (DSP) can implement algorithms for image enhancement prior to display. Current DSP engines are fast enough to give real-time display. Measurement at three, wavelengths, combined into a real-time Red-Green-Blue (RGB) video display with a digital signal processing (DSP) board to implement image algorithms, provides direct visualization of ischemic areas.

Can Real-Time Data Also Be Climate Quality?

NASA Astrophysics Data System (ADS)

Brewer, M.; Wentz, F. J.

2015-12-01

GMI, AMSR-2 and WindSat herald a new era of highly accurate and timely microwave data products. Traditionally, there has been a large divide between real-time and re-analysis data products. What if these completely separate processing systems could be merged? Through advanced modeling and physically based algorithms, Remote Sensing Systems (RSS) has narrowed the gap between real-time and research-quality. Satellite microwave ocean products have proven useful for a wide array of timely Earth science applications. Through cloud SST capabilities have enormously benefited tropical cyclone forecasting and day to day fisheries management, to name a few. Oceanic wind vectors enhance operational safety of shipping and recreational boating. Atmospheric rivers are of import to many human endeavors, as are cloud cover and knowledge of precipitation events. Some activities benefit from both climate and real-time operational data used in conjunction. RSS has been consistently improving microwave Earth Science Data Records (ESDRs) for several decades, while making near real-time data publicly available for semi-operational use. These data streams have often been produced in 2 stages: near real-time, followed by research quality final files. Over the years, we have seen this time delay shrink from months or weeks to mere hours. As well, we have seen the quality of near real-time data improve to the point where the distinction starts to blur. We continue to work towards better and faster RFI filtering, adaptive algorithms and improved real-time validation statistics for earlier detection of problems. Can it be possible to produce climate quality data in real-time, and what would the advantages be? We will try to answer these questions…

Real-time, high frequency QRS electrocardiograph

NASA Technical Reports Server (NTRS)

Schlegel, Todd T. (Inventor); DePalma, Jude L. (Inventor); Moradi, Saeed (Inventor)

2006-01-01

Real time cardiac electrical data are received from a patient, manipulated to determine various useful aspects of the ECG signal, and displayed in real time in a useful form on a computer screen or monitor. The monitor displays the high frequency data from the QRS complex in units of microvolts, juxtaposed with a display of conventional ECG data in units of millivolts or microvolts. The high frequency data are analyzed for their root mean square (RMS) voltage values and the discrete RMS values and related parameters are displayed in real time. The high frequency data from the QRS complex are analyzed with imbedded algorithms to determine the presence or absence of reduced amplitude zones, referred to herein as RAZs. RAZs are displayed as go, no-go signals on the computer monitor. The RMS and related values of the high frequency components are displayed as time varying signals, and the presence or absence of RAZs may be similarly displayed over time.

Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

PubMed

Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

2016-05-01

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical 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.

Connecting real-time data to algorithms and databases: EarthCube's Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS)

NASA Astrophysics Data System (ADS)

Daniels, M. D.; Graves, S. J.; Kerkez, B.; Chandrasekar, V.; Vernon, F.; Martin, C. L.; Maskey, M.; Keiser, K.; Dye, M. J.

2015-12-01

The Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS) project was funded under the National Science Foundation's EarthCube initiative. CHORDS addresses the ever-increasing importance of real-time scientific data in the geosciences, particularly in mission critical scenarios, where informed decisions must be made rapidly. Access to constant streams of real-time data also allow many new transient phenomena in space-time to be observed, however, much of these streaming data are either completely inaccessible or only available to proprietary in-house tools or displays. Small research teams do not have the resources to develop tools for the broad dissemination of their unique real-time data and require an easy to use, scalable, cloud-based solution to facilitate this access. CHORDS will make these diverse streams of real-time data available to the broader geosciences community. This talk will highlight a recently developed CHORDS portal tools and processing systems which address some of the gaps in handling real-time data, particularly in the provisioning of data from the "long-tail" scientific community through a simple interface that is deployed in the cloud, is scalable and is able to be customized by research teams. A running portal, with operational data feeds from across the nation, will be presented. The processing within the CHORDS system will expose these real-time streams via standard services from the Open Geospatial Consortium (OGC) in a way that is simple and transparent to the data provider, while maximizing the usage of these investments. The ingestion of high velocity, high volume and diverse data has allowed the project to explore a NoSQL database implementation. Broad use of the CHORDS framework by geoscientists will help to facilitate adaptive experimentation, model assimilation and real-time hypothesis testing.

Investigations on the frequency of norovirus contamination of ready-to-eat food items in Istanbul, Turkey, by using real-time reverse transcription PCR.

PubMed

Yilmaz, Aysun; Bostan, Kamil; Altan, Eda; Muratoglu, Karlo; Turan, Nuri; Tan, Derya; Helps, Christopher; Yilmaz, Huseyin

2011-05-01

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy.

PubMed

Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Brusin, Martina Rosa; Finotti, Serena; Paderi, Giulia; Gabiano, Clara

2017-04-01

Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log 10 ) ± 7.1(log 10 ) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

Real-time support for high performance aircraft operation

NASA Technical Reports Server (NTRS)

Vidal, Jacques J.

1989-01-01

The feasibility of real-time processing schemes using artificial neural networks (ANNs) is investigated. A rationale for digital neural nets is presented and a general processor architecture for control applications is illustrated. Research results on ANN structures for real-time applications are given. Research results on ANN algorithms for real-time control are also shown.

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities▿

PubMed Central

Steinberg, Lisa M.; Regan, John M.

2009-01-01

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members. PMID:19447957

Real-time video compressing under DSP/BIOS

NASA Astrophysics Data System (ADS)

Chen, Qiu-ping; Li, Gui-ju

2009-10-01

This paper presents real-time MPEG-4 Simple Profile video compressing based on the DSP processor. The programming framework of video compressing is constructed using TMS320C6416 Microprocessor, TDS510 simulator and PC. It uses embedded real-time operating system DSP/BIOS and the API functions to build periodic function, tasks and interruptions etcs. Realize real-time video compressing. To the questions of data transferring among the system. Based on the architecture of the C64x DSP, utilized double buffer switched and EDMA data transfer controller to transit data from external memory to internal, and realize data transition and processing at the same time; the architecture level optimizations are used to improve software pipeline. The system used DSP/BIOS to realize multi-thread scheduling. The whole system realizes high speed transition of a great deal of data. Experimental results show the encoder can realize real-time encoding of 768*576, 25 frame/s video images.

Delayed vaccine virus replication in chickens vaccinated subcutaneously with an immune complex infectious bursal disease vaccine: Quantification of vaccine virus by real-time polymerase chain reaction

PubMed Central

2005-01-01

Abstract The distribution of the immune complex vaccine virus for infectious bursal disease (IBD) in tissue was examined and the viral loads of the organs were quantitatively compared. One-day-old specific pathogen free (SPF) and maternally immune broiler chickens were injected subcutaneously with the vaccine. Lymphoid and non-lymphoid tissues were collected at various time intervals during the experiment to test for infectious bursal disease virus (IBDV)-RNA by using reverse transcriptase-polymerase chain reaction (RT-PCR). Only the bursa of Fabricius was found to be positive with unusually long viral persistence in the broiler group. The positive bursa samples were further investigated by using real-time PCR coupled with a TaqMan probe. The highest amounts of the virus were detected at its first appearance in the bursa: on day 14 post vaccination (PV) in the SPF chickens and on day 17 and day 21 PV in the maternally immune broiler group. The virus then gradually cleared, most likely due to the parallel appearance of the active immune response indicated by seroconversion. PMID:15971678

Real-time ISEE data system

NASA Technical Reports Server (NTRS)

Tsurutani, B. T.; Baker, D. N.

1979-01-01

A real-time ISEE data system directed toward predicting geomagnetic substorms and storms is discussed. Such a system may allow up to 60+ minutes advance warning of magnetospheric substorms and up to 30 minute warnings of geomagnetic storms (and other disturbances) induced by high-speed streams and solar flares. The proposed system utilizes existing capabilities of several agencies (NASA, NOAA, USAF), and thereby minimizes costs. This same concept may be applicable to data from other spacecraft, and other NASA centers; thus, each individual experimenter can receive quick-look data in real time at his or her base institution.

Subsystem real-time time dependent density functional theory.

PubMed

Krishtal, Alisa; Ceresoli, Davide; Pavanello, Michele

2015-04-21

We present the extension of Frozen Density Embedding (FDE) formulation of subsystem Density Functional Theory (DFT) to real-time Time Dependent Density Functional Theory (rt-TDDFT). FDE is a DFT-in-DFT embedding method that allows to partition a larger Kohn-Sham system into a set of smaller, coupled Kohn-Sham systems. Additional to the computational advantage, FDE provides physical insight into the properties of embedded systems and the coupling interactions between them. The extension to rt-TDDFT is done straightforwardly by evolving the Kohn-Sham subsystems in time simultaneously, while updating the embedding potential between the systems at every time step. Two main applications are presented: the explicit excitation energy transfer in real time between subsystems is demonstrated for the case of the Na4 cluster and the effect of the embedding on optical spectra of coupled chromophores. In particular, the importance of including the full dynamic response in the embedding potential is demonstrated.

A distributed agent architecture for real-time knowledge-based systems: Real-time expert systems project, phase 1

NASA Technical Reports Server (NTRS)

Lee, S. Daniel

1990-01-01

We propose a distributed agent architecture (DAA) that can support a variety of paradigms based on both traditional real-time computing and artificial intelligence. DAA consists of distributed agents that are classified into two categories: reactive and cognitive. Reactive agents can be implemented directly in Ada to meet hard real-time requirements and be deployed on on-board embedded processors. A traditional real-time computing methodology under consideration is the rate monotonic theory that can guarantee schedulability based on analytical methods. AI techniques under consideration for reactive agents are approximate or anytime reasoning that can be implemented using Bayesian belief networks as in Guardian. Cognitive agents are traditional expert systems that can be implemented in ART-Ada to meet soft real-time requirements. During the initial design of cognitive agents, it is critical to consider the migration path that would allow initial deployment on ground-based workstations with eventual deployment on on-board processors. ART-Ada technology enables this migration while Lisp-based technologies make it difficult if not impossible. In addition to reactive and cognitive agents, a meta-level agent would be needed to coordinate multiple agents and to provide meta-level control.

Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings.

PubMed

Zheng, Qianwang; Mikš-Krajnik, Marta; Yang, Yishan; Xu, Wang; Yuk, Hyun-Gyun

2014-09-01

Conventional culture detection methods are time consuming and labor-intensive. For this reason, an alternative rapid method combining real-time PCR and immunomagnetic separation (IMS) was investigated in this study to detect both healthy and heat-injured Salmonella Typhimurium on raw duck wings. Firstly, the IMS method was optimized by determining the capture efficiency of Dynabeads(®) on Salmonella cells on raw duck wings with different bead incubation (10, 30 and 60 min) and magnetic separation (3, 10 and 30 min) times. Secondly, three Taqman primer sets, Sal, invA and ttr, were evaluated to optimize the real-time PCR protocol by comparing five parameters: inclusivity, exclusivity, PCR efficiency, detection probability and limit of detection (LOD). Thirdly, the optimized real-time PCR, in combination with IMS (PCR-IMS) assay, was compared with a standard ISO and a real-time PCR (PCR) method by analyzing artificially inoculated raw duck wings with healthy and heat-injured Salmonella cells at 10(1) and 10(0) CFU/25 g. Finally, the optimized PCR-IMS assay was validated for Salmonella detection in naturally contaminated raw duck wing samples. Under optimal IMS conditions (30 min bead incubation and 3 min magnetic separation times), approximately 85 and 64% of S. Typhimurium cells were captured by Dynabeads® from pure culture and inoculated raw duck wings, respectively. Although Sal and ttr primers exhibited 100% inclusivity and exclusivity for 16 Salmonella spp. and 36 non-Salmonella strains, the Sal primer showed lower LOD (10(3) CFU/ml) and higher PCR efficiency (94.1%) than the invA and ttr primers. Moreover, for Sal and invA primers, 100% detection probability on raw duck wings suspension was observed at 10(3) and 10(4) CFU/ml with and without IMS, respectively. Thus, the Sal primer was chosen for further experiments. The optimized PCR-IMS method was significantly (P=0.0011) better at detecting healthy Salmonella cells after 7-h enrichment than traditional PCR

Real-time software receiver

NASA Technical Reports Server (NTRS)

Psiaki, Mark L. (Inventor); Kintner, Jr., Paul M. (Inventor); Ledvina, Brent M. (Inventor); Powell, Steven P. (Inventor)

2007-01-01

A real-time software receiver that executes on a general purpose processor. The software receiver includes data acquisition and correlator modules that perform, in place of hardware correlation, baseband mixing and PRN code correlation using bit-wise parallelism.

Real-time software receiver

NASA Technical Reports Server (NTRS)

Psiaki, Mark L. (Inventor); Ledvina, Brent M. (Inventor); Powell, Steven P. (Inventor); Kintner, Jr., Paul M. (Inventor)

2006-01-01

A real-time software receiver that executes on a general purpose processor. The software receiver includes data acquisition and correlator modules that perform, in place of hardware correlation, baseband mixing and PRN code correlation using bit-wise parallelism.

Real-time PCR to identify variola virus or other human pathogenic orthopox viruses.

PubMed

Scaramozzino, Natale; Ferrier-Rembert, Audrey; Favier, Anne-Laure; Rothlisberger, Corinne; Richard, Stéphane; Crance, Jean-Marc; Meyer, Hermann; Garin, Daniel

2007-04-01

Variola virus (family Poxviridae, genus Orthopoxvirus) and the closely related cowpox, vaccinia, and monkeypox viruses can infect humans. Efforts are mounting to replenish the smallpox vaccine stocks, optimize diagnostic methods for poxviruses, and develop new antivirals against smallpox, because it is feared that variola virus might be used as a weapon of bioterrorism. We developed an assay for the detection of variola virus DNA. The assay is based on TaqMan chemistry targeting the 14-kD protein gene. For the 1st stage of the assay we used genus consensus primers and a mixture of 2 probes (14-kD POX and 14-kD VAR) spanning the 14-kD protein-encoding gene for detection of all human pathogenic orthopoxviruses. We then tested positive samples with the specific orthopoxvirus-specific probe 14-kD POX to identify monkeypox, cowpox, and vaccinia viruses and with the 14-kD VAR probe to identify variola viruses. The assay was established on 4 different PCR cycler platforms. It was assessed in a study with 85 different orthopoxvirus species and strains that included variola, camelpox, cowpox, monkeypox, and vaccinia viruses at concentrations ranging from 100 ng/L to 1 microg/L. The assay detected as little as 0.05 fg of DNA, corresponding to 25 copies of DNA, and enabled differentiation of variola virus from the other orthopoxviruses. This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections.

Real-time fault diagnosis for propulsion systems

NASA Technical Reports Server (NTRS)

Merrill, Walter C.; Guo, Ten-Huei; Delaat, John C.; Duyar, Ahmet

1991-01-01

Current research toward real time fault diagnosis for propulsion systems at NASA-Lewis is described. The research is being applied to both air breathing and rocket propulsion systems. Topics include fault detection methods including neural networks, system modeling, and real time implementations.

Capability of a Mobile Monitoring System to Provide Real-Time Data Broadcasting and Near Real-Time Source Attribution

NASA Astrophysics Data System (ADS)

Erickson, M.; Olaguer, J.; Wijesinghe, A.; Colvin, J.; Neish, B.; Williams, J.

2014-12-01

It is becoming increasingly important to understand the emissions and health effects of industrial facilities. Many areas have no or limited sustained monitoring capabilities, making it difficult to quantify the major pollution sources affecting human health, especially in fence line communities. Developments in real-time monitoring and micro-scale modeling offer unique ways to tackle these complex issues. This presentation will demonstrate the capability of coupling real-time observations with micro-scale modeling to provide real-time information and near real-time source attribution. The Houston Advanced Research Center constructed the Mobile Acquisition of Real-time Concentrations (MARC) laboratory. MARC consists of a Ford E-350 passenger van outfitted with a Proton Transfer Reaction Mass Spectrometer (PTR-MS) and meteorological equipment. This allows for the fast measurement of various VOCs important to air quality. The data recorded from the van is uploaded to an off-site database and the information is broadcast to a website in real-time. This provides for off-site monitoring of MARC's observations, which allows off-site personnel to provide immediate input to the MARC operators on how to best achieve project objectives. The information stored in the database can also be used to provide near real-time source attribution. An inverse model has been used to ascertain the amount, location, and timing of emissions based on MARC measurements in the vicinity of industrial sites. The inverse model is based on a 3D micro-scale Eulerian forward and adjoint air quality model known as the HARC model. The HARC model uses output from the Quick Urban and Industrial Complex (QUIC) wind model and requires a 3D digital model of the monitored facility based on lidar or industrial permit data. MARC is one of the instrument platforms deployed during the 2014 Benzene and other Toxics Exposure Study (BEE-TEX) in Houston, TX. The main goal of the study is to quantify and explain the

Design and Prototyping of Hard Real Time Systems

DTIC Science & Technology

1990-06-01

AD’-A254 097 DESIGNadPROTOTYPING (9 lliIlhIIIIIl/IIIIIliIliiiiliii OF HARD REAL TIME SYSTEMS Electronics Research Laboratory - - __University of...e ...*...*... . e * ... , .. * 311 2.9 Design of Real - Time Systems with Application to Robotics...Right now we are looking into possible techniques and the implications on the system architecture and partitioning. 2.9. Design of Real - Time Systems with

Real-time computational photon-counting LiDAR

NASA Astrophysics Data System (ADS)

Edgar, Matthew; Johnson, Steven; Phillips, David; Padgett, Miles

2018-03-01

The availability of compact, low-cost, and high-speed MEMS-based spatial light modulators has generated widespread interest in alternative sampling strategies for imaging systems utilizing single-pixel detectors. The development of compressed sensing schemes for real-time computational imaging may have promising commercial applications for high-performance detectors, where the availability of focal plane arrays is expensive or otherwise limited. We discuss the research and development of a prototype light detection and ranging (LiDAR) system via direct time of flight, which utilizes a single high-sensitivity photon-counting detector and fast-timing electronics to recover millimeter accuracy three-dimensional images in real time. The development of low-cost real time computational LiDAR systems could have importance for applications in security, defense, and autonomous vehicles.

Real time magnetic resonance guided endomyocardial local delivery

PubMed Central

Corti, R; Badimon, J; Mizsei, G; Macaluso, F; Lee, M; Licato, P; Viles-Gonzalez, J F; Fuster, V; Sherman, W

2005-01-01

Objective: To investigate the feasibility of targeting various areas of left ventricle myocardium under real time magnetic resonance (MR) imaging with a customised injection catheter equipped with a miniaturised coil. Design: A needle injection catheter with a mounted resonant solenoid circuit (coil) at its tip was designed and constructed. A 1.5 T MR scanner with customised real time sequence combined with in-room scan running capabilities was used. With this system, various myocardial areas within the left ventricle were targeted and injected with a gadolinium-diethylenetriaminepentaacetic acid (DTPA) and Indian ink mixture. Results: Real time sequencing at 10 frames/s allowed clear visualisation of the moving catheter and its transit through the aorta into the ventricle, as well as targeting of all ventricle wall segments without further image enhancement techniques. All injections were visualised by real time MR imaging and verified by gross pathology. Conclusion: The tracking device allowed real time in vivo visualisation of catheters in the aorta and left ventricle as well as precise targeting of myocardial areas. The use of this real time catheter tracking may enable precise and adequate delivery of agents for tissue regeneration. PMID:15710717

Rate Monotonic Analysis for Real-Time Systems

DTIC Science & Technology

1991-03-01

The essential goal of the Rate Monotonic Analysis (RMA) for Real - Time Systems Project at the Software Engineering Institute is to catalyze...improvement in the practice of real time systems engineering, specifically by increasing the use of rate monotonic analysis and scheduling algorithms. In this

MISR Level 1 Near Real Time Products

Atmospheric Science Data Center

2016-10-31

Level 1 Near Real Time The MISR Near Real Time Level 1 data products ... km MISR swath and projected onto a Space-Oblique Mercator (SOM) map grid. The Ellipsoid-projected and Terrain-projected top-of-atmosphere (TOA) radiance products provide measurements respectively resampled onto the ...

[Analytical performances of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine].

PubMed

De Monte, Anne; Cannavo, Isabelle; Caramella, Anne; Ollier, Laurence; Giordanengo, Valérie

2016-01-01

Congenital cytomegalovirus (CMV) infection is the leading cause of sensoneurinal disability due to infectious congenital disease. The diagnosis of congenital CMV infection is based on the search of CMV in the urine within the first two weeks of life. Viral culture of urine is the gold standard. However, the PCR is highly sensitive and faster. It is becoming an alternative choice. The objective of this study is the validation of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine. Repeatability, reproducibility, detection limit and inter-sample contamination were evaluated. Urine samples from patients (n=141) were collected and analyzed simultaneously in culture and PCR in order to assess the correlation of these two methods. The sensitivity and specificity of PCR were also calculated. The Abbott RealTime CMV PCR in urine is an automated and sensitive method (detection limit 200 UI/mL). Fidelity is very good (standard deviation of repeatability: 0.08 to 0.15 LogUI/mL and reproducibility 0.18 LogUI/mL). We can note a good correlation between culture and Abbott RealTime CMV PCR (kappa 96%). When considering rapid culture as reference, real-time PCR was highly sensitive (100%) and specific (98.2%). The real-time PCR by Abbott RealTime CMV with m2000 is optimal for CMV detection in urine.

Formal methods for dependable real-time systems

NASA Technical Reports Server (NTRS)

Rushby, John

1993-01-01

The motivation for using formal methods to specify and reason about real time properties is outlined and approaches that were proposed and used are sketched. The formal verifications of clock synchronization algorithms are concluded as showing that mechanically supported reasoning about complex real time behavior is feasible. However, there was significant increase in the effectiveness of verification systems since those verifications were performed, at it is to be expected that verifications of comparable difficulty will become fairly routine. The current challenge lies in developing perspicuous and economical approaches to the formalization and specification of real time properties.

Real Time Computer Graphics From Body Motion

NASA Astrophysics Data System (ADS)

Fisher, Scott; Marion, Ann

1983-10-01

This paper focuses on the recent emergence and development of real, time, computer-aided body tracking technologies and their use in combination with various computer graphics imaging techniques. The convergence of these, technologies in our research results, in an interactive display environment. in which multipde, representations of a given body motion can be displayed in real time. Specific reference, to entertainment applications is described in the development of a real time, interactive stage set in which dancers can 'draw' with their bodies as they move, through the space. of the stage or manipulate virtual elements of the set with their gestures.

Real time sensor for therapeutic radiation delivery

DOEpatents

Bliss, Mary; Craig, Richard A.; Reeder, Paul L.

1998-01-01

The invention is a real time sensor for therapeutic radiation. A probe is placed in or near the patient that senses in real time the dose at the location of the probe. The strength of the dose is determined by either an insertion or an exit probe. The location is determined by a series of vertical and horizontal sensing elements that gives the operator a real time read out dose location relative to placement of the patient. The increased accuracy prevents serious tissue damage to the patient by preventing overdose or delivery of a dose to a wrong location within the body.

Development and evaluation of probe based real time loop mediated isothermal amplification for Salmonella: A new tool for DNA quantification.

PubMed

Mashooq, Mohmad; Kumar, Deepak; Niranjan, Ankush Kiran; Agarwal, Rajesh Kumar; Rathore, Rajesh

2016-07-01

A one step, single tube, accelerated probe based real time loop mediated isothermal amplification (RT LAMP) assay was developed for detecting the invasion gene (InvA) of Salmonella. The probe based RT LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity and rapidity under isothermal conditions with a set of six primers. The whole procedure is very simple and rapid, and amplification can be obtained in 20min. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of DNA binding dye at the end of the reaction results in colour difference and can be visualized under normal day light and in UV. The sensitivity of developed assay was found 10 fold higher than taqman based qPCR. The specificity of the RT LAMP assay was validated by the absence of any cross reaction with other members of enterobacteriaceae family and other gram negative bacteria. These results indicate that the probe based RT LAMP assay is extremely rapid, cost effective, highly specific and sensitivity and has potential usefulness for rapid Salmonella surveillance. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Real-time Avatar Animation from a Single Image.

PubMed

Saragih, Jason M; Lucey, Simon; Cohn, Jeffrey F

2011-01-01

A real time facial puppetry system is presented. Compared with existing systems, the proposed method requires no special hardware, runs in real time (23 frames-per-second), and requires only a single image of the avatar and user. The user's facial expression is captured through a real-time 3D non-rigid tracking system. Expression transfer is achieved by combining a generic expression model with synthetically generated examples that better capture person specific characteristics. Performance of the system is evaluated on avatars of real people as well as masks and cartoon characters.

Real-time Avatar Animation from a Single Image

PubMed Central

Saragih, Jason M.; Lucey, Simon; Cohn, Jeffrey F.

2014-01-01

A real time facial puppetry system is presented. Compared with existing systems, the proposed method requires no special hardware, runs in real time (23 frames-per-second), and requires only a single image of the avatar and user. The user’s facial expression is captured through a real-time 3D non-rigid tracking system. Expression transfer is achieved by combining a generic expression model with synthetically generated examples that better capture person specific characteristics. Performance of the system is evaluated on avatars of real people as well as masks and cartoon characters. PMID:24598812

A multiprocessing architecture for real-time monitoring

NASA Technical Reports Server (NTRS)

Schmidt, James L.; Kao, Simon M.; Read, Jackson Y.; Weitzenkamp, Scott M.; Laffey, Thomas J.

1988-01-01

A multitasking architecture for performing real-time monitoring and analysis using knowledge-based problem solving techniques is described. To handle asynchronous inputs and perform in real time, the system consists of three or more distributed processes which run concurrently and communicate via a message passing scheme. The Data Management Process acquires, compresses, and routes the incoming sensor data to other processes. The Inference Process consists of a high performance inference engine that performs a real-time analysis on the state and health of the physical system. The I/O Process receives sensor data from the Data Management Process and status messages and recommendations from the Inference Process, updates its graphical displays in real time, and acts as the interface to the console operator. The distributed architecture has been interfaced to an actual spacecraft (NASA's Hubble Space Telescope) and is able to process the incoming telemetry in real-time (i.e., several hundred data changes per second). The system is being used in two locations for different purposes: (1) in Sunnyville, California at the Space Telescope Test Control Center it is used in the preflight testing of the vehicle; and (2) in Greenbelt, Maryland at NASA/Goddard it is being used on an experimental basis in flight operations for health and safety monitoring.

An Analysis of Input/Output Paradigms for Real-Time Systems

DTIC Science & Technology

1990-07-01

timing and concurrency aspects of real - time systems . This paper illustrates how to build a mathematical model of the schedulability of a real-time...various design alternatives. The primary characteristic that distinguishes real-time system from non- real - time systems is the importance of time. The

PERTS: A Prototyping Environment for Real-Time Systems

NASA Technical Reports Server (NTRS)

Liu, Jane W. S.; Lin, Kwei-Jay; Liu, C. L.

1993-01-01

PERTS is a prototyping environment for real-time systems. It is being built incrementally and will contain basic building blocks of operating systems for time-critical applications, tools, and performance models for the analysis, evaluation and measurement of real-time systems and a simulation/emulation environment. It is designed to support the use and evaluation of new design approaches, experimentations with alternative system building blocks, and the analysis and performance profiling of prototype real-time systems.

Real-time position reconstruction with hippocampal place cells.

PubMed

Guger, Christoph; Gener, Thomas; Pennartz, Cyriel M A; Brotons-Mas, Jorge R; Edlinger, Günter; Bermúdez I Badia, S; Verschure, Paul; Schaffelhofer, Stefan; Sanchez-Vives, Maria V

2011-01-01

Brain-computer interfaces (BCI) are using the electroencephalogram, the electrocorticogram and trains of action potentials as inputs to analyze brain activity for communication purposes and/or the control of external devices. Thus far it is not known whether a BCI system can be developed that utilizes the states of brain structures that are situated well below the cortical surface, such as the hippocampus. In order to address this question we used the activity of hippocampal place cells (PCs) to predict the position of an rodent in real-time. First, spike activity was recorded from the hippocampus during foraging and analyzed off-line to optimize the spike sorting and position reconstruction algorithm of rats. Then the spike activity was recorded and analyzed in real-time. The rat was running in a box of 80 cm × 80 cm and its locomotor movement was captured with a video tracking system. Data were acquired to calculate the rat's trajectories and to identify place fields. Then a Bayesian classifier was trained to predict the position of the rat given its neural activity. This information was used in subsequent trials to predict the rat's position in real-time. The real-time experiments were successfully performed and yielded an error between 12.2 and 17.4% using 5-6 neurons. It must be noted here that the encoding step was done with data recorded before the real-time experiment and comparable accuracies between off-line (mean error of 15.9% for three rats) and real-time experiments (mean error of 14.7%) were achieved. The experiment shows proof of principle that position reconstruction can be done in real-time, that PCs were stable and spike sorting was robust enough to generalize from the training run to the real-time reconstruction phase of the experiment. Real-time reconstruction may be used for a variety of purposes, including creating behavioral-neuronal feedback loops or for implementing neuroprosthetic control.

Real-Time Position Reconstruction with Hippocampal Place Cells

PubMed Central

Guger, Christoph; Gener, Thomas; Pennartz, Cyriel M. A.; Brotons-Mas, Jorge R.; Edlinger, Günter; Bermúdez i Badia, S.; Verschure, Paul; Schaffelhofer, Stefan; Sanchez-Vives, Maria V.

2011-01-01

Brain–computer interfaces (BCI) are using the electroencephalogram, the electrocorticogram and trains of action potentials as inputs to analyze brain activity for communication purposes and/or the control of external devices. Thus far it is not known whether a BCI system can be developed that utilizes the states of brain structures that are situated well below the cortical surface, such as the hippocampus. In order to address this question we used the activity of hippocampal place cells (PCs) to predict the position of an rodent in real-time. First, spike activity was recorded from the hippocampus during foraging and analyzed off-line to optimize the spike sorting and position reconstruction algorithm of rats. Then the spike activity was recorded and analyzed in real-time. The rat was running in a box of 80 cm × 80 cm and its locomotor movement was captured with a video tracking system. Data were acquired to calculate the rat's trajectories and to identify place fields. Then a Bayesian classifier was trained to predict the position of the rat given its neural activity. This information was used in subsequent trials to predict the rat's position in real-time. The real-time experiments were successfully performed and yielded an error between 12.2 and 17.4% using 5–6 neurons. It must be noted here that the encoding step was done with data recorded before the real-time experiment and comparable accuracies between off-line (mean error of 15.9% for three rats) and real-time experiments (mean error of 14.7%) were achieved. The experiment shows proof of principle that position reconstruction can be done in real-time, that PCs were stable and spike sorting was robust enough to generalize from the training run to the real-time reconstruction phase of the experiment. Real-time reconstruction may be used for a variety of purposes, including creating behavioral–neuronal feedback loops or for implementing neuroprosthetic control. PMID:21808603

Quantitative real-time imaging of glutathione

USDA-ARS?s Scientific Manuscript database

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

The Synthesis of Intelligent Real-Time Systems

DTIC Science & Technology

1990-11-09

Synthesis of Intelligent Real - Time Systems . The purpose of the effort was to develop and extend theories and techniques that facilitate the design and...implementation of intelligent real - time systems . In particular, Teleos has extended situated-automata theory to apply to situations in which the system has

The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F

PubMed Central

Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

2017-01-01

ABSTRACT Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. PMID:28202793

Review of Real-Time Simulator and the Steps Involved for Implementation of a Model from MATLAB/SIMULINK to Real-Time

NASA Astrophysics Data System (ADS)

Mikkili, Suresh; Panda, Anup Kumar; Prattipati, Jayanthi

2015-06-01

Nowadays the researchers want to develop their model in real-time environment. Simulation tools have been widely used for the design and improvement of electrical systems since the mid twentieth century. The evolution of simulation tools has progressed in step with the evolution of computing technologies. In recent years, computing technologies have improved dramatically in performance and become widely available at a steadily decreasing cost. Consequently, simulation tools have also seen dramatic performance gains and steady cost decreases. Researchers and engineers now have the access to affordable, high performance simulation tools that were previously too cost prohibitive, except for the largest manufacturers. This work has introduced a specific class of digital simulator known as a real-time simulator by answering the questions "what is real-time simulation", "why is it needed" and "how it works". The latest trend in real-time simulation consists of exporting simulation models to FPGA. In this article, the Steps involved for implementation of a model from MATLAB to REAL-TIME are provided in detail.

Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex.

PubMed

Kishimoto, Mai; Tsuchiaka, Shinobu; Rahpaya, Sayed Samim; Hasebe, Ayako; Otsu, Keiko; Sugimura, Satoshi; Kobayashi, Suguru; Komatsu, Natsumi; Nagai, Makoto; Omatsu, Tsutomu; Naoi, Yuki; Sano, Kaori; Okazaki-Terashima, Sachiko; Oba, Mami; Katayama, Yukie; Sato, Reiichiro; Asai, Tetsuo; Mizutani, Tetsuya

2017-03-18

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris

PubMed Central

2013-01-01

Background Failure of colony PCRs in green microalga Chlorella vulgaris is typically attributed to the difficulty in disrupting its notoriously rigid cell walls for releasing the genetic materials and therefore the development of an effective colony PCR procedure in C. vulgaris presents a challenge. Results Here we identified that colony PCR results were significantly affected by the accumulated lipids rather than the rigid cell walls of C. vulgaris. The higher lipids accumulated in C. vulgaris negatively affects the effective amplification by DNA polymerase. Based on these findings, we established a simple and extremely effective colony PCR procedure in C. vulgaris. By simply pipetting/votexing the pellets of C. vulgaris in 10 ul of either TE (10 mM Tris/1 mM EDTA) or 0.2% SDS buffer at room temperature, followed by the addition of 10 ul of either hexane or Phenol:Chloroform:Isoamyl Alcohol in the same PCR tube for extraction. The resulting aqueous phase was readily PCR-amplified as genomic DNA templates as demonstrated by successful amplification of the nuclear 18S rRNA and the chloroplast rbcL gene. This colony PCR protocol is effective and robust in C. vulgaris and also demonstrates its effectiveness in other Chlorella species. Conclusions The accumulated lipids rather than the rigid cell walls of C. vulgaris significantly impede the extraction of genetic materials and subsequently the effective colony PCRs. The finding has the potential to aid the isolation of high-quality total RNAs and mRNAs for transcriptomic studies in addition to the genomic DNA isolation in Chlorella. PMID:24219401

HybProbes-based real-time PCR assay for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei, the potato common scab pathogens.

PubMed

Xu, R; Falardeau, J; Avis, T J; Tambong, J T

2016-02-01

The aim of this study was to develop and validate a HybProbes-based real-time PCR assay targeting the trpB gene for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei. Four primer pairs and a fluorescent probe were designed and evaluated for specificity in identifying S. scabies and Streptomyces europaeiscabiei, the potato common scab pathogens. The specificity of the HybProbes-based real-time PCR assay was evaluated using 46 bacterial strains, 23 Streptomyces strains and 23 non-Streptomyces bacterial species. Specific and strong fluorescence signals were detected from all nine strains of S. scabies and Streptomyces europaeiscabiei. No fluorescence signal was detected from 14 strains of other Streptomyces species and all non-Streptomyces strains. The identification was corroborated by the melting curve analysis that was performed immediately after the amplification step. Eight of the nine S. scabies and S. europaeiscabiei strains exhibited a unique melting peak, at Tm of 69·1°C while one strain, Warba-6, had a melt peak at Tm of 65·4°C. This difference in Tm peaks could be attributed to a guanine to cytosine mutation in strain Warba-6 at the region spanning the donor HybProbe. The reported HybProbes assay provides a more specific tool for accurate identification of S. scabies and S. europaeiscabiei strains. This study reports a novel assay based on HybProbes chemistry for rapid and accurate identification of the potato common scab pathogens. Since the HybProbes chemistry requires two probes for positive identification, the assay is considered to be more specific than conventional PCR or TaqMan real-time PCR. The developed assay would be a useful tool with great potential in early diagnosis and detection of common scab pathogens of potatoes in infected plants or for surveillance of potatoes grown in soil environment. © 2015 Her Majesty the Queen in Right of Canada © 2015 The Society for Applied Microbiology.

Real-Time IRI driven by GIRO data

NASA Astrophysics Data System (ADS)

Galkin, Ivan; Huang, Xueqin; Reinisch, Bodo; Bilitza, Dieter; Vesnin, Artem

Real-time extensions of the empirical International Reference Ionosphere (IRI) model are based on assimilative techniques that preserve the IRI formalism which is optimized for the description of climatological ionospheric features. The Global Ionosphere Radio Observatory (GIRO) team has developed critical parts of an IRI Real Time Assimilative Model (IRTAM) for the global ionospheric plasma distribution using measured data available in real time from ~50 ionosondes of the GIRO network, The current assimilation results present global assimilative maps of foF2 and hmF2 that reproduce available data at the sensor sites and smoothly return to the climatological specifications when and where the data are missing, and are free from artificial sharp gradients and short-lived artifacts when viewed in time progression. Animated real-time maps of foF2 and hmF2 are published with a few minutes latency at http://giro.uml.edu/IRTAM/. Our real-time IRI modeling uses morphing, a technique that transforms the climatological ionospheric specifications to match the observations by iteratively computing corrections to the original coefficients of the diurnal/spatial expansions, used in IRI to map the key ionospheric characteristics, while keeping the IRI expansion basis formalism intact. Computation of the updated coefficient set for a given point in time includes analysis of the latest 24-hour history of observations, which allows the morphing technique to sense evolving ionospheric dynamics even with a sparse sensor network. A Non-linear Error Compensation Technique for Associative Restoration (NECTAR), one of the features in our morphing approach, has been in operation at the Lowell GIRO Data Center since 2013. The cornerstone of NECTAR is a recurrent neural network optimizer that is responsible for smoothing the transitions between the grid cells where observations are available. NECTAR has proved suitable for real-time operations that require the assimilation code to be

Real-time Simulation of Turboprop Engine Control System

NASA Astrophysics Data System (ADS)

Sheng, Hanlin; Zhang, Tianhong; Zhang, Yi

2017-05-01

On account of the complexity of turboprop engine control system, real-time simulation is the technology, under the prerequisite of maintaining real-time, to effectively reduce development cost, shorten development cycle and avert testing risks. The paper takes RT-LAB as a platform and studies the real-time digital simulation of turboprop engine control system. The architecture, work principles and external interfaces of RT-LAB real-time simulation platform are introduced firstly. Then based on a turboprop engine model, the control laws of propeller control loop and fuel control loop are studied. From that and on the basis of Matlab/Simulink, an integrated controller is designed which can realize the entire process control of the engine from start-up to maximum power till stop. At the end, on the basis of RT-LAB platform, the real-time digital simulation of the designed control system is studied, different regulating plans are tried and more ideal control effects have been obtained.

Proceedings of the Real-Time Systems Engineering Workshop

DTIC Science & Technology

2001-08-01

real - time systems engineering. The workshop was held as part of the SEI Symposium in...Washington, DC, during September 2000. The objective of the workshop was to identify key issues and obtain feedback from attendees concerning real - time systems engineering...and interoperability. This report summarizes the workshop in terms of foundation, management, and technical topics, and it contains a discussion related to developing a community of interest for real - time systems

Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections.

PubMed

Onyango, Clayton O; Loparev, Vladimir; Lidechi, Shirley; Bhullar, Vinod; Schmid, D Scott; Radford, Kay; Lo, Michael K; Rota, Paul; Johnson, Barbara W; Munoz, Jorge; Oneko, Martina; Burton, Deron; Black, Carolyn M; Neatherlin, John; Montgomery, Joel M; Fields, Barry

2017-07-01

Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites ( Balamuthia mandrillaris and Acanthamoeba ), six bacterial pathogens ( Streptococcus pneumonia e, Haemophilus influenzae , Neisseria meningitidis , Mycoplasma pneumoniae , Mycobacterium tuberculosis , and Bartonella ), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease. Copyright © 2017 American Society for Microbiology.

Real time sensor for therapeutic radiation delivery

DOEpatents

Bliss, M.; Craig, R.A.; Reeder, P.L.

1998-01-06

The invention is a real time sensor for therapeutic radiation. A probe is placed in or near the patient that senses in real time the dose at the location of the probe. The strength of the dose is determined by either an insertion or an exit probe. The location is determined by a series of vertical and horizontal sensing elements that gives the operator a real time read out dose location relative to placement of the patient. The increased accuracy prevents serious tissue damage to the patient by preventing overdose or delivery of a dose to a wrong location within the body. 14 figs.

Real-time inspection by submarine images

NASA Astrophysics Data System (ADS)

Tascini, Guido; Zingaretti, Primo; Conte, Giuseppe

1996-10-01

A real-time application of computer vision concerning tracking and inspection of a submarine pipeline is described. The objective is to develop automatic procedures for supporting human operators in the real-time analysis of images acquired by means of cameras mounted on underwater remotely operated vehicles (ROV) Implementation of such procedures gives rise to a human-machine system for underwater pipeline inspection that can automatically detect and signal the presence of the pipe, of its structural or accessory elements, and of dangerous or alien objects in its neighborhood. The possibility of modifying the image acquisition rate in the simulations performed on video- recorded images is used to prove that the system performs all necessary processing with an acceptable robustness working in real-time up to a speed of about 2.5 kn, widely greater than that the actual ROVs and the security features allow.

RealTime Physics: Active learning laboratory

NASA Astrophysics Data System (ADS)

Thornton, Ronald K.; Sokoloff, David R.

1997-03-01

Our research shows that student learning of physics concepts in introductory physics courses is enhanced by the use of special guided discovery laboratory curricula which embody the results of educational research and which are supported by the use of the Tools for Scientific Thinking microcomputer-based laboratory (MBL) tools. In this paper we first describe the general characteristics of the research-based RealTime Physics laboratory curricula developed for use in introductory physics classes in colleges, universities and high schools. We then describe RealTime Physics Mechanics in detail. Finally we examine student learning of dynamics in traditional physics courses and in courses using RealTime Physics Mechanics, primarily by the use of correlated questions on the Force and Motion Conceptual Evaluation. We present considerable evidence that students who use the new laboratory curricula demonstrate significantly improved learning and retention of dynamics concepts compared to students taught by traditional methods.

Real-Time Parameter Estimation Using Output Error

NASA Technical Reports Server (NTRS)

Grauer, Jared A.

2014-01-01

Output-error parameter estimation, normally a post- ight batch technique, was applied to real-time dynamic modeling problems. Variations on the traditional algorithm were investigated with the goal of making the method suitable for operation in real time. Im- plementation recommendations are given that are dependent on the modeling problem of interest. Application to ight test data showed that accurate parameter estimates and un- certainties for the short-period dynamics model were available every 2 s using time domain data, or every 3 s using frequency domain data. The data compatibility problem was also solved in real time, providing corrected sensor measurements every 4 s. If uncertainty corrections for colored residuals are omitted, this rate can be increased to every 0.5 s.

Coordinating Transit Transfers in Real Time

DOT National Transportation Integrated Search

2016-05-06

Transfers are a major source of travel time variability for transit passengers. Coordinating transfers between transit routes in real time can reduce passenger waiting times and travel time variability, but these benefits need to be contrasted with t...

Applying Real-Time UML: Real-World Experiences

NASA Astrophysics Data System (ADS)

Cooling, Niall; Pachschwoell, Stefan

2004-06-01

This paper presents Austrian Aerospace's experiences of applying UML for the design of an embedded real-time avionics system based on Feabhas' "Pragma Process". It describes the complete lifecycle from adoption of UML, through training, CASE-tool selection, system analysis, and software design and development of the project itself. It concludes by reflecting on the experiences obtained and some lessons learnt.

Head movement compensation in real-time magnetoencephalographic recordings.

PubMed

Little, Graham; Boe, Shaun; Bardouille, Timothy

2014-01-01

Neurofeedback- and brain-computer interface (BCI)-based interventions can be implemented using real-time analysis of magnetoencephalographic (MEG) recordings. Head movement during MEG recordings, however, can lead to inaccurate estimates of brain activity, reducing the efficacy of the intervention. Most real-time applications in MEG have utilized analyses that do not correct for head movement. Effective means of correcting for head movement are needed to optimize the use of MEG in such applications. Here we provide preliminary validation of a novel analysis technique, real-time source estimation (rtSE), that measures head movement and generates corrected current source time course estimates in real-time. rtSE was applied while recording a calibrated phantom to determine phantom position localization accuracy and source amplitude estimation accuracy under stationary and moving conditions. Results were compared to off-line analysis methods to assess validity of the rtSE technique. The rtSE method allowed for accurate estimation of current source activity at the source-level in real-time, and accounted for movement of the source due to changes in phantom position. The rtSE technique requires modifications and specialized analysis of the following MEG work flow steps.•Data acquisition•Head position estimation•Source localization•Real-time source estimation This work explains the technical details and validates each of these steps.

Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS)

NASA Astrophysics Data System (ADS)

Daniels, M. D.; Graves, S. J.; Vernon, F.; Kerkez, B.; Chandra, C. V.; Keiser, K.; Martin, C.

2014-12-01

Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS) Access, utilization and management of real-time data continue to be challenging for decision makers, as well as researchers in several scientific fields. This presentation will highlight infrastructure aimed at addressing some of the gaps in handling real-time data, particularly in increasing accessibility of these data to the scientific community through cloud services. The Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS) system addresses the ever-increasing importance of real-time scientific data, particularly in mission critical scenarios, where informed decisions must be made rapidly. Advances in the distribution of real-time data are leading many new transient phenomena in space-time to be observed, however real-time decision-making is infeasible in many cases that require streaming scientific data as these data are locked down and sent only to proprietary in-house tools or displays. This lack of accessibility to the broader scientific community prohibits algorithm development and workflows initiated by these data streams. As part of NSF's EarthCube initiative, CHORDS proposes to make real-time data available to the academic community via cloud services. The CHORDS infrastructure will enhance the role of real-time data within the geosciences, specifically expanding the potential of streaming data sources in enabling adaptive experimentation and real-time hypothesis testing. Adherence to community data and metadata standards will promote the integration of CHORDS real-time data with existing standards-compliant analysis, visualization and modeling tools.

Real-time hierarchically distributed processing network interaction simulation

NASA Technical Reports Server (NTRS)

Zimmerman, W. F.; Wu, C.

1987-01-01

The Telerobot Testbed is a hierarchically distributed processing system which is linked together through a standard, commercial Ethernet. Standard Ethernet systems are primarily designed to manage non-real-time information transfer. Therefore, collisions on the net (i.e., two or more sources attempting to send data at the same time) are managed by randomly rescheduling one of the sources to retransmit at a later time interval. Although acceptable for transmitting noncritical data such as mail, this particular feature is unacceptable for real-time hierarchical command and control systems such as the Telerobot. Data transfer and scheduling simulations, such as token ring, offer solutions to collision management, but do not appropriately characterize real-time data transfer/interactions for robotic systems. Therefore, models like these do not provide a viable simulation environment for understanding real-time network loading. A real-time network loading model is being developed which allows processor-to-processor interactions to be simulated, collisions (and respective probabilities) to be logged, collision-prone areas to be identified, and network control variable adjustments to be reentered as a means of examining and reducing collision-prone regimes that occur in the process of simulating a complete task sequence.

Time-critical multirate scheduling using contemporary real-time operating system services

NASA Technical Reports Server (NTRS)

Eckhardt, D. E., Jr.

1983-01-01

Although real-time operating systems provide many of the task control services necessary to process time-critical applications (i.e., applications with fixed, invariant deadlines), it may still be necessary to provide a scheduling algorithm at a level above the operating system in order to coordinate a set of synchronized, time-critical tasks executing at different cyclic rates. The scheduling requirements for such applications and develops scheduling algorithms using services provided by contemporary real-time operating systems.

Real-Time Optimization and Control of Next-Generation Distribution

Science.gov Websites

Infrastructure | Grid Modernization | NREL Real-Time Optimization and Control of Next -Generation Distribution Infrastructure Real-Time Optimization and Control of Next-Generation Distribution Infrastructure This project develops innovative, real-time optimization and control methods for next-generation

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

PubMed

Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

2014-07-05

The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the

Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species.

PubMed

Palacio-Bielsa, Ana; Cubero, Jaime; Cambra, Miguel A; Collados, Raquel; Berruete, Isabel M; López, María M

2011-01-01

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being X. arboricola pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in X. arboricola pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10(2) CFU ml(-1), thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for X. arboricola pv. pruni strains from different origins as well as for closely related Xanthomonas species, non-Xanthomonas species, saprophytic bacteria, and healthy Prunus samples. The efficiency of the developed protocol was evaluated with field samples of 14 Prunus species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and X. arboricola pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for X. arboricola pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

Two New Real-Time PCR-based Surveillance Systems for “Candidatus Liberibacter” Species Detection

USDA-ARS?s Scientific Manuscript database

We developed two novel surveillance systems for “Candidatus Liberibacter” (CL) species detection and identification. The first system is called “single tube dual primer Taq-Man PCR” (STDP). The procedure involves two sequential rounds of PCR using the CL asiaticus species-specific outer and inner pr...

Molecular Tracing of the Origin of Six Different Plant Species in Bee Honey Using Real-Time PCR.

PubMed

Wu, Yajun; Yang, Yange; Liu, Mingchang; Wang, Bin; Li, Meige; Chen, Ying

2017-05-01

The quality of honey is significantly influenced by floral origin. Mislabeling floral species occurs frequently in bee honey products. To protect consumers from economic fraud and maintain a fair market environment, methods to identify floral species in honey are necessary. In our study, real-time PCRs were established, targeting six honey types mainly produced in China (canola, Chinese milkvetch, Chinese chaste tree, locust tree, litchi, and longan). Sensitivity testing on DNA from plant tissues exhibited LODs of about 0.5-5 pg/μL. For DNA extracts of pollen sediments from different honey species, LODs ranged from 13.6 to 403.2 pg/μL. In an experiment to determine the practical LODs of honey in which adulterant honey was spiked in the genuine honey, adulterant honey as low as about 0.1-0.5% was detected in 90-100% in 10 parallel tests. Additionally, pollen was spiked in the honey and stored under various conditions to investigate the migration of pollen DNA into the honey supernatant. Finally, the efficiency of our method was investigated by testing honey samples of unknown compositions from different geographic regions. Of the 159 honey samples that were supposed to be monofloral that had been collected in five provinces, a small portion were found to be contaminated with foreign pollen (7%). The methods proved to be specific, sensitive, and reliable in identifying the six plant species in honey, which would be a useful tool during the market supervision and QC of honey products.

Real-Time Interactive Tree Animation.

PubMed

Quigley, Ed; Yu, Yue; Huang, Jingwei; Lin, Winnie; Fedkiw, Ronald

2018-05-01

We present a novel method for posing and animating botanical tree models interactively in real time. Unlike other state of the art methods which tend to produce trees that are overly flexible, bending and deforming as if they were underwater plants, our approach allows for arbitrarily high stiffness while still maintaining real-time frame rates without spurious artifacts, even on quite large trees with over ten thousand branches. This is accomplished by using an articulated rigid body model with as-stiff-as-desired rotational springs in conjunction with our newly proposed simulation technique, which is motivated both by position based dynamics and the typical algorithms for articulated rigid bodies. The efficiency of our algorithm allows us to pose and animate trees with millions of branches or alternatively simulate a small forest comprised of many highly detailed trees. Even using only a single CPU core, we can simulate ten thousand branches in real time while still maintaining quite crisp user interactivity. This has allowed us to incorporate our framework into a commodity game engine to run interactively even on a low-budget tablet. We show that our method is amenable to the incorporation of a large variety of desirable effects such as wind, leaves, fictitious forces, collisions, fracture, etc.

Real-time neutron imaging of gas turbines

NASA Astrophysics Data System (ADS)

Stewart, P. A. E.

1987-06-01

The current status of real-time neutron radiography imaging is briefly reviewed, and results of tests carried out on cold neutron sources are reported. In particular, attention is given to demonstrations of neutron radiography on a running gas turbine engine. The future role of real-time neutron imaging in engineering diagnostics is briefly discussed.

Explaining How to Play Real-Time Strategy Games

NASA Astrophysics Data System (ADS)

Metoyer, Ronald; Stumpf, Simone; Neumann, Christoph; Dodge, Jonathan; Cao, Jill; Schnabel, Aaron

Real-time strategy games share many aspects with real situations in domains such as battle planning, air traffic control, and emergency response team management which makes them appealing test-beds for Artificial Intelligence (AI) and machine learning. End user annotations could help to provide supplemental information for learning algorithms, especially when training data is sparse. This paper presents a formative study to uncover how experienced users explain game play in real-time strategy games. We report the results of our analysis of explanations and discuss their characteristics that could support the design of systems for use by experienced real-time strategy game users in specifying or annotating strategy-oriented behavior.

Real-time color measurement using active illuminant

NASA Astrophysics Data System (ADS)

Tominaga, Shoji; Horiuchi, Takahiko; Yoshimura, Akihiko

2010-01-01

This paper proposes a method for real-time color measurement using active illuminant. A synchronous measurement system is constructed by combining a high-speed active spectral light source and a high-speed monochrome camera. The light source is a programmable spectral source which is capable of emitting arbitrary spectrum in high speed. This system is the essential advantage of capturing spectral images without using filters in high frame rates. The new method of real-time colorimetry is different from the traditional method based on the colorimeter or the spectrometers. We project the color-matching functions onto an object surface as spectral illuminants. Then we can obtain the CIE-XYZ tristimulus values directly from the camera outputs at every point on the surface. We describe the principle of our colorimetric technique based on projection of the color-matching functions and the procedure for realizing a real-time measurement system of a moving object. In an experiment, we examine the performance of real-time color measurement for a static object and a moving object.

A Practical Approach to Implementing Real-Time Semantics

NASA Technical Reports Server (NTRS)

Luettgen, Gerald; Bhat, Girish; Cleaveland, Rance

1999-01-01

This paper investigates implementations of process algebras which are suitable for modeling concurrent real-time systems. It suggests an approach for efficiently implementing real-time semantics using dynamic priorities. For this purpose a proces algebra with dynamic priority is defined, whose semantics corresponds one-to-one to traditional real-time semantics. The advantage of the dynamic-priority approach is that it drastically reduces the state-space sizes of the systems in question while preserving all properties of their functional and real-time behavior. The utility of the technique is demonstrated by a case study which deals with the formal modeling and verification of the SCSI-2 bus-protocol. The case study is carried out in the Concurrency Workbench of North Carolina, an automated verification tool in which the process algebra with dynamic priority is implemented. It turns out that the state space of the bus-protocol model is about an order of magnitude smaller than the one resulting from real-time semantics. The accuracy of the model is proved by applying model checking for verifying several mandatory properties of the bus protocol.

Recent achievements in real-time computational seismology in Taiwan

NASA Astrophysics Data System (ADS)

Lee, S.; Liang, W.; Huang, B.

2012-12-01

Real-time computational seismology is currently possible to be achieved which needs highly connection between seismic database and high performance computing. We have developed a real-time moment tensor monitoring system (RMT) by using continuous BATS records and moment tensor inversion (CMT) technique. The real-time online earthquake simulation service is also ready to open for researchers and public earthquake science education (ROS). Combine RMT with ROS, the earthquake report based on computational seismology can provide within 5 minutes after an earthquake occurred (RMT obtains point source information < 120 sec; ROS completes a 3D simulation < 3 minutes). All of these computational results are posted on the internet in real-time now. For more information, welcome to visit real-time computational seismology earthquake report webpage (RCS).

Fault recovery for real-time, multi-tasking computer system

NASA Technical Reports Server (NTRS)

Hess, Richard (Inventor); Kelly, Gerald B. (Inventor); Rogers, Randy (Inventor); Stange, Kent A. (Inventor)

2011-01-01

System and methods for providing a recoverable real time multi-tasking computer system are disclosed. In one embodiment, a system comprises a real time computing environment, wherein the real time computing environment is adapted to execute one or more applications and wherein each application is time and space partitioned. The system further comprises a fault detection system adapted to detect one or more faults affecting the real time computing environment and a fault recovery system, wherein upon the detection of a fault the fault recovery system is adapted to restore a backup set of state variables.

Multicentre study of Y chromosome microdeletions in 1,808 Chinese infertile males using multiplex and real-time polymerase chain reaction.

PubMed

Zhu, X-B; Gong, Y-H; He, J; Guo, A-L; Zhi, E-L; Yao, J-E; Zhu, B-S; Zhang, A-J; Li, Z

2017-06-01

Azoospermia factor (AZF) genes on the long arm of the human Y chromosome are involved in spermatogenesis, and microdeletions in the AZF region have been recognised to be the second major genetic cause of spermatogenetic failure resulting in male infertility. While screening for these microdeletions can avoid unnecessary medical and surgical treatments, current methods are generally time-consuming. Therefore, we established a new method to detect and analyse microdeletions in the AZF region quickly, safely and efficiently. In total, 1,808 patients with spermatogenetic failure were recruited from three hospitals in southern China, of which 600 patients were randomly selected for screening for Y chromosome microdeletions in AZF regions employing real-time polymerase chain reaction with a TaqMan probe. In our study, of 1,808 infertile patients, 150 (8.3%) were found to bear microdeletions in the Y chromosome using multiplex PCR, while no deletions were found in the controls. Among the AZF deletions detected, two were in AZFa, three in AZFb, 35 in AZFc, three in AZFb+c and two in AZFa+b+c. Our method is fast-it permits the scanning of DNA from a patient in one and a half hours-and reliable, minimising the risk of cross-contamination and false-positive and false-negative results. © 2016 Blackwell Verlag GmbH.

Processor tradeoffs in distributed real-time systems

NASA Technical Reports Server (NTRS)

Krishna, C. M.; Shin, Kang G.; Bhandari, Inderpal S.

1987-01-01

The problem of the optimization of the design of real-time distributed systems is examined with reference to a class of computer architectures similar to the continuously reconfigurable multiprocessor flight control system structure, CM2FCS. Particular attention is given to the impact of processor replacement and the burn-in time on the probability of dynamic failure and mean cost. The solution is obtained numerically and interpreted in the context of real-time applications.

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

PubMed Central

Rodríguez-Lázaro, David; Pla, Maria; Scortti, Mariela; Monzó, Héctor J.; Vázquez-Boland, José A.

2005-01-01

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure. PMID:16332910

Real-time two-dimensional temperature imaging using ultrasound.

PubMed

Liu, Dalong; Ebbini, Emad S

2009-01-01

We present a system for real-time 2D imaging of temperature change in tissue media using pulse-echo ultrasound. The frontend of the system is a SonixRP ultrasound scanner with a research interface giving us the capability of controlling the beam sequence and accessing radio frequency (RF) data in real-time. The beamformed RF data is streamlined to the backend of the system, where the data is processed using a two-dimensional temperature estimation algorithm running in the graphics processing unit (GPU). The estimated temperature is displayed in real-time providing feedback that can be used for real-time control of the heating source. Currently we have verified our system with elastography tissue mimicking phantom and in vitro porcine heart tissue, excellent repeatability and sensitivity were demonstrated.

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Computing Quantitative Characteristics of Finite-State Real-Time Systems

DTIC Science & Technology

1994-05-04

Current methods for verifying real - time systems are essentially decision procedures that establish whether the system model satisfies a given...specification. We present a general method for computing quantitative information about finite-state real - time systems . We have developed algorithms that...our technique can be extended to a more general representation of real - time systems , namely, timed transition graphs. The algorithms presented in this

Evaluation of Open-Source Hard Real Time Software Packages

NASA Technical Reports Server (NTRS)

Mattei, Nicholas S.

2004-01-01

Reliable software is, at times, hard to find. No piece of software can be guaranteed to work in every situation that may arise during its use here at Glenn Research Center or in space. The job of the Software Assurance (SA) group in the Risk Management Office is to rigorously test the software in an effort to ensure it matches the contract specifications. In some cases the SA team also researches new alternatives for selected software packages. This testing and research is an integral part of the department of Safety and Mission Assurance. Real Time operation in reference to a computer system is a particular style of handing the timing and manner with which inputs and outputs are handled. A real time system executes these commands and appropriate processing within a defined timing constraint. Within this definition there are two other classifications of real time systems: hard and soft. A soft real time system is one in which if the particular timing constraints are not rigidly met there will be no critical results. On the other hand, a hard real time system is one in which if the timing constraints are not met the results could be catastrophic. An example of a soft real time system is a DVD decoder. If the particular piece of data from the input is not decoded and displayed to the screen at exactly the correct moment nothing critical will become of it, the user may not even notice it. However, a hard real time system is needed to control the timing of fuel injections or steering on the Space Shuttle; a delay of even a fraction of a second could be catastrophic in such a complex system. The current real time system employed by most NASA projects is Wind River's VxWorks operating system. This is a proprietary operating system that can be configured to work with many of NASA s needs and it provides very accurate and reliable hard real time performance. The down side is that since it is a proprietary operating system it is also costly to implement. The prospect of

Ames Lab 101: Real-Time 3D Imaging

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

Zhang, Song

2010-08-02

Ames Laboratory scientist Song Zhang explains his real-time 3-D imaging technology. The technique can be used to create high-resolution, real-time, precise, 3-D images for use in healthcare, security, and entertainment applications.

Ames Lab 101: Real-Time 3D Imaging

ScienceCinema

Zhang, Song

2017-12-22

Ames Laboratory scientist Song Zhang explains his real-time 3-D imaging technology. The technique can be used to create high-resolution, real-time, precise, 3-D images for use in healthcare, security, and entertainment applications.

High-fidelity real-time maritime scene rendering

NASA Astrophysics Data System (ADS)

Shyu, Hawjye; Taczak, Thomas M.; Cox, Kevin; Gover, Robert; Maraviglia, Carlos; Cahill, Colin

2011-06-01

The ability to simulate authentic engagements using real-world hardware is an increasingly important tool. For rendering maritime environments, scene generators must be capable of rendering radiometrically accurate scenes with correct temporal and spatial characteristics. When the simulation is used as input to real-world hardware or human observers, the scene generator must operate in real-time. This paper introduces a novel, real-time scene generation capability for rendering radiometrically accurate scenes of backgrounds and targets in maritime environments. The new model is an optimized and parallelized version of the US Navy CRUISE_Missiles rendering engine. It was designed to accept environmental descriptions and engagement geometry data from external sources, render a scene, transform the radiometric scene using the electro-optical response functions of a sensor under test, and output the resulting signal to real-world hardware. This paper reviews components of the scene rendering algorithm, and details the modifications required to run this code in real-time. A description of the simulation architecture and interfaces to external hardware and models is presented. Performance assessments of the frame rate and radiometric accuracy of the new code are summarized. This work was completed in FY10 under Office of Secretary of Defense (OSD) Central Test and Evaluation Investment Program (CTEIP) funding and will undergo a validation process in FY11.

A real-time biomimetic acoustic localizing system using time-shared architecture

NASA Astrophysics Data System (ADS)

Nourzad Karl, Marianne; Karl, Christian; Hubbard, Allyn

2008-04-01

In this paper a real-time sound source localizing system is proposed, which is based on previously developed mammalian auditory models. Traditionally, following the models, which use interaural time delay (ITD) estimates, the amount of parallel computations needed by a system to achieve real-time sound source localization is a limiting factor and a design challenge for hardware implementations. Therefore a new approach using a time-shared architecture implementation is introduced. The proposed architecture is a purely sample-base-driven digital system, and it follows closely the continuous-time approach described in the models. Rather than having dedicated hardware on a per frequency channel basis, a specialized core channel, shared for all frequency bands is used. Having an optimized execution time, which is much less than the system's sample rate, the proposed time-shared solution allows the same number of virtual channels to be processed as the dedicated channels in the traditional approach. Hence, the time-shared approach achieves a highly economical and flexible implementation using minimal silicon area. These aspects are particularly important in efficient hardware implementation of a real time biomimetic sound source localization system.

Competitive On-Line Scheduling for Overloaded Real-Time Systems

DTIC Science & Technology

1993-09-01

Real - Time Systems by Gilad Koren a dissertation submitted in partial fulfillment of the requirements...Overloaded Real - Time Systems 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK...1.1 Introduction : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 2 1.1.1 Real - Time Systems : : : : : : : : : : : : : : : : : : : : : : : : : : : :

Real-time operating system timing jitter and its impact on motor control

NASA Astrophysics Data System (ADS)

Proctor, Frederick M.; Shackleford, William P.

2001-12-01

General-purpose microprocessors are increasingly being used for control applications due to their widespread availability and software support for non-control functions like networking and operator interfaces. Two classes of real-time operating systems (RTOS) exist for these systems. The traditional RTOS serves as the sole operating system, and provides all OS services. Examples include ETS, LynxOS, QNX, Windows CE and VxWorks. RTOS extensions add real-time scheduling capabilities to non-real-time OSes, and provide minimal services needed for the time-critical portions of an application. Examples include RTAI and RTL for Linux, and HyperKernel, OnTime and RTX for Windows NT. Timing jitter is an issue in these systems, due to hardware effects such as bus locking, caches and pipelines, and software effects from mutual exclusion resource locks, non-preemtible critical sections, disabled interrupts, and multiple code paths in the scheduler. Jitter is typically on the order of a microsecond to a few tens of microseconds for hard real-time operating systems, and ranges from milliseconds to seconds in the worst case for soft real-time operating systems. The question of its significance on the performance of a controller arises. Naturally, the smaller the scheduling period required for a control task, the more significant is the impact of timing jitter. Aside from this intuitive relationship is the greater significance of timing on open-loop control, such as for stepper motors, than for closed-loop control, such as for servo motors. Techniques for measuring timing jitter are discussed, and comparisons between various platforms are presented. Techniques to reduce jitter or mitigate its effects are presented. The impact of jitter on stepper motor control is analyzed.

Real-time subway information for improving transit ridership.

DOT National Transportation Integrated Search

2016-08-01

In recent years, the standardization of transit schedule information has yielded a dramatic increase in the accessibility of computerized transit schedules and given rise to real-time service schedules. Two such real-time service schedules are the Ge...

Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS)

NASA Astrophysics Data System (ADS)

Daniels, M. D.; Graves, S. J.; Kerkez, B.; Chandrasekar, V.; Vernon, F.; Martin, C. L.; Maskey, M.; Keiser, K.; Dye, M. J.

2015-12-01

The Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS) project, funded as part of NSF's EarthCube initiative, addresses the ever-increasing importance of real-time scientific data, particularly in mission critical scenarios, where informed decisions must be made rapidly. Advances in the distribution of real-time data are leading many new transient phenomena in space-time to be observed, however, real-time decision-making is infeasible in many cases as these streaming data are either completely inaccessible or only available to proprietary in-house tools or displays. This lack of accessibility prohibits advanced algorithm and workflow development that could be initiated or enhanced by these data streams. Small research teams do not have resources to develop tools for the broad dissemination of their valuable real-time data and could benefit from an easy to use, scalable, cloud-based solution to facilitate access. CHORDS proposes to make a very diverse suite of real-time data available to the broader geosciences community in order to allow innovative new science in these areas to thrive. This presentation will highlight recently developed CHORDS portal tools and processing systems aimed at addressing some of the gaps in handling real-time data, particularly in the provisioning of data from the "long-tail" scientific community through a simple interface deployed in the cloud. The CHORDS system will connect these real-time streams via standard services from the Open Geospatial Consortium (OGC) and does so in a way that is simple and transparent to the data provider. Broad use of the CHORDS framework will expand the role of real-time data within the geosciences, and enhance the potential of streaming data sources to enable adaptive experimentation and real-time hypothesis testing. Adherence to community data and metadata standards will promote the integration of CHORDS real-time data with existing standards-compliant analysis, visualization and modeling

Feedback as Real-Time Constructions

ERIC Educational Resources Information Center

Keiding, Tina Bering; Qvortrup, Ane

2014-01-01

This article offers a re-description of feedback and the significance of time in feedback constructions based on systems theory. It describes feedback as internal, real-time constructions in a learning system. From this perspective, feedback is neither immediate nor delayed, but occurs in the very moment it takes place. This article argues for a…

"Internet of Things" Real-Time Free Flap Monitoring.

PubMed

Kim, Sang Hun; Shin, Ho Seong; Lee, Sang Hwan

2018-01-01

Free flaps are a common treatment option for head and neck reconstruction in plastic reconstructive surgery, and monitoring of the free flap is the most important factor for flap survival. In this study, the authors performed real-time free flap monitoring based on an implanted Doppler system and "internet of things" (IoT)/wireless Wi-Fi, which is a convenient, accurate, and efficient approach for surgeons to monitor a free flap. Implanted Doppler signals were checked continuously until the patient was discharged by the surgeon and residents using their own cellular phone or personal computer. If the surgeon decided that a revision procedure or exploration was required, the authors checked the consumed time (positive signal-to-operating room time) from the first notification when the flap's status was questioned to the determination for revision surgery according to a chart review. To compare the efficacy of real-time monitoring, the authors paired the same number of free flaps performed by the same surgeon and monitored the flaps using conventional methods such as a physical examination. The total survival rate was greater in the real-time monitoring group (94.7% versus 89.5%). The average time for the real-time monitoring group was shorter than that for the conventional group (65 minutes versus 86 minutes). Based on this study, real-time free flap monitoring using IoT technology is a method that surgeon and reconstruction team can monitor simultaneously at any time in any situation.

Real-time fMRI: a tool for local brain regulation.

PubMed

Caria, Andrea; Sitaram, Ranganatha; Birbaumer, Niels

2012-10-01

Real-time fMRI permits simultaneous measurement and observation of brain activity during an ongoing task. One of the most challenging applications of real-time fMRI in neuroscientific and clinical research is the possibility of acquiring volitional control of localized brain activity using real-time fMRI-based neurofeedback protocols. Real-time fMRI allows the experimenter to noninvasively manipulate brain activity as an independent variable to observe the effects on behavior. Real-time fMRI neurofeedback studies demonstrated that learned control of the local brain activity leads to specific changes in behavior. Here, the authors describe the implementation and application of real-time fMRI with particular emphasis on the self-regulation of local brain activity and the investigation of brain-function relationships. Real-time fMRI represents a promising new approach to cognitive neuroscience that could complement traditional neuroimaging techniques by providing more causal insights into the functional role of circumscribed brain regions in behavior.

Programming Models for Concurrency and Real-Time

NASA Astrophysics Data System (ADS)

Vitek, Jan

Modern real-time applications are increasingly large, complex and concurrent systems which must meet stringent performance and predictability requirements. Programming those systems require fundamental advances in programming languages and runtime systems. This talk presents our work on Flexotasks, a programming model for concurrent, real-time systems inspired by stream-processing and concurrent active objects. Some of the key innovations in Flexotasks are that it support both real-time garbage collection and region-based memory with an ownership type system for static safety. Communication between tasks is performed by channels with a linear type discipline to avoid copying messages, and by a non-blocking transactional memory facility. We have evaluated our model empirically within two distinct implementations, one based on Purdue’s Ovm research virtual machine framework and the other on Websphere, IBM’s production real-time virtual machine. We have written a number of small programs, as well as a 30 KLOC avionics collision detector application. We show that Flexotasks are capable of executing periodic threads at 10 KHz with a standard deviation of 1.2us and have performance competitive with hand coded C programs.

Enhancements to the EPANET-RTX (Real-Time Analytics) ...

EPA Pesticide Factsheets

Technical brief and software The U.S. Environmental Protection Agency (EPA) developed EPANET-RTX as a collection of object-oriented software libraries comprising the core data access, data transformation, and data synthesis (real-time analytics) components of a real-time hydraulic and water quality modeling system. While EPANET-RTX uses the hydraulic and water quality solvers of EPANET, the object libraries are a self-contained set of building blocks for software developers. “Real-time EPANET” promises to change the way water utilities, commercial vendors, engineers, and the water community think about modeling.

Real-time measurements of jet aircraft engine exhaust.

PubMed

Rogers, Fred; Arnott, Pat; Zielinska, Barbara; Sagebiel, John; Kelly, Kerry E; Wagner, David; Lighty, JoAnn S; Sarofim, Adel F

2005-05-01

Particulate-phase exhaust properties from two different types of ground-based jet aircraft engines--high-thrust and turboshaft--were studied with real-time instruments on a portable pallet and additional time-integrated sampling devices. The real-time instruments successfully characterized rapidly changing particulate mass, light absorption, and polycyclic aromatic hydrocarbon (PAH) content. The integrated measurements included particulate-size distributions, PAH, and carbon concentrations for an entire test run (i.e., "run-integrated" measurements). In all cases, the particle-size distributions showed single modes peaking at 20-40nm diameter. Measurements of exhaust from high-thrust F404 engines showed relatively low-light absorption compared with exhaust from a turboshaft engine. Particulate-phase PAH measurements generally varied in phase with both net particulate mass and with light-absorbing particulate concentrations. Unexplained response behavior sometimes occurred with the real-time PAH analyzer, although on average the real-time and integrated PAH methods agreed within the same order of magnitude found in earlier investigations.

Real-Time Safety Risk Assessment Based on a Real-Time Location System for Hydropower Construction Sites

PubMed Central

Fan, Qixiang; Qiang, Maoshan

2014-01-01

The concern for workers' safety in construction industry is reflected in many studies focusing on static safety risk identification and assessment. However, studies on real-time safety risk assessment aimed at reducing uncertainty and supporting quick response are rare. A method for real-time safety risk assessment (RTSRA) to implement a dynamic evaluation of worker safety states on construction site has been proposed in this paper. The method provides construction managers who are in charge of safety with more abundant information to reduce the uncertainty of the site. A quantitative calculation formula, integrating the influence of static and dynamic hazards and that of safety supervisors, is established to link the safety risk of workers with the locations of on-site assets. By employing the hidden Markov model (HMM), the RTSRA provides a mechanism for processing location data provided by the real-time location system (RTLS) and analyzing the probability distributions of different states in terms of false positives and negatives. Simulation analysis demonstrated the logic of the proposed method and how it works. Application case shows that the proposed RTSRA is both feasible and effective in managing construction project safety concerns. PMID:25114958

Real-time safety risk assessment based on a real-time location system for hydropower construction sites.

PubMed

Jiang, Hanchen; Lin, Peng; Fan, Qixiang; Qiang, Maoshan

2014-01-01

The concern for workers' safety in construction industry is reflected in many studies focusing on static safety risk identification and assessment. However, studies on real-time safety risk assessment aimed at reducing uncertainty and supporting quick response are rare. A method for real-time safety risk assessment (RTSRA) to implement a dynamic evaluation of worker safety states on construction site has been proposed in this paper. The method provides construction managers who are in charge of safety with more abundant information to reduce the uncertainty of the site. A quantitative calculation formula, integrating the influence of static and dynamic hazards and that of safety supervisors, is established to link the safety risk of workers with the locations of on-site assets. By employing the hidden Markov model (HMM), the RTSRA provides a mechanism for processing location data provided by the real-time location system (RTLS) and analyzing the probability distributions of different states in terms of false positives and negatives. Simulation analysis demonstrated the logic of the proposed method and how it works. Application case shows that the proposed RTSRA is both feasible and effective in managing construction project safety concerns.

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

PubMed

Opota, Onya; Jaton, Katia; Branley, James; Vanrompay, Daisy; Erard, Veronique; Borel, Nicole; Longbottom, David; Greub, Gilbert

2015-10-01

Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml(- 1)). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

Real time gamma-ray signature identifier

DOEpatents

Rowland, Mark [Alamo, CA; Gosnell, Tom B [Moraga, CA; Ham, Cheryl [Livermore, CA; Perkins, Dwight [Livermore, CA; Wong, James [Dublin, CA

2012-05-15

A real time gamma-ray signature/source identification method and system using principal components analysis (PCA) for transforming and substantially reducing one or more comprehensive spectral libraries of nuclear materials types and configurations into a corresponding concise representation/signature(s) representing and indexing each individual predetermined spectrum in principal component (PC) space, wherein an unknown gamma-ray signature may be compared against the representative signature to find a match or at least characterize the unknown signature from among all the entries in the library with a single regression or simple projection into the PC space, so as to substantially reduce processing time and computing resources and enable real-time characterization and/or identification.