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Sample records for real-time rt-pcr analysis

  1. A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarray analysis and quantitative real-time RT-PCR are the major high-throughput techniques that are used to study transcript profiles. One of the major limitations in these technologies is the isolation maximum yield of highly-pure RNA from plant tissues rich in complex polysaccharides, polyphen...

  2. Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis.

    PubMed

    Ghorashi, Seyed A; O'Rourke, Denise; Ignjatovic, Jagoda; Noormohammadi, Amir H

    2011-01-01

    Differentiation of infectious bursal disease virus (IBDV) strains is crucial for effective vaccination programs and epidemiological investigations. In this study, a combination of real-time RT-PCR and high resolution melt (HRM) curve analysis was developed for simultaneous detection and differentiation of IBDV strains/isolates. The hypervariable region of VP2 gene was amplified from several IBDV strains and subjected to HRM curve analysis. The method could readily differentiate between classical vaccines/isolates and variants. Analysis of the nucleotide sequence of the amplicons from each strain revealed that each melt curve profile was related to a unique DNA sequence. The real-time RT-PCR HRM curve analysis was also able to differentiate IBDV strains/isolates directly in bursal tissues from field submissions and from vaccinated commercial flocks. The differences between melting peaks generated from IBDV strains were significantly different (P<0.0001) demonstrating the high discriminatory power of this technique. The results presented in this study indicated that real-time RT-PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping IBDV isolates/strains and can contribute to effective control of IBDV outbreaks. PMID:21111004

  3. Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI

    PubMed Central

    2012-01-01

    Background Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. Methods In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. Results A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. Conclusion The real-time RT-PCR HRM

  4. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

    PubMed Central

    Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

    2009-01-01

    Background Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. Results From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. Conclusion In this work, we provided a set of genes that are suitable reference

  5. Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a...

  6. Detection sensitivity and quantitation of Plasmodium falciparum var gene transcripts by real-time RT-PCR in comparison with conventional RT-PCR.

    PubMed

    Gatton, Michelle L; Peters, Jennifer M; Gresty, Karryn; Fowler, Elizabeth V; Chen, Nanhua; Cheng, Qin

    2006-08-01

    Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples. PMID:16896121

  7. Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (Anser anser domestica) Gene Expression

    PubMed Central

    Ji, Hong; Wang, Jianfa; Liu, Juxiong; Guo, Jingru; Wang, Zhongwei; Zhang, Xu; Guo, Li; Yang, Huanmin

    2013-01-01

    Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don’t know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), β-actin (ACTB), β-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese. PMID:25049806

  8. Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

    NASA Astrophysics Data System (ADS)

    Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

    2013-03-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

  9. Real-Time RT-PCR for the Detection of Lyssavirus Species

    PubMed Central

    Deubelbeiss, A.; Zahno, M.-L.; Zanoni, M.; Bruegger, D.; Zanoni, R.

    2014-01-01

    The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used. PMID:26464934

  10. Real-Time RT-PCR for the Detection of Lyssavirus Species.

    PubMed

    Deubelbeiss, A; Zahno, M-L; Zanoni, M; Bruegger, D; Zanoni, R

    2014-01-01

    The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used. PMID:26464934

  11. Type A influenza virus detection from horses by real-time RT-PCR and insulated isothermal RT-PCR.

    PubMed

    Balasuriya, Udeni B R

    2014-01-01

    Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu A(®) test and virus isolation in embryonated hens' eggs. Recently, a TaqMan(®) probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance. PMID:24899448

  12. Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR.

    PubMed

    Kittl, Roman; Sygmund, Christoph; Halada, Petr; Volc, Jindrich; Divne, Christina; Haltrich, Dietmar; Peterbauer, Clemens K

    2008-02-01

    Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation. PMID:18097667

  13. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  14. Detection of Zika virus by SYBR green one-step real-time RT-PCR.

    PubMed

    Xu, Ming-Yue; Liu, Si-Qing; Deng, Cheng-Lin; Zhang, Qiu-Yan; Zhang, Bo

    2016-10-01

    The ongoing Zika virus (ZIKV) outbreak has rapidly spread to new areas of Americas, which were the first transmissions outside its traditional endemic areas in Africa and Asia. Due to the link with newborn defects and neurological disorder, numerous infected cases throughout the world and various mosquito vectors, the virus has been considered to be an international public health emergency. In the present study, we developed a SYBR Green based one-step real-time RT-PCR assay for rapid detection of ZIKV. Our results revealed that the real-time assay is highly specific and sensitive in detection of ZIKV in cell samples. Importantly, the replication of ZIKV at different time points in infected cells could be rapidly monitored by the real-time RT-PCR assay. Specifically, the real-time RT-PCR showed acceptable performance in measurement of infectious ZIKV RNA. This assay could detect ZIKV at a titer as low as 1PFU/mL. The real-time RT-PCR assay could be a useful tool for further virology surveillance and diagnosis of ZIKV. PMID:27444120

  15. Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

    PubMed

    Luan, Huaibiao; Wang, Yixin; Li, Yang; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2016-09-01

    Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers). PMID:27122388

  16. DEVELOPMENT OF MULTIPLEX REAL-TIME RT-PCR AS A DIAGNOSTIC TOOL FOR AVIAN INFLUENZA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex real-time RT-PCR (RRT-PCR) assay for the simultaneous detection of the H5 and H7 hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro tran...

  17. Real-time RT-PCR Assay for Detection and Differentiation of Citrus Tristeza Virus Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiplex one step real time RT-PCR assays using TaqMan probes were developed for detection and strain differentiation of Citrus tristeza virus (CTV). For broad spectrum CTV detection, a TaqMan primer and Cy5-labeled probe were designed using CP gene sequences. An internal control was developed us...

  18. Type-A influenza virus detection and quantitation by real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) is a relatively new technology which has been used for AIV detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of RRT-PCR are: quantitative nature, scalability, cost, high sensitivity, high specificity, and ...

  19. Real time RT-PCR for the H5 HA subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) for type A influenza detection is widely used with subsequent tests for subtype identification. Due to the importance of the H5 HA subtype, rapid and sensitive detection of the H5 HA subtype is particularly useful due to the importance of the recent Asian H5N1 HPAI viruse...

  20. Real-time RT-PCR assay for detection and differentiation of Citrus tristeza virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For universal detection of Citrus tristeza virus (CTV) strains by real time RT-PCR, a protocol was developed based on a set of primers and a Cy5-labeled TaqMan probe. This test included primers and a TET-labeled TaqMan probe selected on the mitochondrial nad5 gene for the simultaneous detection of ...

  1. Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR

    PubMed Central

    Pagliarulo, Vincenzo; George, Ben; Beil, Stephen J; Groshen, Susan; Laird, Peter W; Cai, Jie; Willey, James; Cote, Richard J; Datar, Ram H

    2004-01-01

    Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB). Results The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. Conclusion StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further

  2. A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV)

    PubMed Central

    2013-01-01

    Background Wheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases. Methods In this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and β-actin were selected in order to adjust the veracity of the real-time RT-PCR assay. Results We developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: β-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV. Conclusions The Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods. PMID:23725024

  3. A real-time RT-PCR assay for rapid detection of coxsackievirus A10.

    PubMed

    Mu, C Y; Wang, A Y; Chen, C; Zhao, L; Li, Z

    2015-01-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) have been the primary causative agents of hand, foot, and mouth disease (HFMD) outbreaks in mainland China in the past. Hence, the surveillance of HFMD has mostly focused on these viruses. However, in recent years, coxsackievirus A10 (CA10) has also been associated with the increasing sporadic HFMD cases and outbreaks. Therefore, a sensitive assay for rapid detection of the CA10 RNA is necessary for disease control. Here, we have developed a specific TaqMan real-time RT-PCR assay by analyzing VP1 gene sequences of CA10 strains from different locations. The assay has been shown to be specific, sensitive, and robust through detection of other related viruses, standard curves, and clinical samples, respectively. This is the first report on development of a VP1 gene-based TaqMan real-time RT-PCR assay for rapid diagnosis of CA10 virus. PMID:26782393

  4. Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei.

    PubMed

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

    Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: β-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); β-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions. PMID:26327538

  5. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2013-12-01

    Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies. PMID:24057254

  6. Detection of Langat virus by TaqMan real-time one-step qRT-PCR method

    PubMed Central

    Muhd Radzi, Siti Fatimah; Rückert, Claudia; Sam, Sing-Sin; Teoh, Boon-Teong; Jee, Pui-Fong; Phoon, Wai-Hong; Abubakar, Sazaly; Zandi, Keivan

    2015-01-01

    Langat virus (LGTV), one of the members of the tick-borne encephalitis virus (TBEV) complex, was firstly isolated from Ixodes granulatus ticks in Malaysia. However, the prevalence of LGTV in ticks in the region remains unknown. Surveillance for LGTV is therefore important and thus a tool for specific detection of LGTV is needed. In the present study, we developed a real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for rapid detection of LGTV. Our findings showed that the developed qRT-PCR could detect LGTV at a titre as low as 0.1 FFU/ml. The detection limit of the qRT-PCR assay at 95% probability was 0.28 FFU/ml as determined by probit analysis (p ≤ 0.05). Besides, the designed primers and probe did not amplify ORF of the E genes for some closely related and more pathogenic viruses including TBEV, Louping ill virus, Omsk hemorrhagic fever virus (OHFV), Alkhurma virus (ALKV), Kyasanur Forest Disease virus (KFDV) and Powassan virus (POWV) which showed the acceptable specificity of the developed assay. The sensitivity of the developed method also has been confirmed by determining the LGTV in infected tick cell line as well as LGTV- spiked tick tissues. PMID:26360297

  7. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

    PubMed Central

    Expósito-Rodríguez, Marino; Borges, Andrés A; Borges-Pérez, Andrés; Pérez, José A

    2008-01-01

    Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene

  8. Characterization of two novel pacifastin-like peptide precursor isoforms in the desert locust (Schistocerca gregaria): cDNA cloning, functional analysis and real-time RT-PCR gene expression studies.

    PubMed

    Simonet, Gert; Breugelmans, Bert; Proost, Paul; Claeys, Ilse; Van Damme, Jozef; De Loof, Arnold; Vanden Broeck, Jozef

    2005-05-15

    In the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (Schistocerca gregaria PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed in vitro. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1-3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism. PMID:15631618

  9. Evaluation of a broad range real-time polymerase chain reaction (RT-PCR) assay for the diagnosis of septic synovitis in horses

    PubMed Central

    Elmas, Colette R.; Koenig, Judith B.; Bienzle, Dorothee; Cribb, Nicola C.; Cernicchiaro, Natalia; Coté, Nathalie M.; Weese, J. Scott

    2013-01-01

    Septic synovitis is a potentially debilitating and life-threatening disorder in horses. We hypothesized that a universal bacterial real-time PCR (RT-PCR) assay would have improved sensitivity and decreased turn-around time for detection of bacteria in synovial fluid (SF) samples. Forty-eight SF samples were collected from 36 horses that presented to two referral institutions with suspected septic synovitis. Universal RT-PCR, bacterial culture and SF analysis were performed on all samples, and an interpretation on the sample being septic or not was derived by three board certified specialists from the history, clinical assessment and SF characteristics. RT-PCR results were compared to a composite standard comprised of positive culture and interpretation by all three specialists of samples as “septic”. For 41 of 48 samples (85%), culture and RT-PCR results were concordant. Compared to the composite standard, 83% of samples were correctly classified by RT-PCR (turn-around time of approximately 4 hours). Relative sensitivity and specificity of RT-PCR were 87% and 72% respectively, and 56% and 86% for culture. Hence, universal RT-PCR was a rapid and highly sensitive test, which may accelerate diagnosis and improve outcome for horses with septic synovitis. PMID:24101798

  10. A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle.

    PubMed

    La Rocca, S A; Sandvik, T

    2009-10-01

    A rapid single step real-time duplex TaqMan RT-PCR was developed for detection of bovine viral diarrhoea virus (BVDV)-1, BVDV-2 and border disease virus (BDV). Based on alignment of available and newly generated partial 5'-UTR nucleotide sequences, one forward and two reverse primers were designed, which amplify a 104bp PCR product. Two TaqMan probes labelled with different fluorochromes were designed to detect BVDV-1/BVDV-2 and BDVs, respectively. The assay was able to detect a selection of strains and isolates that represent the genetic diversity of these three viruses, with an analytical sensitivity that corresponded to 3.6, 48 and 4.8 TCID(50) of BVDV-1, BVDV-2 and BDV, respectively. With an overall cycling time of around 70 min, the assay allows rapid diagnosis and efficient use of modern thermocycling machines. Although developed principally for the diagnosis of BVD, the assay should be equally useful for diagnosis of BD in sheep. PMID:19523981

  11. Real time RT-PCR assays for detection and typing of African horse sickness virus.

    PubMed

    Bachanek-Bankowska, Katarzyna; Maan, Sushila; Castillo-Olivares, Javier; Manning, Nicola M; Maan, Narender Singh; Potgieter, Abraham C; Di Nardo, Antonello; Sutton, Geoff; Batten, Carrie; Mertens, Peter P C

    2014-01-01

    Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the 'Orbivirus Reference Collection' (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures. PMID:24721971

  12. Evaluation of an updated real-time RT-PCR test for the identification of the H7 subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of avian influenza (AI) virus and identification of the H5 and H7 subtypes is critical for wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in North America was first reported in 2002. With the recent surveillance in wild birds it was disc...

  13. Characterization of cytokine expression induced by avian influenza virus infection with real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowledge of how birds react to infection from avian influenza virus is critical to understanding disease pathogenesis and host response. The use of real-time (R), reverse-transcriptase (RT), PCR to measure innate immunity, including cytokine and interferon gene expression, has become a standard tec...

  14. Bluetongue virus RNA detection by real-time rt-PCR in post-vaccination samples from cattle.

    PubMed

    De Leeuw, I; Garigliany, M; Bertels, G; Willems, T; Desmecht, D; De Clercq, K

    2015-04-01

    Bluetongue virus serotype 8 (BTV-8) was responsible for a large outbreak among European ruminant populations in 2006-2009. In spring 2008, a massive vaccination campaign was undertaken, leading to the progressive disappearance of the virus. During surveillance programmes in Western Europe in 2010-2011, a low but significant number of animals were found weakly positive using BTV-specific real-time RT-PCR, raising questions about a possible low level of virus circulation. An interference of the BTV-8 inactivated vaccine on the result of the real-time RT-PCR was also hypothesized. Several studies specifically addressed the potential association between a recent vaccination and BTV-8 RNA detection in the blood of sheep. Results were contradictory and cattles were not investigated. To enlighten this point, a large study was performed to determine the risks of detection of bluetongue vaccine-associated RNA in the blood and spleen of cattle using real-time RT-PCR. Overall, the results presented clearly demonstrate that vaccine viral RNA can reach the blood circulation in sufficient amounts to be detected by real-time RT-PCR in cattle. This BTV-8 vaccine RNA carriage appears as short lasting. PMID:23611408

  15. Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus.

    PubMed

    Jiang, Tao; Liu, Juan; Deng, Yong-Qiang; Su, Jing-Liang; Xu, Li-Juan; Liu, Zhi-Hui; Li, Xiao-Feng; Yu, Xue-Dong; Zhu, Shun-Ya; Gao, George Fu; Qin, E-De; Qin, Cheng-Feng

    2012-12-01

    A new duck Tembusu virus (TMUV), also known as BYD virus, has been identified as the causative agent for the emerging duck egg-drop syndrome in mainland China. The rapid spread and wide distribution of the new TMUV in mainland China result in heavy loss to the poultry industry and pose great threats to public health. Rapid and sensitive detection methods are critical for prevention and control of TMUV infections. In this study, a reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) and an SYBR Green-I-based real-time RT-PCR assay specific for the duck TMUV were developed and validated with laboratory and field samples, respectively. The detection limits were 1 × 10(-4) and 1 × 10(-3) PFU per reaction for the RT-LAMP assay and real-time RT-PCR assay, respectively. The specificities were analyzed with other related members of the genus Flavivirus, and no cross-reaction was observed. Furthermore, both assays were evaluated with field samples, and they exhibited high sensitivity and specificity. In addition, the real-time RT-PCR assay worked well in viral load analysis, which revealed that the spleen may be the primary target for the replication of new duck TMUV in ducks. The TMUV-specific RT-LAMP and real-time RT-PCR assays will provide useful tools for the diagnosis and epidemiological surveillance of TMUV infection. PMID:22865206

  16. Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR.

    PubMed

    Li, Lin; Wu, Xiaodong; Liu, Fuxiao; Wang, Zhiliang; Liu, Chunju; Wang, Qinghua; Bao, Jingyue

    2016-09-01

    Peste des petits ruminants virus (PPRV) is the cause agent of peste des petitis ruminants (PPR). A novel lineage IV PPRV has reemerged in China in 2013 and 2014. Mass vaccination was implemented in most provinces in China. In order to detect lineage IV PPRV in clinical samples and to distinguish rapidly it from the other lineages PPRVs, a real-time RT-PCR assay was developed. This assay showed high sensitivity, specificity and efficiency in differentiating the lineage IV PPRV from others. The performance of this assay was evaluated by positive clinical samples of lineage IV viruses. This new real-time RT-PCR assay will facilitate epidemiological investigations and rapid differentiatial diagnosis in areas where lineage IV viruses are circulating. PMID:27260657

  17. Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus

    PubMed Central

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

    2015-01-01

    Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. PMID:26161784

  18. Detection and quantitation of Citrus leaf blotch virus by TaqMan real-time RT-PCR.

    PubMed

    Ruiz-Ruiz, Susana; Ambrós, Silvia; Vives, María del Carmen; Navarro, Luis; Moreno, Pedro; Guerri, José

    2009-09-01

    A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times more sensitive than detection by conventional RT-PCR. An external standard curve using in vitro synthesized RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range (seven logarithmic units of concentration) and very low variation coefficient values. This protocol enabled detection of as little as 100 copies of CLBV RNA in various tissues and citrus varieties infected with CLBV sources from different geographical origins. The new assay greatly improves current detection methods for CLBV and it has been most helpful for the Spanish citrus sanitation, quarantine and certification programs, and fitness evaluation of infectious cDNA clones of CLBV, useful potentially as viral vectors for citrus. PMID:19406167

  19. Detection of a broad range of class I and II Newcastle disease viruses using a multiplex real time RT-PCR assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prompt detection of virulent strains of Newcastle disease virus (vNDV) using real time RT-PCR (rRT-PCR) is challenging due to the broad genetic variability across two clades comprising 18 recognized genotypes. A large proportion of class I low virulence Newcastle disease viruses (loNDV) recently id...

  20. Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil

    PubMed Central

    Yang, Jin-Guang; Wang, Feng-Long; Chen, De-Xin; Shen, Li-Li; Qian, Yu-Mei; Liang, Zhi-Yong; Zhou, Wen-Chang; Yan, Tai-He

    2012-01-01

    Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control. PMID:23211755

  1. Quantitation of Taura syndrome virus by real-time RT-PCR with a TaqMan assay.

    PubMed

    Tang, Kathy F J; Wang, Jun; Lightner, Donald V

    2004-01-01

    A real-time RT-PCR assay was developed using a TaqMan probe to detect and quantify Taura syndrome virus (TSV) in penaeid shrimp. A pair of RT-PCR primers, which amplify a 72 bp DNA fragment, and a TaqMan probe were selected from open reading frame 1 (ORF1) of the TSV genome. The primers and TaqMan probe used in this assay reacted with TSV isolates from Hawaii, Texas, Colombia, Mexico, Belize, Indonesia, and Thailand, but neither with RNA of healthy shrimp nor with an isolate of yellow head virus. A plasmid (pTSV-1) that contains the target TSV sequence was constructed and used to generate positive control RNA through in vitro transcription. The positive control RNA was used to demonstrate that the real-time RT-PCR assay has a detection limit of 100 copies and a log-linear range up to 10(8) copies of TSV RNA. This quantitative method was found to be highly reproducible, with low intra- and inter-assay variation. Coefficient of variation (CVs) values were 0.04-8.9 and 0.05-3.7%, respectively, for replicates within and among assays. This assay was used to quantify TSV in both acutely and chronically infected shrimp in a laboratory experiment. The quantities of TSV in the tissues of pleopods and gills were not significantly different, and there was no difference in TSV levels between the acutely and chronically infected groups. However, in the chronically infected shrimp, the quantities of TSV were one to two orders of magnitude higher in the lymphoid organ than in either gills or pleopods. This assay proved to be specific with high sensitivity, and it can be used to detect and quantify TSV in shrimp samples. PMID:14656468

  2. Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

    PubMed Central

    Takle, Gunnhild W; Toth, Ian K; Brurberg, May B

    2007-01-01

    Background Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae. Results Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh), encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels. Conclusion Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens. PMID:17888160

  3. Tick-borne encephalitis in dogs: application of "nested real-time RT-PCR" for intravital virus detection.

    PubMed

    Hekrlová, Alena; Kubíček, Oldfich; Lány, Petr; Rosenbergová, Kateřina; Schánilec, Pavel

    2015-01-01

    Tick-borne encephalitis (TBE) virus is a tick-transmitted virus causing disorders of the nervous system in humans, monkeys, dogs and horses (rarely). At present the detection of TBE infection in dogs is performed by confirmation of seroconversion in paired samples of serum in clinical practice. The intention of the study was the assessment of the possible application of nested real-time RT-PCR for detection of TBE virus in canine blood. The study was carried out in 2011-2012 using samples originating in the Czech Republic, South Moravian region (region with endemic occurrence of TBE). The dogs were randomly selected from the patients visiting the clinic during this time period. Of the total amount of 159 canine blood samples, 20 samples were tested with a PCR-positive result (12.6%). Out of these 20 animals, the neurological clinical symptoms typical of TBE were detected in seven dogs. PCR-positive results were found between March and November. Three dogs were tested with a competitive ELISA-positive result and a "nested real-time RT-PCR"-positive result concurrently. In the group of 159 dogs the value of seroprevalence was found to be 11.3%. PMID:26591386

  4. Multiplex real-time RT-PCR for the simultaneous detection and quantification of GI, GII and GIV noroviruses.

    PubMed

    Farkas, Tibor; Singh, Amy; Le Guyader, Françoise S; La Rosa, Giuseppina; Saif, Linda; McNeal, Monica

    2015-10-01

    Noroviruses are important causes of acute gastroenteritis and are classified into six genogroups with GI, GII and GIV containing human pathogens. This high genetic diversity represents a significant challenge for diagnostic assay development. Genogroup specific monoplex and multiplex real time RT-PCR assays are widely used for the detection of GI and GII noroviruses. On the other hand, GIV norovirus detection is not part of routine laboratory diagnosis. This study describes the development and evaluation of a one tube, real time RT-PCR assay for the simultaneous detection and quantification of GI, GII and GIV noroviruses, including both GIV.1 (human) and GIV.2 (animal) strains. Assay performance was evaluated on a panel of norovirus positive clinical samples by comparison of monoplex and multiplex standard curves and Ct values. The multiplex assay demonstrated equal sensitivity and specificity to the monoplex assays and was able to detect all GI, GII and GIV noroviruses with Ct values equal to that of the monoplex assays. The multiplex assay described in this study will be instrumental for the better understanding of GIV norovirus epidemiology, including their possible zoonotic nature. PMID:26248055

  5. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

    PubMed Central

    Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong

    2014-01-01

    The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species. PMID:24823940

  6. Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

    PubMed

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

    2016-09-01

    There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. PMID:27180038

  7. Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain.

    PubMed

    Hodneland, K; García, R; Balbuena, J A; Zarza, C; Fouz, B

    2011-03-01

    Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have been classified as free of infection. Clinical signs of nodavirus infection were reproduced in fish infected following bath exposure or intramuscular injection, demonstrating horizontal transmission of the disease. Nodavirus was always detected in the brain of diseased fish but also in many recovered fish. The new assay enables us to confirm the presence of the virus at an early phase in the production cycle and may represent a useful tool to prevent or slow down the spread of nodavirus to new locations. PMID:21306586

  8. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    PubMed

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR. PMID:24509829

  9. Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

    PubMed

    Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

    2009-05-01

    Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins. PMID:18612846

  10. Detection of influenza A(H1N1)v virus by real-time RT-PCR.

    PubMed

    Panning, M; Eickmann, M; Landt, O; Monazahian, M; Olschläger, S; Baumgarte, S; Reischl, U; Wenzel, J J; Niller, H H; Günther, S; Hollmann, B; Huzly, D; Drexler, J F; Helmer, A; Becker, S; Matz, B; Eis-Hübinger, Am; Drosten, C

    2009-09-10

    Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A virus H5N1 and porcine influenza A virus H1N1, H1N2 and H3N2 samples. The real-time RT-PCR assay for the influenza A matrix gene recommended in 2007 by the World Health Organization was modified to work under the same reaction conditions as the influenza A(H1N1)v virus-specific test. Both assays were equally sensitive. Clinical applicability of both assays was demonstrated by screening of almost 2,000 suspected influenza (H1N1)v specimens, which included samples from the first cases of pandemic H1N1 influenza imported to Germany. Measuring influenza A(H1N1)v virus concentrations in 144 laboratory-confirmed samples yielded a median of 4.6 log RNA copies/ml. The new methodology proved its principle and might assist public health laboratories in the upcoming influenza pandemic. PMID:19758541

  11. Development of strand-specific real-time RT-PCR to distinguish viral RNAs during Newcastle disease virus infection.

    PubMed

    Qiu, Xusheng; Yu, Yang; Yu, Shengqing; Zhan, Yuan; Wei, Nana; Song, Cuiping; Sun, Yingjie; Tan, Lei; Ding, Chan

    2014-01-01

    Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between 5.5 × 10(2) and 1.1 × 10(9) copies/μL of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10'h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection. Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV. PMID:25379553

  12. 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. PMID:26709100

  13. Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection.

    PubMed

    Baert, Leen; Uyttendaele, Mieke; Debevere, Johan

    2008-03-31

    Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform. PMID:18258325

  14. Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples.

    PubMed

    Abera, Tsegalem; Thangavelu, Ardhanary

    2014-12-01

    A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/μl with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50/ml and 1TCID50/ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids. PMID:25194891

  15. A rapid real-time qRT-PCR assay for ovine beta-actin mRNA.

    PubMed

    Bjarnadottir, Helga; Jonsson, Jon J

    2005-05-01

    Beta-Actin mRNA is often used for normalization in gene expression experiments. We describe a sensitive, rapid and specific quantitative assay for the cytoplasmic ovine beta-actin mRNA. The assay was based on the polymerase chain reaction (PCR) with real-time fluorescence resonance energy transfer (FRET) measurements to amplify cDNA products reverse transcribed from mRNA. A part of the ovine beta-actin sequence was amplified from cDNA from fetal ovine synovial (FOS) cells with mRNA-specific primers and cloned into a plasmid clone. The assay standard curve was constructed with dilutions of this plasmid. The assay was linear over five orders of magnitude and detected down to 600 copies per reaction of target DNA. Intraassay coefficient of variation was 12%. Detection of the beta-actin gene was eliminated by designing FRET probes at splice junctions and detection of putative processed pseudogenes was minimized by using FRET assay design with four oligonucleotides. We measured 0.2 copies per cell in RNA preparations without reverse transcription and DNase digestion. This might represent processed pseudogenes. In constrast, we measured 1400 beta-actin mRNA copies per cell in RNA preparations after the RT and DNase steps. The assay should, therefore, be sensitive enough to measure beta-actin from a single individual cell. Dilution of target DNA in murine RNA or ovine cDNA preparations did not effect efficiency of PCR or linearity of the assay. The quantitative assay described in this work can be used to correct for variations in various real-time qRT-PCR experiments in ovine cells with diverse goals, including gene expression studies, quantitation of viral load in infected cells and in various gene therapy experiments measuring vector load and expression in transduced cells. PMID:15823406

  16. Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa.

    PubMed

    Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua; Wang, Hongzhi

    2016-01-01

    The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT-PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation. PMID:27300480

  17. A Tool Set for the Genome-Wide Analysis of Neurospora crassa by RT-PCR.

    PubMed

    Hurley, Jennifer H; Dasgupta, Arko; Andrews, Peter; Crowell, Alexander M; Ringelberg, Carol; Loros, Jennifer J; Dunlap, Jay C

    2015-10-01

    Neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. Although the community-accumulated tool set for the molecular analysis of Neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time PCR (RT-PCR). To address the first of these we have identified genes that are optimal for use as reference genes in RT-PCR across a wide range of expression levels; the mRNA/transcripts from these genes have potential for use as reference noncycling transcripts outside of Neurospora. In addition, we have generated a genome-wide set of RT-PCR primers, thereby streamlining the analysis of gene expression. In validation studies these primers successfully identified target mRNAs arising from 70% (34 of 49) of all tested genes and from all (28) of the moderately to highly expressed tested genes. PMID:26248984

  18. Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

    PubMed Central

    2009-01-01

    Background HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas. Methods To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. Results The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350). Conclusion Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene. PMID:19309522

  19. Utility of automated real-time RT-PCR for the detection of foot-and-mouth disease virus excreted in milk.

    PubMed

    Reid, Scott M; Parida, Satya; King, Donald P; Hutchings, Geoffrey H; Shaw, Andrew E; Ferris, Nigel P; Zhang, Zhidong; Hillerton, J Eric; Paton, David J

    2006-01-01

    Foot-and-mouth disease virus (FMDV) can be excreted in milk and thereby spread infection to susceptible animals in other holdings. The feasibility of using real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic tool for detection of FMDV in milk was assessed by studying the excretion of virus from experimentally-infected cattle. Fore- and machine milk samples were collected over a 4-week period from two dairy cows infected with FMDV and from two in-contact cows held in the same pen. The whole, skim, cream and cellular debris components of the milks were tested by automated rRT-PCR and results compared to virus isolation (VI) in cell culture. The onset of clinical signs of FMD in all four cows correlated with viraemia, and the presence of FMDV in other clinical samples. rRT-PCR results matched closely with VI in detecting FMDV in all milk components and generally coincided with, but did not consistently precede, the onset of clinical signs. rRT-PCR detected FMDV in milk up to 23 days post inoculation which was longer than VI. Furthermore, the detection limit of FMDV in milk was greater by rRT-PCR than VI and, in contrast to VI, rRT-PCR detected virus genome following heat treatment that simulated pasteurisation. rRT-PCR was also able to detect FMDV in preservative-treated milk. In conclusion, this study showed that automated rRT-PCR is quicker and more sensitive than VI and can be used to detect FMDV in whole milk as well as milk fractions from infected animals. PMID:16336929

  20. Evaluation of Xpert® Norovirus Assay performance in comparison with real-time RT-PCR in hospitalized adult patients with acute gastroenteritis.

    PubMed

    Rovida, Francesca; Premoli, Marta; Campanini, Giulia; Sarasini, Antonella; Baldanti, Fausto

    2016-08-01

    Xpert® Norovirus Assay (Cepheid, Sunnyvale, CA) was compared with a laboratory-developed real-time RT-PCR assay for the detection of Norovirus GI and GII in hospitalized patients with acute gastroenteritis. The two assays showed a high level of concordance but Xpert® Norovirus Assay allowed faster detection of Norovirus and a simpler sample handling. PMID:27233425

  1. Development of a One-Step Immunocapture Real-Time TaqMan RT-PCR Assay for the Broad Spectrum Detection of Pepino Mosaic Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A Real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse PepMV isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single co...

  2. Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conser...

  3. Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Efforts to analyze the replicative RNA produced by Maize fine streak virus (MFSV) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

  4. Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

  5. Analytical validation of a real-time RT-PCR test for Pan-American lineage H7 subtype avian influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of avian influenza virus and identification of the H5 and H7 hemagglutinin subtypes some of which are associated with high pathogenicity in poultry is critical for clinical diagnosis and wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in N...

  6. Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid.

    PubMed

    Papayiannis, Lambros C

    2014-02-01

    Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes. PMID:24252553

  7. Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis.

    PubMed

    Wang, Hai-Bo; Mo, Qiu-Hua; Wang, Qi; Wu, Bi-Mei; Feng, Zi-Li; Lin, Ji-Can; Yang, Ze

    2016-09-01

    Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10(0), 10(2), 10(0) and 10(3) copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens. PMID:27461241

  8. Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar).

    PubMed

    Snow, M; McKay, P; McBeath, A J A; Black, J; Doig, F; Kerr, R; Cunningham, C O; Nylund, A; Devold, M

    2006-01-01

    Infectious salmon anaemia (ISA) is a disease of cultured Atlantic salmon (Salmo salar) which was successfully eradicated from Scotland following its emergence in 1998. The rapid deployment of sensitive diagnostic methods for the detection of ISA virus (ISAV) was fundamental to the swift eradication of ISA disease in Scotland and continues to be of crucial importance to surveillance of the aquaculture industry. This study reports the development, validation, application and interpretation of two independent, highly sensitive and specific semi-quantitative Taqman real-time RT-PCR (qRT-PCR) methods for the detection of ISAV. Such technology offers considerable advantages over conventional RT-PCR methods in current routine use for ISAV surveillance. These include an increased sensitivity, enhanced specificity, semi-quantification using endogenous controls, a lack of subjectivity in results interpretation, speed of processing and improved contamination control. PMID:17058489

  9. Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Hart, S. Alexandra; Kurath, Gael; Winton, James R.

    2006-01-01

    The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.

  10. New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

    PubMed Central

    Kawashima, Kenji; Lucero, Ginger; Ackermann, Mark R.

    2005-01-01

    We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead. PMID:16136226

  11. Factors Affecting Detection of Hepatitis E Virus on Canadian Retail Pork Chops and Pork Livers Assayed Using Real-Time RT-PCR.

    PubMed

    Wilhelm, B J; Leblanc, D; Avery, B; Pearl, D L; Houde, A; Rajić, A; McEwen, S A

    2016-03-01

    We collected 599 Canadian retail pork chops and 283 pork livers routinely (usually weekly) from April 2011 to March 2012 using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) retail sampling platform. Samples were assayed using validated real-time (q) reverse transcriptase polymerase chain reaction (RT-PCR) and nested classical RT-PCR for the detection of hepatitis E virus (HEV), porcine enteric calicivirus (PEC) and rotavirus (RV). The presence of Escherichia coli, Salmonella spp. and Campylobacter spp. was measured on a subset of our samples. Exact logistic regression models were fitted for predictors for HEV detection, for each assay. For both assays, sample type (pork chop versus liver) was a significant predictor for HEV RNA detection. For nested classical RT-PCR but not qRT-PCR, region of sample collection was a significant predictor (P = 0.008) of HEV detection. Odds of HEV detection were greatest in spring relative to other seasons. E. coli was a significant predictor for HEV RNA detection using the qRT-PCR (P = 0.03). Overall, the prevalence of E. coli, Salmonella spp. and Campylobacter spp. was significantly greater than HEV, PEC or RV on our retail pork samples. Our sparse data set for the detection of PEC and RV precluded modelling of risk factors for the detection of these viruses. PMID:26192650

  12. Comparison of Luminex xTAG® RVP fast assay and real time RT-PCR for the detection of respiratory viruses in adults with community-acquired pneumonia.

    PubMed

    Luchsinger, Vivian; Prades, Yara; Ruiz, Mauricio; Pizarro, Rolando; Rossi, Patricio; Lizama, Luis; Garmendia, María Luisa; Meza, Angela; Larrañaga, Carmen; Avendaño, Luis F

    2016-07-01

    Community-acquired pneumonia (CAP) is the third cause of death worldwide. Viruses are frequently detected in adult CAP. Highly sensitive diagnostic techniques should be used due to poor viral shedding. Different sampling methods can affect viral detection, being necessary to establish the optimal type of sample for identifying respiratory viruses in adults. The detection rates of respiratory viruses by Luminex xTAG® RVP fast assay, real time RT-PCR (rtRT-PCR) (Sacace®), and immunofluorescence assay (IFA) in adult CAP were performed in nasopharyngeal swabs (NPS) and aspirates (NPA) from 179 hospitalized adults. Positivity was 47.5% for Luminex®, 42.5% for rtRT-PCR (P = 0.3), and 2.7% for IFA (2.7%) (P < 0.0). The sensitivity, specificity, and kappa coefficient of xTAG® RVP compared with rtRT-PCR were 84.2%, 79.6%, and 0.62%, respectively. Luminex® and rtRT-PCR detected 65 (58.0%) and 57 (50.9%) viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P < 0.01). xTAG® RVP is appropriate for detecting respiratory viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs. J. Med. Virol. 88:1173-1179, 2016. © 2016 Wiley Periodicals, Inc. PMID:27061405

  13. A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus

    PubMed Central

    2014-01-01

    Background Peste des petits ruminants (PPR) is an economically important disease of small ruminants such as sheep and goats. The disease is characterized by severe pyrexia, oculo-nasal discharge, pneumonia, necrosis and ulceration of the mucous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhea. A SYBR Green I based real time RT-PCR targeting the N gene of PPRV has not been established for PPRV detection. Thus, the objective of present study was to develop highly sensitive N gene target SYBR Green I real time RT-PCR for specific detection and quantification of PPRV in clinical samples. A set of primers was designed to detect the nucleocapsid (N) gene of PPRV. Results The assay exhibited high specificity as all the viruses which have clinical and structural similarities to PPRV including Canine distemper virus (CDV), Measles virus (MV), Bluetongue virus (BTV) and Newcastle disease virus (NDV) failed to show an amplification signal. The lower detection limit of the assay was 5.11 copies/μl (Ct value of 33.67 ± 0.5) and 0.001 TCID50/ml (Ct value of 34.7 ± 0.5) based on plasmid copy number and tissue culture infectivity titre. The assay was 3-log more sensitive than the conventional RT-PCR. The coefficient of variation (CV) values for intra- and inter-assay variability were low, ranging from 0.32% - 2.31%, and 0.71% - 5.32%, respectively. To evaluate the performance of the newly developed assay, a total of 36 clinical samples suspected of PPR were screened for the presence of PPRV in parallel with conventional RT-PCR. The real time RT-PCR assay detected PPRV in 30 (83.3%) of clinical samples compared to 16 (44.4%) by conventional RT-PCR. Conclusions The two-step SYBR Green I based real time RT-PCR assay reported here is highly sensitive, specific, reproducible and rapid for detection and quantification of PPRV nucleic acids. PMID:24423231

  14. Detection of West Nile viral RNA from field-collected mosquitoes in tropical regions by conventional and real-time RT-PCR.

    PubMed

    González-Reiche, Ana Silvia; Monzón-Pineda, María de Lourdes; Johnson, Barbara W; Morales-Betoulle, María Eugenia

    2010-01-01

    West Nile virus (WNV) is an emerging mosquito-borne flavivirus, which has rapidly spread and is currently widely distributed. Therefore, efforts for WNV early detection and ecological surveillance of this disease agent have been increased around the world. Although virus isolation is known to be the standard method for detection and identification of viruses, the use of RT-PCR assays as routine laboratory tests provides a rapid alterative suitable for the detection of viral RNA on field-collected samples. A method for WNV RNA genome detection in field-collected mosquitoes is presented in this chapter. This method has been designed for virus surveillance in tropical regions endemic for other flaviviruses. Reverse Transcriptase-PCR (RT-PCR) assays, both standard and real time, to detect WNV and other flaviviruses are described. A first screening for flavivirus RNA detection is performed using a conventional RT-PCR with two different sets of flavivirus consensus primers. Mosquito samples are then tested for WNV RNA by a real-time (TaqMan) RT-PCR assay. Sample preparation and RNA extraction procedures are also described. PMID:20300994

  15. Whole genome alignment based one-step real-time RT-PCR for universal detection of avian orthoreoviruses of chicken, pheasant and turkey origins.

    PubMed

    Tang, Yi; Lu, Huaguang

    2016-04-01

    Newly emerging avian orthoreovirus (ARV) variants have been continuously detected in Pennsylvania poultry since 2011. In this paper, we report our recent diagnostic assay development of one-step real-time RT-PCR (rRT-PCR) for the rapid and universal detection of all ARVs or reference strains of chicken, pheasant and turkey origins and six σC genotypes of the newly emerging field ARV variants in Pennsylvania (PA) poultry. Primers and probes for the rRT-PCR were designed from the conserved region of the M1 genome segment 5' end based on the whole-genome alignment of various ARV strains, including six field variants or novel strains obtained in PA poultry. The detection limit of the newly developed rRT-PCR for ARV was as low as 10 copies/reaction of viral RNA, and 10(0.50)-10(0.88) tissue culture infectious dose (TCID50)/100 μL of viruses. This new rRT-PCR detected all six σC genotypes from the 66 ARV field variant strains and reference strains tested in this study. There were no cross-reactions with other avian viruses. Reproducibility of the assay was confirmed by intra- and inter-assay tests with variability from 0.12% to 2.19%. Sensitivity and specificity of this new rRT-PCR for ARV were achieved at 100% and 88%, respectively, in comparison with virus isolation as the "gold standard" in testing poultry tissue specimen. PMID:26812128

  16. Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease.

    PubMed

    Gorna, K; Relmy, A; Romey, A; Zientara, S; Blaise-Boisseau, S; Bakkali-Kassimi, L

    2016-09-01

    Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (FMDV) were established and validated. Each RT-PCR test consists of a ready-to-use master mix for simultaneous detection of the well established 3D or IRES FMDV targets and incorporates the host β-actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/β-actin and IRES/β-actin tests are highly sensitive and able to detect up to 7TCID50/ml of FMDV and 10 copies/1μl of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/β-actin test and 97% (95% CI; 87-100%) for the IRES/β-actin test. The diagnostic specificity was 100% (95% CI; 95-100%) for both RT-PCRs. In addition, the two protocols proved to be robust, showing inter-assay coefficients of variation ranging from 1.94% to 6.73% for the IRES target and from 2.33% to 5.42% for the 3D target for different RNA extractions and different RT-PCR conditions. The internally controlled one-step real-time RT-PCR protocols described in this study provide a rapid, effective and reliable method for the detection of FMDV and thus may improve the routine diagnosis for foot-and-mouth disease. PMID:27317973

  17. Detection, discrimination and quantitation of 22 bluetongue virus serotypes using real-time RT-PCR with TaqMan MGB probes.

    PubMed

    Feng, Yufei; Yang, Tao; Xu, Qingyuan; Sun, Encheng; Li, Junping; Lv, Shuang; Wang, Haixiu; Zhang, Qin; Zhang, Jikai; Wu, Donglai

    2015-09-01

    Bluetongue virus (BTV) is the etiological agent of bluetongue (BT) disease, a noncontagious insect-transmitted disease of international importance. To date, 26 BTV serotypes have been recognized worldwide. Methods to discriminate BTV serotypes in clinical samples are essential to epidemiological surveillance efforts and BTV vaccination programs. The BTV VP2 major outer capsid protein, encoded by genomic segment 2 (Seg-2), is the most highly variable BTV protein and is the primary determinant of the virus serotype. Here, we report the development of rapid and reliable real-time RT-PCR assays to detect and discriminate 22 BTV serotypes on the basis of VP2-encoding genomic sequences. Serotype-specific primers and probes detected only the targeted BTV serotype and displayed no cross-amplification of off-target BTV serotypes or other closely related Reoviridae and Bunyaviridae family members. The real-time RT-PCR assays developed were highly sensitive, and the majority of serotype-specific reactions could detect template when present at ≥10 copies. These BTV serotype-specific real-time RT-PCR assays represent a rapid, sensitive, and reliable method for the identification, differentiation and quantification of 22 BTV serotypes. PMID:26115692

  18. Quantitative RT-PCR analysis of the MOZ-CBP fusion transcript in therapy-related acute myeloid leukemia with t(8;16)(p11;p13).

    PubMed

    Fujiki, Atsushi; Imamura, Toshihiko; Furutani, Akiyo; Hatano, Waka; Asai, Daisuke; Hirashima, Yoshifumi; Miyachi, Mitsuru; Tamura, Shinichi; Tsuchiya, Kunihiko; Iehara, Tomoko; Ishida, Hiroyuki; Yoshihara, Takao; Hosoi, Hajime

    2012-07-01

    We developed a real time reverse transcriptase polymerase chain reaction (RT-PCR) assay system for detecting the MOZ-CBP fusion transcript and used it to monitor minimal residual disease (MRD) status in a patient with therapy related acute myeloid leukemia (t-AML) harboring t(8;16)(p11;p13). Expression of the MOZ-CBP fusion transcript was determined by RT-PCR analysis of the patient's bone marrow at the time of diagnosis. Thereafter, real time RT-PCR was used to evaluate MRD levels throughout the entire course of treatment. The sensitivity of quantitative RT-PCR for the MOZ-CBP fusion transcript was 10(-5). Below this level, MRD was classified as negative. Real time RT-PCR of the bone marrow after induction therapy showed the reduction of MOZ-CBP transcript to approximately 10(-3) level when compared to the diagnostic sample. MRD was classified as negative (< 10(-5) compared with that in the bone marrow at diagnosis) after 5 courses of chemotherapy, a level that was maintained post-allo-hematopoietic stem cell transplantation. Real time RT-PCR of the MOZ-CBP transcript is a useful tool for assessing MRD status for a patient with therapy related acute myeloid leukemia who was initially predicted to have a poor prognosis. PMID:22278196

  19. Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production

    PubMed Central

    Shcherbik, Svetlana; Sergent, Sheila B.; Davis, William G.; Shu, Bo; Barnes, John; Kiseleva, Irina; Larionova, Natalie; Klimov, Alexander; Bousse, Tatiana

    2015-01-01

    Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012–2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8 EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1 EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate. PMID:24056261

  20. 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.

  1. Validation of a real-time RT-PCR method to quantify Newcastle Disease Virus (NDV) titer and comparison with other quantifiable methods.

    PubMed

    Jang, Juno; Hong, Sung-Hwan; Kim, Ik-Hwan

    2011-01-01

    A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity. PMID:21301199

  2. Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

    PubMed

    Chen, Lida; Li, Wenli; Zhang, Kuo; Zhang, Rui; Lu, Tian; Hao, Mingju; Jia, Tingting; Sun, Yu; Lin, Guigao; Wang, Lunan; Li, Jinming

    2016-01-01

    Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses. PMID:26612712

  3. Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa

    PubMed Central

    Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua

    2016-01-01

    The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT–PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation. PMID:27300480

  4. Identification of CD70 as a diagnostic biomarker for clear cell renal cell carcinoma by gene expression profiling, real-time RT-PCR and immunohistochemistry.

    PubMed

    Diegmann, Julia; Junker, Kerstin; Gerstmayer, Bernhard; Bosio, Andreas; Hindermann, Winfried; Rosenhahn, Julia; von Eggeling, Ferdinand

    2005-08-01

    The underlying molecular mechanisms of renal cell carcinoma (RCC) are poorly understood and more reliable markers for early diagnosis are needed. Hence, alternative strategies for biomarker discovery with appropriate validation technologies have to be performed. To elucidate genesis and progression of RCC we used high parallel chip based gene expression profiling comparing normal and tumour tissues. We compared corresponding control and tumour tissue samples from 10 patients with clear cell RCC. We isolated RNA from histologically well characterised tissue sections and performed reverse transcription, labelling and linear RNA amplification. Samples were hybridised on microarrays containing 642 human cDNAs. Of the 352 differentially expressed genes found, CD70 and FRA2 were selected for further evaluation by real-time RT-PCR. The analysis all showed a high potential to discriminate between normal and tumour tissue. Moreover, increased CD70 mRNA expression in tumour cells could be correlated to its expression at the protein level. Immunohistochemistry (IHC) showed very strong expression of CD70 in all tumour samples but no expression in adjacent normal kidney tissue. With our combined approach we were able to identify CD70 as a new marker for RCC, which may be useful in the future for improved immunohistochemical diagnosis. PMID:16043348

  5. A SYBR Green-based real-time RT-PCR assay for simple and rapid detection and differentiation of highly pathogenic and classical type 2 porcine reproductive and respiratory syndrome virus circulating in China.

    PubMed

    Chai, Zheng; Ma, Wenjun; Fu, Fang; Lang, Yuekun; Wang, Wei; Tong, Guangzhi; Liu, Qinfang; Cai, Xuehui; Li, Xi

    2013-02-01

    SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID(50) for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China. PMID:23070137

  6. Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

    PubMed Central

    Kamboj, Aman; Pateriya, Atul Kumar; Mishra, Anamika; Ranaware, Pradip; Kulkarni, Diwakar D.; Raut, Ashwin Ashok

    2014-01-01

    Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF. PMID:24877102

  7. Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1

    PubMed Central

    Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence

    2015-01-01

    This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes. PMID:25785274

  8. Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1.

    PubMed

    Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence

    2015-01-01

    This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R (2) values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes. PMID:25785274

  9. Simultaneous detection of influenza viruses A, B, and swine origin influenza A using multiplex one-step real-time RT-PCR assay.

    PubMed

    Monavari, S H R; Mollaie, H R; Fazlalipour, M

    2014-01-01

    Every year, seasonal epidemics of influenza viruses are causing considerable morbidity and mortality worldwide. Also infrequent novel and rearranged strains of influenza viruses have caused quick, acute universal pandemics resulting in millions of mortalities. The usage of efficient and accurate detection is superior for infection control, effective treatment, and epidemiological supervision. Therefore, evaluation of useful real-time PCR molecular tests for the detection of pandemic viruses is important before the next wave of the pandemic. A novel quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers was used successfully for detection and monitoring of the influenza A, B, and swine influenza. The newly designed primers target highly conserved regions in influenza viruses. Our qRT-PCR assay is highly specific for detecting influenza A, B, and swine influenza viruses. The cutoff CT value was determined <38 for domestic human diagnostic test, under conditions of FDA emergency, and the reaction efficiency of the InfA, swInfA, and InfB assays were thereby estimated to be 97.9 % (R2 = 0.998), 98.3 % (R2 = 0.986), and 99.5 % (R2 = 0.995), respectively. Interestingly, based on our finding, there is no cross reactivity of detecting other viruses. PMID:24142356

  10. Biosurveillance of avian influenza and Newcastle disease viruses in the Barda region of Azerbaijan using real time RT-PCR and hemagglutination inhibition

    PubMed Central

    Zeynalova, Shalala; Guliyev, Fizuli; Vatani, Mahira; Abbasov, Bahruz

    2015-01-01

    The Azerbaijan State Veterinary Control Service (SVCS) has conducted active serological surveillance for avian influenza (AI) in poultry since 2006, when the first outbreak of AI H5N1 occurred in Azerbaijan. Samples are collected from September to May annually and tested using a hemagglutination inhibition (HI) assay to detect antibodies against H5 AI viruses. HI testing is also performed for Newcastle disease virus (NDV) upon request, but since this method cannot distinguish between natural infections and immune responses to vaccination, all positive results require follow-up epidemiological investigations. Furthermore, blood collection for the surveillance program is time-intensive and can be stressful to birds. In order to improve the national surveillance program, alternative sampling and testing methodologies were applied among a population of birds in the Barda region and compared with results of the national surveillance program. Tracheal and cloacal swabs were collected instead of blood. Rather than testing individual samples, RNA was pooled to conserve resources and time, and pools were tested by real-time reverse transcription polymerase chain reaction (rRT-PCR). Environmental sampling at a live bird market was also introduced as another surveillance mechanism. A total of 1,030 swabs were collected, comprising tracheal, and cloacal samples from 441 birds and 148 environmental surface samples from farms or the live bird market. During the same time, 3,890 blood samples were collected nationally for the surveillance program; 400 of these samples originated in the Barda region. Birds sampled for rRT-PCR were likely different than those tested as part of national surveillance. All swab samples tested negative by rRT-PCR for both AI and NDV. All blood samples tested negative for H5 by HI, while 6.2% of all samples and 5% of the Barda samples tested positive for exposure to NDV. Follow-up investigations found that positive samples were from birds vaccinated in

  11. Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples.

    PubMed

    Cabrerizo, María; Calvo, Cristina; Rabella, Nuria; Muñoz-Almagro, Carmen; del Amo, Eva; Pérez-Ruiz, Mercedes; Sanbonmatsu-Gámez, Sara; Moreno-Docón, Antonio; Otero, Almudena; Trallero, Gloria

    2014-11-01

    Human enteroviruses (EVs) and parechoviruses (HPeVs) are important etiological agents causing infections such as meningitis, encephalitis and sepsis-like disease in neonates and young children. We have developed a real-time RT-PCR for simultaneous detection of EV and HPeV in clinical samples. Primers and probe sets were designed from the conserved 5'-noncoding region of the genomes. The sensitivity, specificity and reproducibility of the technique were measured using a set of 25 EV and 6 HPeV types. All EVs but no HPeVs were detected with the EV primers-probe set. The HPeV primers-probe set detected only the 6 HPeV types. The lower detection limit was found to be 4 and 40CCID50/ml for HPeV and EV respectively, demonstrating high sensitivity of the technique for both viruses. The threshold cycle values were highly reproducible on repeat testing of positive controls among assay runs. The assay was evaluated in 53 clinical samples of suspected meningitis, sepsis or febrile syndromes from children under 3 years. In 11 of these (21%) EVs were detected, while 4, i.e. 7.5%, were HPeV positive. Molecular typing was carried out for 73% of the viruses. In summary, the RT-PCR method developed demonstrated effectively both EV and HPeV detection, which can cause similar clinical symptoms in infants. PMID:25152526

  12. Comparison of three magnetic-bead-based RNA extraction methods for detection of cucumber green mottle mosaic virus by real-time RT-PCR.

    PubMed

    Zhao, Xiaoli; Zhou, Qi; Zhang, Lijie; Yan, Wenlong; Sun, Ning; Liang, Xinmiao; Deng, Congliang

    2015-07-01

    To determine the efficiency of RNA extraction methods based on magnetic beads, three different bead-based methods (one using silica-coated magnetic beads [SMNP], one using immunomagnetic beads conjugated to a specific antibody [IMB], and one using magnetic beads to nonspecifically adsorb virions [MNP]) were compared with the TRIzol method for the extraction of cucumber green mottle mosaic virus (CGMMV) RNA from cucumber leaves by real-time RT-PCR. The results indicated that the extraction efficiency of the SMNP method was 10 times higher than those of the IMB and MNP methods and 100 times higher than that of the TRIzol method. Therefore, the SMNP method could be considered for use in quarantine measures for the prevention and control of the disease caused by CGMMV. PMID:25951973

  13. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma.

    PubMed

    Hue, Kien Duong Thi; Tuan, Trung Vu; Thi, Hanh Tien Nguyen; Bich, Chau Tran Nguyen; Anh, Huy Huynh Le; Wills, Bridget A; Simmons, Cameron P

    2011-11-01

    Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam. PMID:21843553

  14. Change your conventional practice of qRT-PCR: A simple robust quality control standard for yeast mRNA quantification analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has been realized to be the assay of choice among available techniques for mRNA quantification analysis. For conventional practice, housekeeping genes have been applied as internal reference for data normalization and a...

  15. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

    PubMed Central

    Reid, Karen E; Olsson, Niclas; Schlosser, James; Peng, Fred; Lund, Steven T

    2006-01-01

    Background Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents. Results We describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, α-tubulin, β-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series. Conclusion In order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-α and SAND as the most relevant reference

  16. 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. PMID:27210549

  17. Detection and absolute quantitation of Tomato torrado virus (ToTV) by real time RT-PCR.

    PubMed

    Herrera-Vásquez, José Angel; Rubio, Luis; Alfaro-Fernández, Ana; Debreczeni, Diana Elvira; Font-San-Ambrosio, Isabel; Falk, Bryce W; Ferriol, Inmaculada

    2015-09-01

    Tomato torrado virus (ToTV) causes serious damage to the tomato industry and significant economic losses. A quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) method using primers and a specific TaqMan(®) MGB probe for ToTV was developed for sensitive detection and quantitation of different ToTV isolates. A standard curve using RNA transcripts enabled absolute quantitation, with a dynamic range from 10(4) to 10(10) ToTV RNA copies/ng of total RNA. The specificity of the RT-qPCR was tested with twenty-three ToTV isolates from tomato (Solanum lycopersicum L.), and black nightshade (Solanum nigrum L.) collected in Spain, Australia, Hungary and France, which covered the genetic variation range of this virus. This new RT-qPCR assay enables a reproducible, sensitive and specific detection and quantitation of ToTV, which can be a valuable tool in disease management programs and epidemiological studies. PMID:25956672

  18. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics. PMID:25746154

  19. The use of relative quantitative RT-PCR for expression analysis in azalea flower color sports.

    PubMed

    De Keyser, E; De Riek, J; Van Bockstaele, E

    2003-01-01

    The fastest way to create new azalea (Rhododendron simsii hybrids) cultivars is by making use of flower colour sports, which appear spontaneously on azalea plants. Unfortunately, there is still very little known on how bud sport induction occurs. Therefore, genes coding for two key enzymes of the azalea flavonoid biosynthesis pathway, chalcon synthase (chs) and dihydroflavonol 4-reductase (dfr) that were reported before to be apt for modification by the action of bud sporting, were isolated and characterized. The expression of these two flower colour genes in the petals of azalea flowers will be compared between all 'Hellmut Vogel' flower colour sports. To measure the expression levels of both genes, relative quantitative RT-PCR analysis will be worked out on a real-time PCR machine. The expression of housekeeping genes, which is expected to be the same for all sports, will be used to calculate the relative expression level of the two genes of interest. The optimisation of this technique will be discussed. PMID:24757769

  20. High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

    PubMed Central

    Renn, Lynnsey A.; Theisen, Terence C.; Navarro, Maria B.; Mane, Viraj P.; Schramm, Lynnsie M.; Kirschman, Kevin D.; Fabozzi, Giulia; Hillyer, Philippa; Puig, Montserrat; Verthelyi, Daniela; Rabin, Ronald L.

    2015-01-01

    Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts. PMID:25867042

  1. Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards.

    PubMed

    Lopez-Vazquez, C; Bandín, I; Dopazo, C P

    2015-05-21

    In the present study, 2 systems of real-time RT-PCR-one based on SYBR Green and the other on TaqMan-were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 100 TCID50 ml-1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always <5. This fact, together with the high efficiency and R2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies. PMID:25993885

  2. Development and Validation of a Multiplex, Real-Time RT PCR Assay for the Simultaneous Detection of Classical and African Swine Fever Viruses

    PubMed Central

    Haines, Felicity J.; Hofmann, Martin A.; King, Donald P.; Drew, Trevor W.; Crooke, Helen R.

    2013-01-01

    A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping. PMID:23923045

  3. Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs

    PubMed Central

    SEHATA, Go; SATO, Hiroaki; ITO, Toshihiro; IMAIZUMI, Yoshitaka; NORO, Taichi; OISHI, Eiji

    2015-01-01

    We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

  4. Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

    PubMed

    Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

    2015-07-01

    We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

  5. Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision.

    PubMed

    Goris, Nesya; Vandenbussche, Frank; Herr, Cécile; Villers, Jérôme; Van der Stede, Yves; De Clercq, Kris

    2009-09-01

    Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays are being used routinely for diagnosing foot-and-mouth disease virus (FMDV). Although most laboratories determine analytical and diagnostic sensitivity and specificity, a thorough validation in terms of establishing optimal RNA-extraction conditions, matrix effect, uncertainty of measurement and precision is not performed or reported generally. In this study, different RNA-extraction procedures were compared for two FMDV rRT-PCRs. The NucleoSpin columns available commercially combined high extraction efficiency with ease-of-automation. Furthermore, six different FMDV-negative matrices were spiked with a dilution series of FMDV SAT1 ZIM 25/89. Compared to cell-culture-spiked viral control samples, no matrix effect on the analytical sensitivity was found for blood or foot epithelium. Approximately 1log(10) reduction in detection limit was noted for faecal and tongue epithelium samples, whereas a 3log(10) decrease was observed for spleen samples. By testing the same dilution series in duplicate on 10 different occasions, an estimation of uncertainty of measurement and precision was obtained using blood as matrix. Both rRT-PCRs produced highly precise results emphasising their potential to replace conventional virological methods. The uncertainty measurement, as described in this study, proved to be a useful tool to evaluate the probability of making a wrong decision. PMID:19447138

  6. Rapid detection of foot-and-mouth disease virus, influenza A virus and classical swine fever virus by high-speed real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Beer, Martin; Hoffmann, Bernd

    2013-10-01

    High sensitivity, minor risk of cross-contamination and in particular the rapid reaction time make quantitative real-time polymerase chain reaction (qPCR) assays well suited for outbreak investigations as well as for monitoring epidemics of pathogens. In this study qPCR assays for three highly contagious animal diseases, namely foot-and-mouth-disease (FMD), influenza A (IA) and classical swine fever (CSF) have been developed. Furthermore, an amplification control targeting 18S ribosomal RNA was included. Each assay was validated with samples from infected animals using three different standard qPCR-machines in two thermal profiles: one standard and one high-speed approach, respectively. The high-speed PCR assays allowed the reliable diagnosis of FMD, influenza A and CSF in less than 28 min with an analytical sensitivity of at least 200 genome copies/μl in every case, with slight differences regarding reaction time and sensitivity for the individual PCR-cycler instruments. Therefore, the newly established rapid RT-PCR systems will be a valuable method for the monitoring and control of these three important viruses and will be a robust option for the development of novel molecular pen-side tests. PMID:23702025

  7. Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR

    PubMed Central

    2013-01-01

    Background The diseases caused by Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) have been occurring epidemically in China and southeastern Asia in recent years. A sensitive, reliable and quantitative method is required to detect and distinguish for RBSDV and SRBSDV in rice and vector insects. Results We developed a sensitive and lineage-specific duplex real time RT-qPCR for detection of RBSDV and SRBSDV in a single or/and double infection in rice samples. The duplex RT-qPCR was optimized using standard samples transcribed by T7 Large Scale RNA Production System in vitro. We developed a reliable system for duplex RT-qPCR, in which its co-efficiency of RBSDV and SRBSDV, were 91.6% and 90.7%, respectively. The coefficient of determination was more than 0.990; the slope of linear equation was −3.542, and −3.567, respectively. Out of 30 samples collected in North and Central China, which were suspected to be infected with these two viruses, 10 samples were detected RBSDV positive by RT-PCR and 12 samples by RT-qPCR. No mixed infections were found. Simultaneously, out of total 60 samples collected from Southern China, which were also suspected to be infected with these two viruses, 41 samples were determined SRBSDV positive by RT-PCR and 47 samples by RT-qPCR. Also in this case no mixed infections were found. The rice genes eEF-1a and UBQ5 were selected as internal controls for quantification assay also performed as good expression stability. Conclusion The duplex RT-qPCR assay provided as a sufficiently sensitive, specific, accurate, reproducible and rapid tool for the detection and differentiation of RBSDV and SRBSDV. The RT-qPCR assay can be used in routine diagnostic of these two viruses in order to study the disease epidemiology in rice crops. PMID:23331990

  8. Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges

    PubMed Central

    De Regge, Nick; Madder, Maxime; Deblauwe, Isra; Losson, Bertrand; Fassotte, Christiane; Demeulemeester, Julie; Smeets, François; Tomme, Marie; Cay, Ann Brigitte

    2014-01-01

    Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21–24 and 33–36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV. PMID:24466312

  9. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  10. Development and bench validation of real time RT-PCR protocols for rapid detection of the subtypes H6, H9 and H11 of avian influenza viruses in experimental samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time RT-PCR (RRT-PCR) is commonly used for the rapid detection of avian influenza viruses (AIV) from clinical samples. Samples are typically screened for type A influenza by targeting the matrix gene, and then positive samples are further tested for hemagglutinin (HA) and neuraminidase (NA) su...

  11. Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

    NASA Astrophysics Data System (ADS)

    Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

    2014-11-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

  12. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    PubMed

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, p<0.001). hTR levels however, were high in all tumors and independently of the presence of germ cells also in the benign tissue control group. hTERT mRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype. PMID:12168080

  13. A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.

    PubMed

    Maan, Narender S; Maan, Sushila; Belaganahalli, Manjunatha; Pullinger, Gillian; Montes, Antonio J Arenas; Gasparini, Marcela R; Guimera, Marc; Nomikou, Kyriaki; Mertens, Peter P C

    2015-03-01

    Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes). PMID:25486080

  14. 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

  15. Assessment of Preparation of Samples Under the Field Conditions and a Portable Real-Time RT-PCR Assay for the Rapid On-Site Detection of Newcastle Disease Virus.

    PubMed

    Liu, L; Benyeda, Z; Zohari, S; Yacoub, A; Isaksson, M; Leijon, M; LeBlanc, N; Benyeda, J; Belák, S

    2016-04-01

    Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV-1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real-time RT-PCR method in the portable T-COR4 platform for the rapid, on-site detection of NDV on a farm. In the laboratory setting, the portable real-time RT-PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real-time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real-time RT-PCR detection was performed with the portable thermocycler T-COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures. PMID:25209697

  16. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time reverse transcriptase polymerase chain reaction (RRT-PCR) is routinely used for the rapid detection of Avian Influenza virus (AIV) in clinical samples. The usefulness of diagnostic RRT-PCR can be limited, in part, by the inhibitory substances present in some clinical specimens, which can ...

  17. Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches.

    PubMed

    Zhang, Shu-Qin; Tan, Bin; Li, Peng; Wang, Feng-Xue; Guo, Li; Yang, Yong; Sun, Na; Zhu, Hong-Wei; Wen, Yong-Jun; Cheng, Shi-Peng

    2014-10-01

    Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 10(1) copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials. PMID:25019170

  18. Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix® and RotaTeq® vaccine strains in stool samples

    PubMed Central

    Gautam, Rashi; Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Ian Tam, Ka; Gentsch, Jon R; Bowen, Michael D

    2014-01-01

    Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix® and RotaTeq® are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix® and RotaTeq® vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix® and RotaTeq® vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix® (NSP2, VP4) and RotaTeq® (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix® NSP2 and VP4 qRT-PCR assays exhibited 92–100% sensitivity, 99–100% specificity, 94–105% efficiency, and a limit of detection of 2–3 copies per reaction. RotaTeq® VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94–100% specificity, 91–102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix® and RotaTeq® vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE. PMID:24342877

  19. Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples.

    PubMed

    Gautam, Rashi; Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Ian Tam, Ka; Gentsch, Jon R; Bowen, Michael D

    2014-01-01

    Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix(®) and RotaTeq(®) are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix(®) and RotaTeq(®) vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix(®) and RotaTeq(®) vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix(®) (NSP2, VP4) and RotaTeq(®) (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix(®) NSP2 and VP4 qRT-PCR assays exhibited 92-100% sensitivity, 99-100% specificity, 94-105% efficiency, and a limit of detection of 2-3 copies per reaction. RotaTeq(®) VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94-100% specificity, 91-102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix(®) and RotaTeq(®) vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE. PMID:24342877

  20. Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Che Omar, Sarena; Bentley, Michael A.; Morieri, Giulia; Preston, Gail M.; Gurr, Sarah J.

    2016-01-01

    The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-α) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG

  1. Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae.

    PubMed

    Che Omar, Sarena; Bentley, Michael A; Morieri, Giulia; Preston, Gail M; Gurr, Sarah J

    2016-01-01

    The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-α) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG

  2. Performance of a commercial assay for the diagnosis of influenza A (H1N1) infection in comparison to the Centers for Disease Control and Prevention protocol of real time RT-PCR.

    PubMed

    Barbás, María G; Gallego, Sandra V; Castro, Gonzalo M; Baumeister, Elsa; Kademian, Silvia; De Leon, Juan; Cudolá, Analía

    2012-01-01

    At the time of influenza A (H1N1) emergency, the WHO responded with remarkable speed by releasing guidelines and a protocol for a real-time RT-PCR assay (rRT-PCR). The aim of the present study was to evalúate the performance of the "Real Time Ready Influenza A/H1N1 Detection Set" (June 2009)-Roche kit in comparison to the CDC reference rRT-PCR protocol. The overall sensitivity of the Roche assay for detection of the Inf A gene in the presence or absence of the H1 gene was 74.5 %. The sensitivity for detecting samples that were only positive for the Inf A gene (absence of the H1 gene) was 53.3 % whereas the sensitivity for H1N1-positive samples (presence of the Inf A gene and any other swine gene) was 76.4 %. The specificity of the assay was 97.1 %. A new version of the kit (November 2009) is now available, and a recent evaluation of its performance showed good sensitivity to detect pandemic H1N1 compared to other molecular assays. PMID:22610294

  3. Molecular analysis of dolphin morbillivirus: A new sensitive detection method based on nested RT-PCR.

    PubMed

    Centelleghe, Cinzia; Beffagna, Giorgia; Zanetti, Rossella; Zappulli, Valentina; Di Guardo, Giovanni; Mazzariol, Sandro

    2016-09-01

    Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply. PMID:27220282

  4. Quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.

    PubMed

    Olbrich, Maren; Gerstner, Elke; Welzl, Gerhard; Fleischmann, Frank; Osswald, Wolfgang; Bahnweg, Günther; Ernst, Dieter

    2008-01-01

    Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), a-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions. PMID:18811005

  5. The advantage of absolute quantification in comparative hormone research as indicated by a newly established real-time RT-PCR: GH, IGF-I, and IGF-II gene expression in the tilapia, Oreochromis niloticus.

    PubMed

    Eppler, Elisabeth; Caelers, Antje; Berishvili, Giorgi; Reinecke, Manfred

    2005-04-01

    We have developed a real-time RT-PCR that absolutely quantifies the gene expression of hormones using the standard curve method. The method avoids cloning procedures by using primer extension to create templates containing a T7 promoter gene sequence. It is rapid since neither separate reverse transcriptions nor postamplification steps are necessary, and its low detection level (2 pg/mug total RNA) allows precise absolute quantification. Using the method, we have quantified the gene expression of GH, IGF-I, and IGF-II in the tilapia. PMID:15891047

  6. Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

  7. 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

  8. 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

  9. Down-Regulation of MiR-193a-3p Dictates Deterioration of HCC: A Clinical Real-Time qRT-PCR Study

    PubMed Central

    Liu, Yongru; Ren, Fanghui; Luo, Yihuan; Rong, Minhua; Chen, Gang; Dang, Yiwu

    2015-01-01

    Background Although some recent reports have shown that the expression level of miR-193a varied in different cancers, its role in hepatocellular carcinoma (HCC) remains unidentified. The aim of the current study was to validate the relationship between miR-193a-3p and clinicopathological characteristics in HCC patients. Material/Methods Expression of miR-193a-3p in 95 HCC cases and their corresponding peritumoral tissues (PT) was examined by using quantitative reverse transcription polymerase chain reaction (qRT-PCR). miR-193a-3p expression and its correlation with a variety of clinicopathological features and patient recurrence were analyzed. Results The relative level of miR-193a-3p was 3.2028±1.1951 in PT, significantly higher than its expression in HCC tissues (1.5941±0.7079, P<0.001). The area under the curve of underexpression of miR-193a-3p was 0.906 to distinguish HCC from normal liver (95% CI: 0.864–0.948, P<0.001). Expression of miR-193a-3p was negatively correlated to metastasis (r=−0.371, P=0.000), TNM (r=−0.321, P=0.002), respectively. Additionally, the recurrence time was 50.271±2.631 months for the low miR-193a-3p level group and 60.132±3.626 months for the high miR-193a-3p level group. However, no significant difference between them was found (chi-square=0.354, P=0.552). Conclusions MiR-193a-3p may be a tumor-suppressive miRNA which is down-regulated in HCC tissues. It could be regarded as a predictor for the deterioration of HCC patients. PMID:26263159

  10. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

    PubMed Central

    Ma, Yue-jiao; Sun, Xiao-hong; Xu, Xiao-yan; Zhao, Yong; Pan, Ying-jie; Hwang, Cheng-An; Wu, Vivian C. H.

    2015-01-01

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus. PMID:26659406

  11. Development of an In-House TaqMan Real Time RT-PCR Assay to Quantify Hepatitis C Virus RNA in Serum and Peripheral Blood Mononuclear Cells in Patients With Chronic Hepatitis C Virus Infection

    PubMed Central

    Khalvati Fahlyani, Bahman; Behzad-Behbahani, Abbas; Taghavi, Seiied Alireza; Farhadi, Ali; Salehi, Saeede; Adibzadeh, Setare; Aboualizadeh, Farzaneh; Alavi, Parniyan; Nikouyan, Negin; Okhovat, Mohammad Ali; Ranjbaran, Reza; Rafiei Dehbidi, Gholam Reza; Shakibzadeh, Arash

    2015-01-01

    Background: Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCV-genomes in serum samples of patients after sustained virological response. However, in some patients, HCV viral load in serum samples is too low to be detected by PCR, especially after treatment. Objectives: The aim of this study was to develop a highly specific, sensitive, and reproducible in-house quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 - 4. Materials and Methods: In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5’-non-coding (5’NCR) of four HCV genotypes were used. Using plasmid containing 5’NCR region of HCV, standard curve, threshold, and threshold cycle (CT) values were determined. Real-time and nested PCR were performed on HCV genotypes 1 - 4 extracted from plasma and peripheral blood mononuclear cells (PBMCs) samples collected from patients with chronic HCV infection. Results: The lower limit detection of this in-house HCV real-time RT-PCR was determined as 100 RNA copies/mL. Inter- and intra-assay coefficient of variation (CV) of this in-house HCV real-time RT-PCR ranged from 0.9% to 1.8% and 1.76% to 3.94%, respectively. The viral load of the genotyped samples ranged from 2.0 × 106 ± 0.31 to 2.7 × 105 ± 0.46 copies/mL in serum samples and 5 × 102 ± 0.36 to 4.0 × 103 ± 0.51 copies/106 cells/mL of PBMCs. Conclusions: The quite sensitive in-house TaqMan real time RT-PCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran. PMID:26425128

  12. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  13. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

    PubMed Central

    2010-01-01

    Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene

  14. Real-time RT-PCR systems for CTC detection from blood samples of breast cancer and gynaecological tumour patients (Review).

    PubMed

    Andergassen, Ulrich; Kölbl, Alexandra C; Mahner, Sven; Jeschke, Udo

    2016-04-01

    Cells, which detach from a primary epithelial tumour and migrate through lymphatic vessels and blood stream are called 'circulating tumour cells'. These cells are considered to be the main root of remote metastasis and are correlated to a worse prognosis concerning progression-free and overall survival of the patients. Therefore, the detection of the minimal residual disease is of great importance regarding therapeutic decisions. Many different detection strategies are already available, but only one method, the CellSearch® system, reached FDA approval. The present review focusses on the detection of circulating tumour cells by means of real-time PCR, a highly sensitive method based on differences in gene expression between normal and malignant cells. Strategies for an enrichment of tumour cells are mentioned, as well as a large panel of potential marker genes. Drawbacks and advantages of the technique are elucidated, whereas, the greatest advantage might be, that by selection of appropriate marker genes, also tumour cells, which have already undergone epithelial to mesenchymal transition can be detected. Finally, the application of real-time PCR in different gynaecological malignancies is described, with breast cancer being the most studied cancer entity. PMID:26848098

  15. Evaluation of the Xpert Flu test and comparison with in-house real-time RT-PCR assays for detection of influenza virus from 2008 to 2011 in Marseille, France.

    PubMed

    Salez, N; Ninove, L; Thirion, L; Gazin, C; Zandotti, C; de Lamballerie, X; Charrel, R N

    2012-04-01

    Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert(®) Flu assay (Cepheid) with three real-time RT-PCR assays using 127 nasopharyngeal samples, of which 75 were positive for influenza A (with 52 identified as A/H1N1-2009) and 52 were positive for influenza B. The Xpert(®) Flu assay presented a quasi-absence of non-interpretable tests, and showed sensitivity and specificity of 100% and 100% for Flu A, 98.4% and 100% for A/H1N1-2009, and 80.7% and 100% for Flu B. PMID:22360446

  16. Molecular diagnosis of Papaya meleira virus (PMeV) from leaf samples of Carica papaya L. using conventional and real-time RT-PCR.

    PubMed

    Abreu, Paolla M V; Piccin, João G; Rodrigues, Silas P; Buss, David S; Ventura, José A; Fernandes, Patricia M B

    2012-03-01

    Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease. This study describes two methods for molecular diagnosis of PMeV using conventional and real-time PCR. These methods were shown to be more efficient than current methods of viral detection using extraction of PMeV dsRNA and observation of symptoms in the field. The methods described here were used to evaluate the effect of inoculation of papaya plants with purified PMeV dsRNA on the progress of PMeV infection. A single inoculation with PMeV dsRNA was observed to delay the progress of the virus infection by several weeks. The possibility of vertical transmission of PMeV was also investigated. No evidence was found for PMeV transmission through seeds collected from diseased fruit. The implications of these results for the epidemiology of PMeV and the management of papaya sticky disease are discussed. PMID:22193169

  17. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

    PubMed Central

    2012-01-01

    Background Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD. Results A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT). Conclusions The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD. PMID:22455597

  18. Expression of human membrane skeleton protein genes for protein 4.1 and betaIISigma2-spectrin assayed by real-time RT-PCR.

    PubMed

    Taylor-Harris, Pamela M; Felkin, Leanne E; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Barton, Paul J R; Pinder, Jennifer C

    2005-01-01

    The proteins, spectrin and 4.1 confer support and resilience to animal cell membranes, and promote assembly of multimeric, membrane-bound signalling complexes. Protein 4.1 also plays important roles in tumour suppression and the regulation of cell proliferation. To assess relative tissue expression of the four genes encoding human protein 4.1, we measured mRNA levels using quantitative real-time polymerase chain reaction. We compared 4.1 expression with that of a major splice variant of spectrin, betaIISigma2 that has a shortened C-terminus lacking a pleckstrin homology domain. mRNA for 4.1R is four-fold higher in bone marrow than in tissues with the next highest prevalence: cerebellum, lung, testis and thymus. 4.1G mRNA is highly expressed in brain, spinal cord and testis; 4.1N in brain, spinal cord and adrenal gland; 4.1B in testis, brain, spinal cord, and kidney. Thus, 4.1N, 4.1B and 4.1G all show high accumulation in nervous tissues. mRNA for betaIISigma2-spectrin is ubiquitous, but most abundant in cardiac and nervous tissues. Comparative transcript abundance was analysed in heart and brain. betaIISigma2-spectrin was the most abundant transcript in heart with levels 5 fold greater than 4.1G or 4.1N and at least 9 fold greater than 4.1B. In brain, 4.1N was the most abundant transcript, with levels 2.4 fold greater than 4.1B and at least 4 fold greater than 4.1G or betaIISigma2-spectrin. 4.1R abundance was very low in both tissues. Whilst we expected that 4.1 mRNAs would feature highly in muscle and nerve, we note their high abundance in testis, indicating previously unsuspected functions in reproduction. PMID:15809685

  19. RT-PCR analysis of Tecta, Coch, Eya4 and Strc in mouse cochlear explants.

    PubMed

    Maeda, Yukihide; Fukushima, Kunihiro; Kakiuchi, Masashi; Orita, Yorihisa; Nishizaki, Kazunori; Smith, Richard J H

    2005-03-15

    Tecta, Coch, Eya4 and Strc are mouse orthologs of four human deafness-associated genes. Their expression is markedly restricted to specific cell types in cochleae. Cochleae were dissected on embryonic day 15 and cultured in vitro. Relative messenger RNA abundance of each gene was quantified by RT-PCR and compared in-vivo cochleae of equivalent embryonic age. After 48 h in culture, in-vivo and explant Strc expression levels were equivalent, Eya4 level reduced in explanted tissues, and expression of Tecta and Coch did not show the expected temporal rise. Expression of these genes was detectable even after 96 h. These results suggest that it is feasible to test the expression of inner ear specific genes in explanted cochleae. PMID:15729138

  20. Reference Gene Selection for qRT-PCR Analysis in the Sweetpotato Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

    PubMed Central

    Li, Rumei; Xie, Wen; Wang, Shaoli; Wu, Qingjun; Yang, Nina; Yang, Xin; Pan, Huipeng; Zhou, Xiaomao; Bai, Lianyang; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

    2013-01-01

    Background Accurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of “classical” reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly Bemisia tabaci, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated. Results In this study, 15 candidate reference genes from B. tabaci were evaluated using two Excel-based algorithms geNorm and Normfinder under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, ribosomal protein L29 was the most stable reference gene. In contrast, the expression of elongation factor 1 alpha, peptidylprolyl isomerase A, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein 40 were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility. Conclusion Our finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in B. tabaci. PMID:23308130

  1. Establishment of a real-time RT-PCR for the determination of absolute amounts of IGF-I and IGF-II gene expression in liver and extrahepatic sites of the tilapia.

    PubMed

    Caelers, Antje; Berishvili, Giorgi; Meli, Marina L; Eppler, Elisabeth; Reinecke, Manfred

    2004-06-01

    We developed a one-tube two-temperature real-time RT-PCR that allows to absolutely quantify the gene expression of hormones using the standard curve method. As our research focuses on the expression of the insulin-like growth factors (IGFs) in bony fish, we established the technique for IGF-I and IGF-II using the tilapia (Oreochromis niloticus) as model species. As approach, we used primer extension adding a T7 phage polymerase promoter (21 nt) to the 5' end of the antisense primers. This procedure avoids the disadvantages arising from plasmids. Total RNA extracted from liver was subjected to conventional RT-PCR to create templates for in vitro transcription of IGF-I and IGF-II cRNA. Correct template sizes including the T7 promoter were verified (IGF-I: 91 nt; IGF-II: 94 nt). The PCR products were used to create IGF-I and IGF-II cRNAs which were quantified in dot blot by comparison with defined amounts of standardised kanamycin mRNA. Standardised threshold cycle (Ct) values for IGF-I and IGF-II mRNA were achieved by real-time RT-PCR and used to create standard curves. To allow sample normalisation the standard curve was also established for beta-actin as internal calibrator (template: 86 nt), and validation experiments were performed demonstrating similar amplification efficiencies for target and reference genes. Based on the standard curves, the absolute amounts of IGF-I and IGF-II mRNA were determined for liver (IGF-I: 8.90+/-1.90 pg/microg total RNA, IGF-II: 3.59+/-0.98 pg/microg total RNA) and extrahepatic sites, such as heart, kidney, intestine, spleen, gills, gonad, and brain considering the different lengths of cRNAs and mRNAs by correction factors. The reliability of the method was confirmed in additional experiments. The amplification of descending dilutions of cRNA and total liver RNA resulted in parallel slopes of the amplification curves. Furthermore, amplification plots of the standard cRNA and the IGF-I and IGF-II mRNAs showed signals starting at the

  2. Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis.

    PubMed

    Naveen, Kuttanda A; Singh, Shambhu Dayal; Kataria, Jag Mohan; Barathidasan, Rajamani; Dhama, Kuldeep

    2013-06-01

    Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level. PMID:23334380

  3. RT-PCR analysis of dystrophin mRNA in DND/BMD patients

    SciTech Connect

    Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D.

    1994-09-01

    Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.

  4. RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor).

    PubMed

    Yue, Constanze; Genersch, Elke

    2005-12-01

    Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding. PMID:16298989

  5. Proteomic Analysis and qRT-PCR Verification of Temperature Response to Arthrospira (Spirulina) platensis

    PubMed Central

    Huili, Wang; Xiaokai, Zhao; Meili, Lin; Dahlgren, Randy A.; Wei, Chen; Jaiopeng, Zhou; Chengyang, Xu; Chunlei, Jin; Yi, Xu; Xuedong, Wang; Li, Ding; Qiyu, Bao

    2013-01-01

    Arthrospira (Spirulina) platensis (ASP) is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C) using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328). The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones), energy metabolism (photosynthesis, respiratory electron transport), translation (ribosomal structure and biogenesis) and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and translation levels. The

  6. Lymphoid follicles of the ileal Peyer's patch of lambs express low levels of PrP, as demonstrated by quantitative real-time RT-PCR on microdissected tissue compartments, in situ hybridization and immunohistochemistry.

    PubMed

    Austbø, Lars; Espenes, Arild; Olsaker, Ingrid; Press, Charles McL; Skretting, Grethe

    2006-11-01

    The expression level of normal cellular prion protein (PrP(C)) is thought to influence the transmission of transmissible spongiform encephalopathies (TSEs) from the peripheral entry site to the site of pathological changes in the central nervous system. In many TSEs, the clinical disease is preceded by a period in which the agent accumulates in lymphoid organs, particularly in association with follicular dendritic cells of lymphoid follicles. As the probable route of entry of the TSE agent is via the gut, the expression profile of PrP was examined in well-developed gut-associated lymphoid tissue of lambs, the ileal Peyer's patch, by laser microdissection and real-time RT-PCR. Lymphoid follicles were found to have very low levels of expression, whilst highest levels were detected in the outer submucosa and the muscular layer. These findings were supported by in situ hybridization and immunohistochemistry, which showed specific labelling in nerve cells in ganglia of the submucosal (Meissner's) and myenteric (Auerbach's) plexi of the enteric nervous system. Based on the assumption that potential sites for conversion to the scrapie-related prion protein (PrP(Sc)) should display high levels of expression of PrP(C), this study suggests that the accumulation of PrP(Sc) in the lymphoid follicles of the Peyer's patch is not preceded by PrP conversion in the same tissue compartment. PMID:17030883

  7. Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time q...

  8. Nucleic acid extraction from polluted estuarine water for detection of viruses and bacteria by PCR and RT-PCR analysis.

    PubMed

    Petit, F; Craquelin, S; Guespin-Michel, J; Buffet-Janvresse, C

    1999-03-01

    We describe an extraction protocol for genomic DNA and RNA of both viruses and bacteria from polluted estuary water. This procedure was adapted to the molecular study of microflora of estuarine water where bacteria and viruses are found free, forming low-density biofilms, or intimately associated with organo-mineral particles. The sensitivity of the method was determined with seeded samples for RT-PCR and PCR analysis of viruses (10 virions/mL), and bacteria (1 colony-forming unit mL). We report an example of molecular detection of both poliovirus and Salmonella in the Seine estuary (France) and an approach to studying their association with organo-mineral particles. PMID:10209769

  9. Overexpression of vascular endothelial growth factor and its receptors in bronchial dypslasia demonstrated by quantitative RT-PCR analysis.

    PubMed

    Merrick, Daniel T; Haney, Jerry; Petrunich, Sheila; Sugita, Michio; Miller, York E; Keith, Robert L; Kennedy, Tim C; Franklin, Wilbur A

    2005-04-01

    Neoangiogenesis is required for the growth of invasive lung carcinoma, however, the role of angiogenesis in the progression of premalignant changes to carcinoma of the lung is less clear. We have evaluated vascular endothelial growth factor (VEGF) expression and microvessel densities (MVDs) in 62 bronchoscopic biopsies from normal, reactive (basal cell hyperplasia (BCH)) and dysplastic bronchial epithelium and in tissue from twenty-seven invasive lung carcinomas in an effort to demonstrate angiogenic activity in these preneoplastic lesions and determine whether it is associated with increased bronchial epithelial VEGF expression. MVDs and VEGF RNA expression measured by quantitative RT-PCR were found to be elevated in comparison to normal bronchial tissue in bronchial dysplasias and invasive lung carcinomas but not in basal cell hyperplasias. Immunohistochemical (IHC) analyses revealed that expression of VEGF arose predominantly from bronchial epithelium. ELISA analysis of lung tumor tissue showed that elevated VEGF protein expression correlated with VEGF RNA levels (r=0.59, p=0.004). Increased expression of VEGF RNA was also found in histologically normal bronchial mucosa from patients with either dysplasia at other sites or a history of heavy tobacco use suggesting a possible field effect in regard to the elaboration of VEGF. Furthermore, analysis of VEGF isoforms and VEGF receptors by semi-quantitative RT-PCR in dysplastic and invasive lesions revealed characteristic altered patterns of expression in dysplasia and early cancer as compared to normal tissue. These results indicate that angiogenesis develops early in lung carcinogenesis and is associated with overexpression of VEGF. PMID:15777969

  10. Expression of Two Basic mRNA Biomarkers in Peripheral Blood of Patients with Non-Small Cell Lung Cancer Detected by Real-Time RT-PCR, Individually and Simultaneously

    PubMed Central

    Karimi, Shirin; Mohamadnia, Abdolreza; Nadji, Seyed Alireza; Yadegarazari, Reza; Khosravi, Adnan; Bahrami, Naghmeh; Saidijam, Massoud

    2015-01-01

    Introduction: Although extensive research has been conducted on lung cancer markers, a singular clinically applicable marker has not been found yet. The objective of this study was to evaluate the sensitivity and the specificity of carcinoembryonic antigen (CEA) mRNA and lung-specific X protein (LUNX) mRNA biomarkers in peripheral blood to detect lung cancer individually and simultaneously. Methods: Thirty patients affected by lung cancer and 30 healthy individuals were studied in this research. Three vials of cDNA were made from each sample after taking peripheral blood samples and extracting total RNA. Each sample was examined by the real-time RT-PCR technique. The result from each vial was then compared with the sensitivity of overall marker. Results: The CEA mRNA was positive in 24 out of 30 lung cancer patients. Hence, its sensitivity was determined at 80%, differing significantly from that observed in healthy individuals, where 11 positive cases were seen. The overall sensitivity of this marker was significantly associated with positivity in vials 2 and 3 but not in vial 1. The LUNX mRNA was positive in 21 out of 30 patients, indicating 70% sensitivity. This finding significantly differed from that in healthy individuals. The overall sensitivity of this marker was significantly associated with positivity in vials 1 and 3, but not in vial 2. In 93.3% of the patients, at least one positive marker was observed. Conclusion: The mentioned mRNA could be suggested as sensitive and specific markers in peripheral blood for primary diagnosis of lung cancer. PMID:25605485

  11. Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony.

    PubMed

    Blanchard, Philippe; Ribière, Magali; Celle, Olivier; Lallemand, Perrine; Schurr, Frank; Olivier, Violaine; Iscache, Anne Laure; Faucon, Jean Paul

    2007-04-01

    A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9 x 10(10) CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 x 10(6) CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9 x 10(13) CBPV copies) than in forager, drones and house bees (up to 3.4 x 10(6) CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection. PMID:17166598

  12. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  13. Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia

    PubMed Central

    Pagani, Ilaria S.; Spinelli, Orietta; Mattarucchi, Elia; Pirrone, Cristina; Pigni, Diana; Amelotti, Elisabetta; Lilliu, Silvia; Boroni, Chiara; Intermesoli, Tamara; Giussani, Ursula; Caimi, Luigi; Bolda, Federica; Baffelli, Renata; Candi, Eleonora; Pasquali, Francesco; Lo Curto, Francesco; Lanfranchi, Arnalda; Porta, Fulvio; Rambaldi, Alessandro; Porta, Giovanni

    2014-01-01

    Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy. PMID:25594053

  14. Real-time analysis keratometer

    NASA Technical Reports Server (NTRS)

    Adachi, Iwao P. (Inventor); Adachi, Yoshifumi (Inventor); Frazer, Robert E. (Inventor)

    1987-01-01

    A computer assisted keratometer in which a fiducial line pattern reticle illuminated by CW or pulsed laser light is projected on a corneal surface through lenses, a prismoidal beamsplitter quarterwave plate, and objective optics. The reticle surface is curved as a conjugate of an ideal corneal curvature. The fiducial image reflected from the cornea undergoes a polarization shift through the quarterwave plate and beamsplitter whereby the projected and reflected beams are separated and directed orthogonally. The reflected beam fiducial pattern forms a moire pattern with a replica of the first recticle. This moire pattern contains transverse aberration due to differences in curvature between the cornea and the ideal corneal curvature. The moire pattern is analyzed in real time by computer which displays either the CW moire pattern or a pulsed mode analysis of the transverse aberration of the cornea under observation, in real time. With the eye focused on a plurality of fixation points in succession, a survey of the entire corneal topography is made and a contour map or three dimensional plot of the cornea can be made as a computer readout in addition to corneal radius and refractive power analysis.

  15. IGF-I is distinctly located in the bony fish pituitary as revealed for Oreochromis niloticus, the Nile tilapia, using real-time RT-PCR, in situ hybridisation and immunohistochemistry.

    PubMed

    Eppler, Elisabeth; Shved, Natallia; Moret, Olivier; Reinecke, Manfred

    2007-01-01

    In bony fish, IGF-I released from the liver under the control of pituitary GH is the main endocrine regulator of growth, maintenance and development, and the amount of circulating IGF-I regulates synthesis and release of GH. In mammals and amphibia, evidence indicates that anterior pituitary endocrine cells also contain IGF-I. However, only preliminary and conflicting data exist on IGF-I gene expression in bony fish pituitary. Thus, we investigated the presence of IGF-I in the tilapia (Oreochromis niloticus) pituitary by quantitative real-time RT-PCR, in situ hybridisation and immunohistochemistry. The absolute amount of IGF-I mRNA in the whole pituitary (7.4+/-3.3 x 10(-3)pg/microg total RNA) was 1000-times lower than in liver (7.5+/-3.1 pg/microg total RNA). IGF-I peptide occurred in both neuro- and adenohypophysis but IGF-I gene expression was mainly restricted to the adenohypophysis. In the neurohypophysis, only few cells, probably pituicytes, contained IGF-I mRNA whereas IGF-I peptide was found also in numerous axons in the pars nervosa. In the adenohypophysis, both IGF-I mRNA and peptide were present in the majority of ACTH cells in all individuals investigated. In alpha-MSH cells, only IGF-I mRNA but no IGF-I peptide was detected likely suggesting an immediate release of IGF-I after synthesis. IGF-I mRNA and peptide were further observed in GH cells but their presence showed pronounced inter-individual differences likely due to the physiological, e.g., nutritional, status of the individual. IGF-I released from the GH cells may serve as auto/paracrine mediator of a negative feedback mechanism in addition to liver-derived endocrine IGF-I. Generally, the constitutive synthesis of IGF-I in ACTH cells and the varying content in GH and alpha-MSH cells suggest particular roles for IGF-I. Local IGF-I may regulate synthesis and release of pituitary hormones in an autocrine and/or paracrine manner as well as prevent apoptosis and stimulate proliferation of endocrine

  16. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    PubMed

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. PMID:26611227

  17. Application of cDNA microarray technology to in vitro toxicology and the selection of genes for a real-time RT-PCR-based screen for oxidative stress in Hep-G2 cells.

    PubMed

    Morgan, Kevin T; Ni, Hong; Brown, H Roger; Yoon, Lawrence; Qualls, Charles W; Crosby, Lynn M; Reynolds, Randall; Gaskill, Betty; Anderson, Steven P; Kepler, Thomas B; Brainard, Tracy; Liv, Nik; Easton, Marilyn; Merrill, Christine; Creech, Don; Sprenger, Dirk; Conner, Gary; Johnson, Paul R; Fox, Tony; Sartor, Maureen; Richard, Erika; Kuruvilla, Sabu; Casey, Warren; Benavides, Gina

    2002-01-01

    Large-scale analysis of gene expression using cDNA microarrays promises the rapid detection of the mode of toxicity for drugs and other chemicals. cDNA microarrays were used to examine chemically induced alterations of gene expression in HepG2 cells exposed to a diverse group of toxicants at an equitoxic exposure concentration. The treatments were ouabain (43 microM), lauryl sulfate (260 microM), dimethylsulfoxide (1.28 M), cycloheximide (62.5 microM), tolbutamide (12.8 mM), sodium fluoride (3 mM), diethyl maleate (1.25 mM), buthionine sulfoximine (30 mM), potassium bromate (2.5 mM), sodium selenite (30 microM), alloxan (130 mM), adriamycin (40 microM), hydrogen peroxide (4 mM), and heat stress (45 degrees C x 30 minutes). Patterns of gene expression were correlated with morphologic and biochemical indicators of toxicity. Gene expression responses were characteristically different for each treatment. Patterns of expression were consistent with cell cycle arrest, DNA damage, diminished protein synthesis, and oxidative stress. Based upon these results, we concluded that gene expression changes provide a useful indicator of oxidative stress, as assessed by the GSH:GSSG ratio. Under the conditions of this cell culture test system, oxidative stress upregulated 5 genes, HMOX1, p21(waf1/cip1), GCLM, GR, TXNR1 while downregulating CYP1A1 and TOPO2A. Primers and probes for these genes were incorporated into the design of a 7-gene plate for RT-PCR. The plate design permitted statistical analysis and allowed clear discrimination between chemicals inducing oxidative vs nonoxidative stress. A simple oxidative stress score (0-1), based on the responses by the 7 genes (including p-value) on the RT-PCR plate, was correlated with the GSH:GSSG ratio using linear regression and ranking (Pearson product) procedures. These analyses yielded correlation coefficients of 0.74 and 0.87, respectively, for the treatments tested (when 1 outlier was excluded), indicating a good correlation

  18. Identification of commonly dysregulated genes in colorectal cancer by integrating analysis of RNA-Seq data and qRT-PCR validation.

    PubMed

    Xiao, W H; Qu, X L; Li, X M; Sun, Y L; Zhao, H X; Wang, S; Zhou, X

    2015-05-01

    The progression of colorectal cancer (CRC) is a multistep process and metastatic CRC is always incurable; consequently, CRC is the leading cause of cancer-related deaths. There is therefore an urgent need for identifying useful biomarkers with enough sensitivity and specificity to detect this disease at early stages, which will significantly reduce the mortality for this malignancy. In this study, we performed an integrating analysis of different RNA-Seq data sets to find new candidate biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of CRC carcinogenesis. We identified 883 differentially expressed genes (DEGs) across the studies between CRC and normal control (NC) tissues by combining five RNA-Seq data sets. Gene function analysis revealed high correlation with carcinogenesis. The top 10 most significantly DEGs were further evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in both rectal cancer (RC) and colon cancer (CC), and the results matched well with integrating data, suggesting that the method of integrating analysis of different RNA-seq data sets is acceptable. Therefore, integrating analysis of different RNA-seq data sets may be a useful way to overcome the limitation of small sample size in a single RNA-seq study. In addition, our study showed that some genes, such as SIM2, ADAMTS6, FOXD4L4 and DNAH5, may have an important role in the development of CRC, which could be applied for diagnosis, prognosis and as therapy for this malignancy. Our findings would also help to understand the pathology of CRC. PMID:25908452

  19. Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis

    PubMed Central

    Chen, Yongxin; Gelfond, Jonathan AL; McManus, Linda M; Shireman, Paula K

    2009-01-01

    Background MicroRNAs (miRNAs) have critical functions in various biological processes. MiRNA profiling is an important tool for the identification of differentially expressed miRNAs in normal cellular and disease processes. A technical challenge remains for high-throughput miRNA expression analysis as the number of miRNAs continues to increase with in silico prediction and experimental verification. Our study critically evaluated the performance of a novel miRNA expression profiling approach, quantitative RT-PCR array (qPCR-array), compared to miRNA detection with oligonucleotide microchip (microarray). Results High reproducibility with qPCR-array was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array. Conclusion Our studies demonstrated high reproducibility of TaqMan qPCR-array. Comparison between different reverse transcription reactions and qPCR-arrays performed on different days indicated that reverse transcription reactions did not introduce significant variation in the results. The use of cDNA pre-amplification increased the sensitivity of miRNA detection. Although there was variability associated with pre-amplification in low abundance miRNAs, the latter did not involve any systemic bias in the estimation of miRNA expression. Comparison between microarray and q

  20. Differentiation of citrus Hop stunt viroid variants by real-time RT-PCR and high resolution melting analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viroids are small, infectious, single-stranded RNA molecules that cause several important citrus diseases. Viroids are transmitted primarily in budwood, however, spread can also occur mechanically on pruning equipment, budding knives, hedging and topping equipment. Exocortis and cachexia are two we...

  1. Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

    PubMed

    Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

    2016-01-01

    Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures. PMID:26983735

  2. Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the...

  3. Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples.

    PubMed

    Ummul Haninah, A; Vasan, S S; Ravindran, T; Chandru, A; Lee, H L; Shamala Devi, S

    2010-12-01

    This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999. PMID:21399603

  4. Real-time flutter analysis

    NASA Technical Reports Server (NTRS)

    Walker, R.; Gupta, N.

    1984-01-01

    The important algorithm issues necessary to achieve a real time flutter monitoring system; namely, the guidelines for choosing appropriate model forms, reduction of the parameter convergence transient, handling multiple modes, the effect of over parameterization, and estimate accuracy predictions, both online and for experiment design are addressed. An approach for efficiently computing continuous-time flutter parameter Cramer-Rao estimate error bounds were developed. This enables a convincing comparison of theoretical and simulation results, as well as offline studies in preparation for a flight test. Theoretical predictions, simulation and flight test results from the NASA Drones for Aerodynamic and Structural Test (DAST) Program are compared.

  5. Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

    PubMed

    Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

    2015-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

  6. Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

    PubMed Central

    Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.

    2015-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

  7. A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR

    PubMed Central

    Sun, Hao; Olsen, Tim; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A.; Brenner, David J.; Lin, Qiao

    2015-01-01

    Gene expression analysis at the single-cell level is critical to understanding variations among cells in heterogeneous populations. Microfluidic reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is well suited to gene expression assays of single cells. We present a microfluidic approach that integrates all functional steps for RT-qPCR of a single cell, including isolation and lysis of the cell, as well as purification, reverse transcription and quantitative real-time PCR of messenger RNA in the cell lysate. In this approach, all reactions in the multi-step assay of a single lysed cell can be completed on microbeads, thereby simplifying the design, fabrication and operation of the microfluidic device, as well as facilitating the minimization of sample loss or contamination. In the microfluidic device, a single cell is isolated and lysed; mRNA in the cell lysate is then analyzed by RT-qPCR using primers immobilized on microbeads in a single microchamber whose temperature is controlled in closed loop via an integrated heater and temperature sensor. The utility of the approach was demonstrated by the analysis of the effects of the drug (methyl methanesulfonate, MMS) on the induction of the cyclin-dependent kinase inhibitor 1a (CDKN1A) in single human cancer cells (MCF-7), demonstrating the potential of our approach for efficient, integrated single-cell RT-qPCR for gene expression analysis. PMID:25883782

  8. Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR

    PubMed Central

    Goerke, Christiane; Bayer, Manfred G.; Wolz, Christiane

    2001-01-01

    Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated α-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 104 (gyr) and 103 (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples. PMID:11238208

  9. Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus.

    PubMed

    Kong, Lih Ling; Omar, Abdul Rahman; Hair Bejo, Mohd; Ideris, Aini; Tan, Sheau Wei

    2009-11-01

    A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P<0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV. PMID:19591873

  10. Radiolabeled semi-quantitative RT-PCR assay for the analysis of alternative splicing of interleukin genes.

    PubMed

    Shakola, Felitsiya; Byrne, Stephen; Javed, Kainaat; Ruggiu, Matteo

    2014-01-01

    Alternative splicing evolved as a very efficient way to generate proteome diversity from a limited number of genes, while at the same time modulating posttranscriptional events of gene expression-such as stability, turnover, subcellular localization, binding properties, and general activity of both mRNAs and proteins. Since the vast majority of human genes undergo alternative splicing, it comes to no surprise that interleukin genes also show extensive alternative splicing. In fact, there is a growing body of evidence indicating that alternative splicing plays a central role in modulating the pleiotropic functions of cytokines, and aberrant expression of alternatively spliced interleukin mRNAs has been linked to disease. However, while several interleukin splice variants have been described, their function is still poorly understood. This is particularly relevant, since alternatively spliced cytokine isoforms can act both as disease biomarkers and as candidate entry points for therapeutic intervention. In this chapter we describe a protocol that uses radiolabeled semi-quantitative RT-PCR to efficiently detect, analyze, and quantify alternative splicing patterns of cytokine genes. PMID:24908320

  11. Evaluation of reference genes for quantitative RT-PCR in Lolium perenne

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time RT-PCR provides an important tool for analyzing gene expression if proper internal standards are used. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR in perennial ryegrass (Lolium perenne L.) during plant developmen...

  12. Absolute mRNA quantification of Pseudomonas fluorescens Pf-5 by qRT-PCR using universal RNA controls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time quantitative RT-PCR is considered as standard for gene expression and mRNA estimate. As a calibration standard, a conserved control gene such as housekeeping genes is commonly used for data normalization and analysis. A significant problem has been observed with increased applications; h...

  13. 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.

  14. Development and evaluation of a real-time RT-PCR assay for the detection of Ebola virus (Zaire) during an Ebola outbreak in Guinea in 2014-2015.

    PubMed

    Dedkov, V G; Magassouba, N' F; Safonova, M V; Deviatkin, A A; Dolgova, A S; Pyankov, O V; Sergeev, A A; Utkin, D V; Odinokov, G N; Safronov, V A; Agafonov, A P; Maleev, V V; Shipulin, G A

    2016-02-01

    In early February 2014, an outbreak of the Ebola virus disease caused by Zaire ebolavirus (EBOV) occurred in Guinea; cases were also recorded in other West African countries with a combined population of approximately 25 million. A rapid, sensitive and inexpensive method for detecting EBOV is needed to effectively control such outbreak. Here, we report a real-time reverse-transcription PCR assay for Z. ebolavirus detection used by the Specialized Anti-epidemic Team of the Russian Federation during the Ebola virus disease prevention mission in the Republic of Guinea. The analytical sensitivity of the assay is 5 × 10(2) viral particles per ml, and high specificity is demonstrated using representative sampling of viral, bacterial and human nucleic acids. This assay can be applied successfully for detecting the West African strains of Z. ebolavirus as well as on strains isolated in the Democratic Republic of the Congo in 2014. PMID:26597659

  15. Identification by a Digital Gene Expression Displayer (DGED) and test by RT-PCR analysis of new mRNA candidate markers for colorectal cancer in peripheral blood.

    PubMed

    Lauriola, Mattia; Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone; Montroni, Isacco; Manaresi, Alessio; Zattoni, Davide; Rivetti, Stefano; Mattei, Gabriella; Coppola, Domenico; Strippoli, Pierluigi; Taffurelli, Mario; Solmi, Rossella

    2010-08-01

    Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the

  16. Real-Time Quantitative RT-PCR of Defense-Associated Gene Transcripts of Rhizoctonia solani-Infected Bean Seedlings in Response to Inoculation with a Nonpathogenic Binucleate Rhizoctonia Isolate.

    PubMed

    Wen, Kui; Seguin, Philippe; St-Arnaud, Marc; Jabaji-Hare, Suha

    2005-04-01

    ABSTRACT Certain isolates of nonpathogenic binucleate Rhizoctonia spp. (np-BNR) are effective biocontrol agents against seedling root rot and damping-off. Inoculation of bean seed with np-BNR strain 232-CG at sowing reduced disease symptoms in bean (Phaseolus vulgaris) seedlings caused by R. solani. Molecular analyses of the spatial expression of three defense-associated genes were carried out using real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays. This method allowed accurate quantitative evaluation of transcript levels of pG101 encoding for 1,3-beta-D-glucanase, gPAL1 encoding for phenylalanine ammonia lyase, and CHS17 encoding for chalcone synthase in 1- and 2-week-old bean seedlings that were inoculated simultaneously with np-BNR and infected with R. solani, and in seedlings that were singly inoculated with either fungi or not inoculated. In the seedlings that were infected with R. solani only, results revealed that, following infection, activation of all defense-associated gene transcripts was achieved with significant increases ranging from 7- to 40-fold greater than the control, depending on the defense gene and tissue analyzed. Seedlings that were treated with np-BNR and infected with R. solani had expression similar to those that were treated with np-BNR only, but the levels were significantly down-regulated compared with those that were infected with R. solani only. These findings indicate that disease suppression by np-BNR isolate is not correlated to pG101, gPAL1, and CHS17 gene activation. PMID:18943035

  17. APPLICATION OF CDNA MICROARRAY TECHNOLOGY TO IN VITRO TOXICOLOGY AND THE SELECTION OF GENES FOR A REAL TIME RT-PCR-BASED SCREEN FOR OXIDATIVE STRESS IN HEP-G2 CELLS

    EPA Science Inventory

    Large-scale analysis of gene expression using cDNA microarrays promises the
    rapid detection of the mode of toxicity for drugs and other chemicals. cDNA
    microarrays were used to examine chemically-induced alterations of gene
    expression in HepG2 cells exposed to oxidative ...

  18. Real time analysis of voiced sounds

    NASA Technical Reports Server (NTRS)

    Hong, J. P. (Inventor)

    1976-01-01

    A power spectrum analysis of the harmonic content of a voiced sound signal is conducted in real time by phase-lock-loop tracking of the fundamental frequency, (f sub 0) of the signal and successive harmonics (h sub 1 through h sub n) of the fundamental frequency. The analysis also includes measuring the quadrature power and phase of each frequency tracked, differentiating the power measurements of the harmonics in adjacent pairs, and analyzing successive differentials to determine peak power points in the power spectrum for display or use in analysis of voiced sound, such as for voice recognition.

  19. RT-PCR-DGGE Analysis to Elucidate the Dominant Bacterial Species of Industrial Spanish-Style Green Table Olive Fermentations.

    PubMed

    Benítez-Cabello, Antonio; Bautista-Gallego, Joaquín; Garrido-Fernández, Antonio; Rantsiou, Kalliopi; Cocolin, Luca; Jiménez-Díaz, Rufino; Arroyo-López, Francisco N

    2016-01-01

    This paper describes the dominant bacterial species metabolically active through the industrial production of Spanish-style Manzanilla and Gordal olives. For this purpose, samples (brines and fruits) obtained at 0, 15, and 90 fermentation days were analyzed by a culture-independent approach to determine viable cells by reverse transcription of RNA and further PCR-DGGE analysis, detecting at least 7 different species. Vibrio vulnificus, Lactobacillus plantarum group, and Lactobacillus parafarraginis were present in samples from both cultivars; Lactobacillus sanfranciscensis and Halolactobacillus halophilus were detected only in Gordal samples, while Staphylococcus sp. was exclusively found at the onset of Manzanilla fermentations. Physicochemical data showed a typical fermentation profile while scanning electron microscopy confirmed the in situ biofilm formation on the olive epidermis. Different Bacillus, Staphylococcus, and Enterococcus species, not detected during the fermentation process, were also found in the solid marine salt used by the industry for preparation of brines. Elucidation of these non-lactic acid bacteria species role during fermentation is then an appealingly challenge, particularly regarding safety issues. PMID:27582739

  20. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

    Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

  1. A bacterial community analysis using reverse transcription (RT) PCR which detects the bacteria with high activity in a wastewater treatment reactor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research used reverse transcription polymerase chain reaction (RT-PCR) method to help detect active bacteria in a single-tank deammonification reactor combining partial nitritation and anammox. The single-tank aerobic deammonification reactor effectively removed the ammonia in anaerobically di...

  2. Circulating Tumor Cells (CTCs) Detected by RT-PCR and Its Prognostic Role in Gastric Cancer: A Meta-Analysis of Published Literature

    PubMed Central

    Cheng, Boran; Chen, Fangfang; Wang, Zhenmeng; Chen, Yuanyuan; Wang, You; Xiong, Bin

    2014-01-01

    Objective The prognostic significance of circulating tumor cells (CTCs) is controversial in gastric cancer (GC). We performed a meta-analysis of available studies to assess its prognostic value detected by RT-PCR for patients diagnosed with GC. Methods EMBase, PubMed, Ovid, Web of Science, Cochrane library and Google Scholar database search was conducted on all studies reporting the outcomes of interest. The studies were set up according to the inclusion/exclusion criteria. Meta-analysis was performed by using a random-effects model; hazard ratio (HR), risk ratio (RR) and their 95% confidence intervals (95% CIs) were set as effect measures. The information about trial design, results from the data was independently extracted. Heterogeneity of the studies was tested for each pooled analysis. Results Nineteen studies published matched the selection criteria and were included in this meta-analysis. CTCs positivity was significantly associated with poor relapse free survival (RFS) (HR 2.42, 95% CI: [1.94–3.02]; P<0.001) and poor overall survival (OS) (HR 2.42, 95% CI: [1.94–3.02]; P<0.001). CTCs positivity were also significantly associated with regional lymph nodes (RLNs) metastasis (RR 1.42, 95% CI: [1.20–1.68]; p<0.0001), depth of infiltration (RR 1.51, 95% CI: [1.27–1.79]; p<0.0001), vascular invasion (RR  = 1.43, 95% CI: [1.18–1.74], p = 0.0002) and TNM stage(I,II versus III) (RR 0.63, 95% CI [0.48–0.84]; p = 0.001). Conclusion Preoperative CTCs positivity indicates poor prognosis in patients with gastric cancer, and associated with poor clinicopathological prognostic factors. PMID:24901848

  3. A robust standard for absolute mRNA quantification of Saccharomyces cerevisiae by qRT-PCR using the universal RNA controls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently developed universal external RNA controls allow comparison of expression data generated from microarray and real time qRT-PCR, including SYBR Green and TaqMan-probe based chemistry. It provides reliable controls for data normalization and analysis. In this study, we further developed stra...

  4. Trends and advances in food analysis by real-time polymerase chain reaction.

    PubMed

    Salihah, Nur Thaqifah; Hossain, Mohammad Mosharraf; Lubis, Hamadah; Ahmed, Minhaz Uddin

    2016-05-01

    Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling. PMID:27407185

  5. Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions

    PubMed Central

    Letting, Heidi; Hadrup, Niels; Hass, Ulla; Vinggaard, Anne Marie

    2015-01-01

    In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA. PMID:25825680

  6. Non-Invasive Analysis of Recombinant mRNA Stability in Escherichia coli by a Combination of Transcriptional Inducer Wash-Out and qRT-PCR

    PubMed Central

    Kucharova, Veronika; Strand, Trine Aakvik; Almaas, Eivind; Naas, Adrian E.; Brautaset, Trygve; Valla, Svein

    2013-01-01

    mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research. PMID:23840466

  7. Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae).

    PubMed

    Shi, Caihua; Yang, Fengshan; Zhu, Xun; Du, Erxia; Yang, Yuting; Wang, Shaoli; Wu, Qingjun; Zhang, Youjun

    2016-01-01

    The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga. PMID:27399679

  8. Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae)

    PubMed Central

    Shi, Caihua; Yang, Fengshan; Zhu, Xun; Du, Erxia; Yang, Yuting; Wang, Shaoli; Wu, Qingjun; Zhang, Youjun

    2016-01-01

    The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga. PMID:27399679

  9. Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay.

    PubMed

    Rubio-Guerri, Consuelo; Melero, Mar; Rivera-Arroyo, Belén; Bellière, Edwige Nina; Crespo, Jose Luis; García-Párraga, Daniel; Esperón, Fernando; Sánchez-Vizcaíno, Jose Manuel

    2013-07-26

    A highly sensitive and specific real-time (rt) RT-PCR assay has been developed for rapid, simultaneous detection of three strains of cetacean morbillivirus (CeMV). In this assay, two PCR primers and a hydrolysis probe from a commercially available Universal Probe Library (UPL) are used to amplify a highly conserved region within the fusion protein gene. RT-PCR is carried out on the same sample using two primer sets in parallel: one set detects the more virulent strains, dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), and the other set detects the least virulent and least common strain, pilot whale morbillivirus (PWMV). Sensitivity analysis using dilute samples containing purified DMV, PMV and PWMV showed that viral RNA detection limits in this UPL RT-PCR assay were lower than in a conventional RT-PCR assay. Our method gave no amplification signal with field samples positive for viruses related and unrelated to CeMV, such as phocine distemper virus (PDV). The reliability and robustness of the UPL RT-PCR assay were verified using tissue samples previously analyzed by conventional methods, as well as a panel of clinical samples suspected of containing CeMV. Using the UPL RT-PCR assay, we were able to associate DMV with a mass stranding of striped dolphins in the Spanish Mediterranean in 2011 with greater reliability than was possible with a conventional RT-PCR method. These results suggest that this UPL RT-PCR method is more sensitive and specific than the conventional approach, and that it may be an affordable and rapid test for routine diagnosis of three CeMV strains. PMID:23380457

  10. Real-Time Principal-Component Analysis

    NASA Technical Reports Server (NTRS)

    Duong, Vu; Duong, Tuan

    2005-01-01

    A recently written computer program implements dominant-element-based gradient descent and dynamic initial learning rate (DOGEDYN), which was described in Method of Real-Time Principal-Component Analysis (NPO-40034) NASA Tech Briefs, Vol. 29, No. 1 (January 2005), page 59. To recapitulate: DOGEDYN is a method of sequential principal-component analysis (PCA) suitable for such applications as data compression and extraction of features from sets of data. In DOGEDYN, input data are represented as a sequence of vectors acquired at sampling times. The learning algorithm in DOGEDYN involves sequential extraction of principal vectors by means of a gradient descent in which only the dominant element is used at each iteration. Each iteration includes updating of elements of a weight matrix by amounts proportional to a dynamic initial learning rate chosen to increase the rate of convergence by compensating for the energy lost through the previous extraction of principal components. In comparison with a prior method of gradient-descent-based sequential PCA, DOGEDYN involves less computation and offers a greater rate of learning convergence. The sequential DOGEDYN computations require less memory than would parallel computations for the same purpose. The DOGEDYN software can be executed on a personal computer.

  11. CRANS - CONFIGURABLE REAL-TIME ANALYSIS SYSTEM

    NASA Technical Reports Server (NTRS)

    Mccluney, K.

    1994-01-01

    In a real-time environment, the results of changes or failures in a complex, interconnected system need evaluation quickly. Tabulations showing the effects of changes and/or failures of a given item in the system are generally only useful for a single input, and only with regard to that item. Subsequent changes become harder to evaluate as combinations of failures produce a cascade effect. When confronted by multiple indicated failures in the system, it becomes necessary to determine a single cause. In this case, failure tables are not very helpful. CRANS, the Configurable Real-time ANalysis System, can interpret a logic tree, constructed by the user, describing a complex system and determine the effects of changes and failures in it. Items in the tree are related to each other by Boolean operators. The user is then able to change the state of these items (ON/OFF FAILED/UNFAILED). The program then evaluates the logic tree based on these changes and determines any resultant changes to other items in the tree. CRANS can also search for a common cause for multiple item failures, and allow the user to explore the logic tree from within the program. A "help" mode and a reference check provide the user with a means of exploring an item's underlying logic from within the program. A commonality check determines single point failures for an item or group of items. Output is in the form of a user-defined matrix or matrices of colored boxes, each box representing an item or set of items from the logic tree. Input is via mouse selection of the matrix boxes, using the mouse buttons to toggle the state of the item. CRANS is written in C-language and requires the MIT X Window System, Version 11 Revision 4 or Revision 5. It requires 78K of RAM for execution and a three button mouse. It has been successfully implemented on Sun4 workstations running SunOS, HP9000 workstations running HP-UX, and DECstations running ULTRIX. No executable is provided on the distribution medium; however

  12. Expression analysis of Type 1 and 2 Metallothionein genes in Rape (Brassica napus L.) during short-term stress using sqRT-PCR analysis.

    PubMed

    Abdelmigid, Hala M

    2016-03-01

    With the extent of contamination in water and soil today, possibility of presence of toxic heavy metals in plants in everyday life can not be ruled out. In this context, understanding the influence of exogenenous factors on such plants gains importance. Here, we investigated expression of metallothioneins genes MT1 and MT2 in Rape Brassica napus L. as representatives of MT gene type 1, type 2 (BnMT1 and BnMT2), respectively to explore such influence, if there any. Seedlings of 7-day-old were exposed to various exogenous factors including plant hormones, heavy metals, abiotic and biotic stresses. The basal expression levels of two BnMT genes were determined using water-treated samples (control). Each treatment was replicated 3 times for statistical validity. SPSS computer software was used for statistical analyses. Expression profiles of BnMT1 and BnMT2 were generated by semi-quantitative RT-PCR (sqRT-PCR) to monitor stress-response gene expression of both genes. The BnMT1 and BnMT2 genes were expressed at the same level in control samples. In general, BnMT1 gene was better expressed in most treatments compared to BnMT2 throughout the 48 h experimental period. Moreover, BnMT2 expression was not affected by heavy metal stress. The results provide considerable insights into the molecular mechanism of MTs responses to environmental stress in B. napus which can be utilized for future plant manipulations to improve its ability to accumulate higher metal concentration from the soil. PMID:27145635

  13. Real-time Forensic Disaster Analysis

    NASA Astrophysics Data System (ADS)

    Wenzel, F.; Daniell, J.; Khazai, B.; Mühr, B.; Kunz-Plapp, T.; Markus, M.; Vervaeck, A.

    2012-04-01

    The Center for Disaster Management and Risk Reduction Technology (CEDIM, www.cedim.de) - an interdisciplinary research center founded by the German Research Centre for Geoscience (GFZ) and Karlsruhe Institute of Technology (KIT) - has embarked on a new style of disaster research known as Forensic Disaster Analysis. The notion has been coined by the Integrated Research on Disaster Risk initiative (IRDR, www.irdrinternational.org) launched by ICSU in 2010. It has been defined as an approach to studying natural disasters that aims at uncovering the root causes of disasters through in-depth investigations that go beyond the reconnaissance reports and case studies typically conducted after disasters. In adopting this comprehensive understanding of disasters CEDIM adds a real-time component to the assessment and evaluation process. By comprehensive we mean that most if not all relevant aspects of disasters are considered and jointly analysed. This includes the impact (human, economy, and infrastructure), comparisons with recent historic events, social vulnerability, reconstruction and long-term impacts on livelihood issues. The forensic disaster analysis research mode is thus best characterized as "event-based research" through systematic investigation of critical issues arising after a disaster across various inter-related areas. The forensic approach requires (a) availability of global data bases regarding previous earthquake losses, socio-economic parameters, building stock information, etc.; (b) leveraging platforms such as the EERI clearing house, relief-web, and the many sources of local and international sources where information is organized; and (c) rapid access to critical information (e.g., crowd sourcing techniques) to improve our understanding of the complex dynamics of disasters. The main scientific questions being addressed are: What are critical factors that control loss of life, of infrastructure, and for economy? What are the critical interactions

  14. Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Anoectochilus roxburghii.

    PubMed

    Zhang, Gang; Zhao, Mingming; Song, Chao; Luo, Anxiong; Bai, Jianfa; Guo, Shunxing

    2012-05-01

    Accurate quantification of transcript profiling with quantitative real time polymerase chain reaction (qRT-PCR) relies on the reliable normalization of an appropriate reference gene. This study reported the identification and validation of nine reference genes, including β-tubulin (β-TUB), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), actin 1/2(ACT-1 and ACT-2), 18S rRNA, and 26S rRNA, from Anoectochilus roxburghii (Wall.) Lindl., a valuable herb remedy widely used for various diseases treatment in traditional Chinese medicine. Transcriptional levels of the candidate reference genes were examined using qRT-PCR analysis and revealed differential expression of the genes in the leaf, stem, root, flower, and peduncle tissues. The relative quantities data were subjected to geNorm software for ranking the expression stability of the reference genes and the results showed that EF-1β and ACT-2 were the two best stable genes whereas GAPDH and 26S rRNA did not favor normalization of qRT-PCR in these tissues. The expression pattern of a squalene synthase encoding gene (SS) was also determined in parallel. The analyses were in great consistency when the qRT-PCR data was normalized to the expression of each or both of EF-1β and ACT-2 as the internal control, further confirming the reliability of EF-1β and ACT-2 as the best internal control. The present study provided the first important clues for accurate data normalization in transcript profiling in A. roxburghii, which will be essential to further functional genomics study in the valuable medicinal plant. PMID:22201024

  15. Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

  16. Group specific quantitative real-time polymerase chain reaction (qRT-PCR) analysis of methanogenic archaea in stored swine manure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consolidated storage of swine manure is associated with the production of a variety of odors and emissions which result from anaerobic digestion of materials present in the manure. Methanogenic archaea are a diverse group of anaerobic microorganisms responsible for the production of methane. In th...

  17. Rapid differentiation of citrus Hop stunt viroid variants by use of real-time RT-PCR and high resolution melting analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The RNA genome of Hop stunt viroid (HSVd) contains five to six nucleotides in a variable (V) domain, called the cachexia expression motif, which is associated with pathogenic and non-pathogenic variants in citrus. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological i...

  18. WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

    NASA Technical Reports Server (NTRS)

    Parra, Macarena; Almeida, Eduardo; Boone, Travis; Jung, Jimmy; Schonfeld, Julie

    2014-01-01

    The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be

  19. Gene expression analysis by quantitative real-time PCR for floral tissues.

    PubMed

    Bustamante, Mariana; Jin, Jian; Casagran, Oriol; Nolan, Tania; Riechmann, José Luis

    2014-01-01

    Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler(®) 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment. PMID:24395270

  20. Combination of reverse transcription real-time polymerase chain reaction and antigen capture enzyme-linked immunosorbent assay for the detection of animals persistently infected with Bovine viral diarrhea virus.

    PubMed

    Yan, Lifang; Zhang, Shuping; Pace, Lanny; Wilson, Floyd; Wan, Henry; Zhang, Michael

    2011-01-01

    Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle. A successful control program requires early detection and removal of persistently infected (PI) animals. The objective of the current study was to develop, validate, and apply a cost-effective testing scheme for the detection of BVDV PI animals in exposed herds. Pooled samples were screened by using a real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and individual positives were identified with an antigen capture enzyme-linked immunosorbent assay (ACE). The detection limits of the optimized real-time RT-PCR were 10 and 100 RNA copies per reaction for BVDV-1 and BVDV-2, respectively. The semiquantitative results of real-time RT-PCR and ACE or real-time RT-PCR and immunohistochemistry were moderately correlated. The threshold cycle of real-time RT-PCR performed on pooled samples was significantly correlated with the pool size (R(2)  =  0.993). The least-cost pool sizes were 50 at a prevalence of 0.25-0.5% and 25 at a prevalence of 0.75-2.0%. By using the combined real-time RT-PCR and ACE procedure, 111 of 27,932 samples (0.4%) tested positive for BVDV. At this prevalence, cost reduction associated with the application of real-time RT-PCR and ACE ranged from 61% to 94%, compared with testing individual samples by ACE, immunohistochemistry, or real-time RT-PCR. Real-time RT-PCR screening also indicated that 92.94% of PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'-untranslated region of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a. PMID:21217023

  1. Identification of reference genes for quantitative RT-PCR analysis of microRNAs and mRNAs in castor bean (Ricinus communis L.) under drought stress.

    PubMed

    Cassol, Daniela; Cruz, Fernanda P; Espindola, Kauê; Mangeon, Amanda; Müller, Caroline; Loureiro, Marcelo Ehlers; Corrêa, Régis L; Sachetto-Martins, Gilberto

    2016-09-01

    Quantitative real-time PCR (RT-qPCR) is one of the most powerful and sensitive techniques to the study of gene expression. Several factors influence RT-qPCR performance though, including the stability of the reference genes used for data normalization. While the selection of appropriate reference genes is crucial for accurate and reliable gene expression analysis, no suitable reference genes have been previously identified in castor bean under drought stress. In this study, the expression stability of eleven mRNAs, thirteen microRNAs (miRNAs) and one small nuclear RNA were analyzed in roots and leaves across different levels of water deficit. Three different algorithms were employed to analyze the RT-qPCR data, and the resulting outputs were merged using a non-weighted unsupervised rank aggregation method. Our analysis indicated that the Elongation factor 1-beta (EF1B), Protein phosphatase 2A (PP2A) and ADP-ribosylation factor (ADP) ranked as the best candidates across diverse samples submitted to different levels of drought conditions. EF1B and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and EF1B and SKP1/ASK-interacting protein 16 (SKIP16) were found as the most suitable reference genes for expression analysis in roots and leaves, respectively. In addition, miRNAs miR168, miR160 and miR397 were selected as optimal reference genes across all tissues and treatments. miR168 and miR156 were recommended as reference for roots, while miR168 and miR160 were recommended for leaves. Together, our results constitute the first attempt to identify and validate the most suitable reference genes for accurate normalization of gene expression in castor bean under drought stress. PMID:27156134

  2. RT-PCR analysis of Candida albicans ALS gene expression in a hyposalivatory rat model of oral candidiasis and in HIV-positive human patients

    PubMed Central

    GREEN, CLAYTON B.; MARRETTA, SANDRA MANFRA; CHENG, GEORGINA; FADDOUL, FADY F.; EHRHART, E. J.; HOYER, LOIS L.

    2007-01-01

    Summary ALS gene expression was studied in the hyposalivatory rat model of oral candidiasis and in clinical specimens collected from HIV-positive patients to assess similarities in expression patterns between the model system and clinical isolates. Two C. albicans strains, SC5314 and OY-2-76, were used in the rat model system and infection progressed for 3 or 5 days. The strains produced similar oral lesions at 3 days. At 5 days, strain OY-2-76 produced more superficial lesions containing relatively more yeast forms compared to invasive hyphal forms observed for strain SC5314. For all infections, the most severe lesions were observed on the tongue and gingiva overlying the mandible. ALS transcripts were easier to detect by RT-PCR later in infection and under other conditions where more fungal cells were present. Expression of ALS1, ALS2, ALS3 and ALS4 was observed in rats infected for 3 days with ALS5 and ALS9 transcripts detected after 5 days of infection. Expression of ALS6 was observed in a single specimen from a 5-day infection while ALS7 transcript was never found. Expression of all ALS genes was observed in oral clinical material collected from HIV-positive patients although ALS6 and ALS7 transcripts required an extra PCR amplification step to be detected. Overall, the patterns of ALS gene expression were similar between the rat model and human clinical specimens, suggesting that the model would be useful for studying the phenotype of als/als mutant strains. PMID:16519012

  3. Identification of differentially expressed proteins of Arthrospira (Spirulina) plantensis-YZ under salt-stress conditions by proteomics and qRT-PCR analysis

    PubMed Central

    2013-01-01

    Arthrospira (Spirulina) platensis as a representative species of cyanobacteria has been recognized and used worldwide as a source of protein in the food, which possesses some unusual and valuable physiological characteristics, such as alkali and salt tolerance. Based on complete genome sequencing of Arthrospira (Spirulina) plantensis-YZ, we compared the protein expression profiles of this organism under different salt-stress conditions (i.e. 0.02 M, 0.5 M and 1.0 M NaCl, respectively), using 2-D electrophoresis and peptide mass fingerprinting, and retrieved 141 proteins showing significantly differential expression in response to salt-stress. Of the 141 proteins, 114 Arthrospira (Spirulina) plantensis-YZ proteins were found with significant homology to those found in Arthrospira (76 proteins in Arthrospira platensis str. Paraca and 38 in Arthrospira maxima CS-328). The remaining 27 proteins belong to other bacteria. Subsequently, we determined the transcriptional level of 29 genes in vivo in response to NaCl treatments and verified them by qRT-PCR. We found that 12 genes keep consistency at both transcription and protein levels, and transcription of all of them but one were up-regulated. We classified the 141 differentially expressed proteins into 18 types of function categories using COG database, and linked them to their respective KEGG metabolism pathways. These proteins are involved in 31 metabolism pathways, such as photosynthesis, glucose metabolism, cysteine and methionine metabolism, lysine synthesis, fatty acid metabolism, glutathione metabolism. Additionally, the SRPs, heat shock protein and ABC transporter proteins were identified, which probably render Arthrospira (Spirulina) plantensis’s resistance against high salt stress. PMID:23363438

  4. Space Shuttle Main Engine real time stability analysis

    NASA Astrophysics Data System (ADS)

    Kuo, F. Y.

    1993-06-01

    The Space Shuttle Main Engine (SSME) is a reusable, high performance, liquid rocket engine with variable thrust. The engine control system continuously monitors the engine parameters and issues propellant valve control signals in accordance with the thrust and mixture ratio commands. A real time engine simulation lab was installed at MSFC to verify flight software and to perform engine dynamic analysis. A real time engine model was developed on the AD100 computer system. This model provides sufficient fidelity on the dynamics of major engine components and yet simplified enough to be executed in real time. The hardware-in-the-loop type simulation and analysis becomes necessary as NASA is continuously improving the SSME technology, some with significant changes in the dynamics of the engine. The many issues of interfaces between new components and the engine can be better understood and be resolved prior to the firing of the engine. In this paper, the SSME real time simulation Lab at the MSFC, the SSME real time model, SSME engine and control system stability analysis, both in real time and non-real time is presented.

  5. Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita

    PubMed Central

    Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun

    2016-01-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea. PMID:27314014

  6. Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita.

    PubMed

    Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun

    2016-01-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea. PMID:27314014

  7. Vector processing enhancements for real-time image analysis.

    SciTech Connect

    Shoaf, S.; APS Engineering Support Division

    2008-01-01

    A real-time image analysis system was developed for beam imaging diagnostics. An Apple Power Mac G5 with an Active Silicon LFG frame grabber was used to capture video images that were processed and analyzed. Software routines were created to utilize vector-processing hardware to reduce the time to process images as compared to conventional methods. These improvements allow for more advanced image processing diagnostics to be performed in real time.

  8. Prompt detection of influenza A and B viruses using the BD Veritor™ System Flu A+B, Quidel® Sofia® Influenza A+B FIA, and Alere BinaxNOW® Influenza A&B compared to real-time reverse transcription-polymerase chain reaction (RT-PCR).

    PubMed

    Dunn, Jim; Obuekwe, Joy; Baun, Traci; Rogers, Justin; Patel, Twinkle; Snow, Linda

    2014-05-01

    The performance characteristics of rapid influenza diagnostic tests vary widely. This study evaluated the BD Veritor™ System Flu A+B (Veritor; BD Diagnostics, Sparks, MD, USA), Quidel® Sofia® Influenza A+B FIA (Sofia; Quidel Corp., San Diego, CA, USA), and Alere BinaxNOW® Influenza A&B (Binax; Alere Scarborough, Inc., Scarborough, ME, USA) compared to reverse transcription-polymerase chain reaction (RT-PCR) for detection of influenza viruses in nasal wash specimens from 240 pediatric patients. Positive percent agreements for influenza A and B virus detection were 93.8% and 94.2%, 95.8% and 98.1%, and 79.2% and 80.8% for Veritor, Sofia, and Binax, respectively. The Veritor and Binax tests demonstrated negative percent agreements >97.9% for detection of both influenza viruses, but the negative percent agreement of the Sofia test was 91.1% for influenza A and 70.7% for influenza B virus. Overall, the Veritor and Sofia tests were nearly as sensitive as RT-PCR and considerably more sensitive than Binax for detection of influenza viruses. However, the accuracy of the Sofia test was significantly lower than either Veritor or Binax. PMID:24582581

  9. Real-time linear predictive analysis of speech using multimicroprocessors

    SciTech Connect

    Seethardman, S.; Radhakrishnan, T.; Suen, C.Y.

    1982-01-01

    Many applications of linear predictive coding (often known as LPC) of speech signals require a system capable of performing the complete LPC analysis in real time. This paper describes a pipeline network consisting of several general purpose microprocessors, primarily suitable for complete LPC analysis of a 10-pole model with a sampling frequency of 10 khz and a frame rate of 100 hz in real time. The proposed system is different from the previous systems, which either employed special purpose hardware or produced an analysis at a lower frame rate. 27 references.

  10. Toward Real Time Data Analysis for Smart Grids

    SciTech Connect

    Yin, Jian; Gorton, Ian; Sharma, Poorva

    2012-11-10

    This paper describes the architecture and design of a novel system for supporting large-scale real-time data analysis for future power grid systems. The widespread deployment of renewable generation, smart grid controls, energy storage, plug-in hybrids, and new conducting materials will require fundamental changes in the operational concepts and principal components of the grid. As a result, the whole system becomes highly dynamic and requires constant adjusting based on real time data. Even though millions of sensors such as phase measurement units (PMU) and smart meters are being widely deployed, a data layer that can analyze this amount of data in real time is needed. Unlike the data fabric in other cloud services, the data layer for smart grids has some unique design requirements. First, this layer must provide real time guarantees. Second, this layer must be scalable to allow a large number of applications to access the data from millions of sensors in real time. Third, reliability is critical and this layer must be able to continue to provide service in face of failures. Fourth, this layer must be secure. We address these challenges though a scalable system architecture that integrates the I/O and data processing capability in a devise set of devices. Data process operations can be placed anywhere from sensors, data storage devices, to control centers. We further employ compression to improve performance. We design a lightweight compression customized for power grid data. Our system can reduce end-to-end response time by reduce I/O overhead through compression and overlap compression operations with I/O. The initial prototype of our system was demonstrated with several use cases from PNNL’s FPGI and show that our system can provide real time guarantees to a diverse set of applications.

  11. Real-time oriented image analysis in microcirculatory research

    NASA Astrophysics Data System (ADS)

    Pries, Axel R.; Eriksson, S. E.; Jepsen, H.

    1990-11-01

    A digital video image analysis system is presented which consists of a personal computer equipped with a real-time video digitizer and a graphic tablet controlled by a modular set of programs aimed at performing a number of real-time oriented measuring tasks in microcirculatory research. Such tasks comprize the continuous recording of vessel diameters flow velocities or light intensity profiles from video recordings obtained during intravital microscopy of the terminal vascular bed either in research animals or in human beings. Two outstanding features of the presented systems are (A) the spatial correlation module for velocity measurement and (B) the automatic background movement correction. A: The spatial correlation velocity measurement module combined with an asymmetric illumination or gating process for image generation allows measurement of flow velocities from video microscopic images up to 20 mm/sec. This is about 10 to 20 times faster than the maximum. velocities which can be measured using conventional video based techniques. B: The automatic background movement correction is designed to track translational movements of background image structures in a reference window in real time (with respect to the video system). The translational vector of the image background is then used to adjust the position of the individual measuring lines or windows used in the different application modules to their original position relative to the tissue structures which are investigated. Such an automatic real-time tracking system isvery often a

  12. Real-Time Earthquake Analysis for Disaster Mitigation (READI) Network

    NASA Astrophysics Data System (ADS)

    Bock, Y.

    2014-12-01

    Real-time GNSS networks are making a significant impact on our ability to forecast, assess, and mitigate the effects of geological hazards. I describe the activities of the Real-time Earthquake Analysis for Disaster Mitigation (READI) working group. The group leverages 600+ real-time GPS stations in western North America operated by UNAVCO (PBO network), Central Washington University (PANGA), US Geological Survey & Scripps Institution of Oceanography (SCIGN project), UC Berkeley & US Geological Survey (BARD network), and the Pacific Geosciences Centre (WCDA project). Our goal is to demonstrate an earthquake and tsunami early warning system for western North America. Rapid response is particularly important for those coastal communities that are in the near-source region of large earthquakes and may have only minutes of warning time, and who today are not adequately covered by existing seismic and basin-wide ocean-buoy monitoring systems. The READI working group is performing comparisons of independent real time analyses of 1 Hz GPS data for station displacements and is participating in government-sponsored earthquake and tsunami exercises in the Western U.S. I describe a prototype seismogeodetic system using a cluster of southern California stations that includes GNSS tracking and collocation with MEMS accelerometers for real-time estimation of seismic velocity and displacement waveforms, which has advantages for improved earthquake early warning and tsunami forecasts compared to seismic-only or GPS-only methods. The READI working group's ultimate goal is to participate in an Indo-Pacific Tsunami early warning system that utilizes GNSS real-time displacements and ionospheric measurements along with seismic, near-shore buoys and ocean-bottom pressure sensors, where available, to rapidly estimate magnitude and finite fault slip models for large earthquakes, and then forecast tsunami source, energy scale, geographic extent, inundation and runup. This will require

  13. Inactivation conditions for human norovirus measured by an in situ capture-qRT-PCR method.

    PubMed

    Wang, Dapeng; Tian, Peng

    2014-02-17

    Human norovirus (HuNoV) is a leading cause of foodborne gastroenteritis. Unfortunately, the inactivation parameters for HuNoV in clinical, food and environmental samples have not been established. Due to the inability to cultivate HuNoV in vitro, quantitative real-time RT-PCR (qRT-PCR) is widely-used for detecting HuNoVs. However, qRT-PCR does not indicate viral infectivity. Our method employs histo-blood group antigens (HBGAs) as viral receptors/co-receptors and container-affixed capture agents to concentrate HuNoVs. The captured viruses are denatured and its genome is amplified in the same module by in situ capture qRT-PCR (ISC-qRT-PCR). Greater than three log10 reduction in the receptor-captured viral genomic signal (RCVGS) was observed when HuNoV was treated by heat at 72 °C for 4 min, by chlorine at a final concentration of 16 ppm in less than 1 min, and by UV irradiation at 1J/cm². Treatment of low-titer HuNoV (<10³ copies/sample) with 70% ethanol for 20 s reduced the RCVGS of HuNoV by two log10. However, ethanol had a limited effect on high-titer samples of HuNoV (>10³ copies/sample). The results demonstrate that ISC-qRT-PCR method could be used as an alternative method to measure encapsidated viral RNA and indirectly indicate the inactivation status of HuNoV caused by physical treatment such as heat, and chemical treatment such as chlorine, that damage the ability of the virus to bind to its receptor. PMID:24361836

  14. CD44 variant exon 6 expressions in colon cancer assessed by quantitative analysis using real time reverse transcriptase-polymerase chain reaction.

    PubMed

    Yamada, Yoichi; Itano, Naoki; Narimatsu, Hisashi; Kudo, Takashi; Hirohashi, Setsuo; Ochiai, Atsushi; Tohnai, Iwai; Ueda, Minoru; Kimata, Koji

    2003-01-01

    CD44 is a family of transmembrane glycoproteins that serve as a major receptor for hyaluronate and the splice variants play a very important role in tumor progression and metastasis. We examined the relationship between cancer progression and mRNA levels of CD44 variant exon 6 (CD44v6) in specimens of colon cancer at different diagnostic stages from 31 patients using real time RT-PCR analysis. Increased mRNA levels of CD44v6 were observed in 82% of the specimens in comparison with those in the corresponding non-cancerous tissue specimens. A statistically significant correlation between the CD44v6 expression and the cancerous state was found in most specimens at all Dukes stages. None of the other parameters were related to the expression in the cancerous specimens. Quantitative real time RT-PCR analysis showed that there was no correlation of CD44v6 expression with tumor progression, although CD44v6 is upregulated in transformation. Thus, CD44v6 expression may be a clinically useful indicator of colon cancer. PMID:14534719

  15. Utilizing real-time and near real-time data in the iNtegrated Space Weather Analysis System

    NASA Astrophysics Data System (ADS)

    Maddox, M. M.; Mullinix, R. E.; Rastaetter, L.; Pulkkinen, A.; Zheng, Y.; Berrios, D.; Hesse, M.; Kuznetsova, M. M.; Taktakishvili, A.; Chulaki, A.; Shim, J.; Bakshi, S. S.; Patel, K. D.; Jain, P.

    2010-12-01

    Access to near real-time and real-time space weather data is essential to accurately specifying and forecasting the space environment. The Space Weather Desk at NASA Goddard Space Flight Center's Space Weather Laboratory provides vital space weather forecasting services primarily to NASA robotic mission operators, as well as external space weather stakeholders including the Air Force Weather Agency. A key component in this activity is the iNtegrated Space Weather Analysis System which is a joint development project at NASA GSFC between the Space Weather Laboratory, Community Coordinated Modeling Center, Applied Engineering & Technology Directorate, and NASA HQ Office Of Chief Engineer. The iSWA system was developed to address technical challenges in acquiring and disseminating space weather environment information. A key design driver for the iSWA system was to generate and present vast amounts of space weather resources in an intuitive, user-configurable, and adaptable format - thus enabling users to respond to current and future space weather impacts as well as enabling post-impact analysis. Having access to near real-time and real-time data is essential to not only ensuring that relevant observational data is available for analysis - but also in ensuring that models can be driven with the requisite input parameters at proper and efficient temporal and spacial resolutions. The iSWA system currently manages over 250 unique near-real and real-time data feeds from various sources consisting of both observational and simulation data. A comprehensive suite of actionable space weather analysis tools and products are generated and provided utilizing a mixture of the ingested data - enabling new capabilities in quickly assessing past, present, and expected space weather effects. This paper will highlight current and future iSWA system capabilities and also discuss some of the challenges and lessons-learned in dealing with diverse real-time and near-real time space

  16. Complete chemical analysis of aerosol particles in real-time

    SciTech Connect

    Yang, Mo; Reilly, P.T.A.; Gieray, R.A.; Whitten, W.B.; Ramsey, J.M.

    1996-12-31

    Real-time mass spectrometry of individual aerosol particles using an ion trap mass spectrometer is described. The microparticles are sampled directly from the air by a particle inlet system into the vacuum chamber. An incoming particle is detected as it passes through two CW laser beams and a pulsed laser is triggered to intercept the particle for laser ablation ionization at the center of the ion trap. The produced ions are analyzed by the ion trap mass spectrometer. Ions of interest are selected and dissociated through collision with buffer gas atoms for further fragmentation analysis. Real-time chemical analyses of inorganic, organic, and bacterial aerosol articles have been demonstrated. It has been confirmed that the velocity and the size of the incoming particles highly correlate to each other. The performance of the inlet system, particle detection, and preliminary results are discussed.

  17. Analysis of Three Real-Time Dst Indices

    NASA Astrophysics Data System (ADS)

    Carranza-Fulmer, T. L.; Gannon, J. L.; Love, J. J.

    2010-12-01

    The Dst is commonly used to specify geomagnetic disturbance periods and characterize the resulting ring current enhancements from ground-based horizontal magnetic field intensity measurements. Real-time versions of the Dst index are produced for operational purposes, and are of interest to many users, including the US military, airline industry, and power companies. USGS Real time Dst, Kyoto Quicklook Dst, and Space Environment Corporation RDst use preliminary data and use a variety of contributing observatories and processing methods. Both USGS and RDst use a combined time-and-frequency domain method and Kyoto uses a time domain only method in creating the Dst index. We perform an analysis of these three real time Dst indices for the time period of October 1, 2009 to May 31, 2010. The USGS 3, using three observatories instead of the standard four, and the Kyoto Sym-H index, are introduced in the analysis for comparison of observatory location with the three main Dst indices. We present a statistical study of the differences due to algorithm, output time resolution, and location of contributing observatories. Higher time resolution shows higher frequency fluctuations during disturbances and more defined storm features. There were small differences in mid- to low-latitude observatories during quiet to moderate storm time periods. The average impact on the index due to the different algorithms used was approximately 9 nT, and greater for individual storms.

  18. Forensic Disaster Analysis in Near-real Time

    NASA Astrophysics Data System (ADS)

    Kunz, Michael; Zschau, Jochen; Wenzel, Friedemann; Khazai, Bijan; Kunz-Plapp, Tina; Trieselmann, Werner

    2014-05-01

    The impacts of extreme hydro-meteorological and geophysical events are controlled by various factors including severity of the event (intensity, duration, spatial extent), amplification with other phenomena (multihazard or cascading effects), interdependencies of technical systems and infrastructure, preparedness and resilience of the society. The Center for Disaster Management and Risk Reduction Technology (CEDIM) has adopted the comprehensive understanding of disasters and develops methodologies of near real-time FDA as a complementing component of the FORIN program of IRDR. The new research strategy 'Near Real-Time Forensic Disaster Analysis (FDA)' aims at scrutinizing disasters closely with a multi-disciplinary approach in order to assess the various aspects of disasters and to identify mechanisms most relevant for an extreme event to become a disaster (e.g., causal loss analysis). Recent technology developments - which have opened unprecedented opportunities for real-time hazard, vulnerability and loss assessment - are used for analyzing disasters and their impacts in combination with databases of historical events. The former covers modern empirical and analytical methods available in engineering and remote sensing for rapid impact assessments, rapid information extraction from crowd sourcing as well as rapid assessments of socio-economic impacts and economic losses. The event-driven science-based assessments of CEDIM are compiled based on interdisciplinary expertise and include the critical evaluation, assessment, validation, and quantification of an event. An important component of CEDIM's FDA is the near real-time approach which is expected to significantly speed up our understanding of natural disasters and be used to provide timely, relevant and valuable information to various user groups within their respective contexts. Currently, CEDIM has developed models and methodologies to assess different types of hazard. These approaches were applied to several

  19. Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

    PubMed Central

    Pizeta Semighini, Camile; Marins, Mozart; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2002-01-01

    The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background. PMID:11872487

  20. Padlock probe-mediated qRT-PCR for DNA computing answer determination

    PubMed Central

    Xiong, Fusheng; Frasch, Wayne D.

    2011-01-01

    Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (Ct value) and target content. The Ct values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology. PMID:21691417

  1. ISTAR: Intelligent System for Telemetry Analysis in Real-time

    NASA Technical Reports Server (NTRS)

    Simmons, Charles

    1994-01-01

    The intelligent system for telemetry analysis in real-time (ISTAR) is an advanced vehicle monitoring environment incorporating expert systems, analysis tools, and on-line hypermedia documentation. The system was developed for the Air Force Space and Missile Systems Center (SMC) in Los Angeles, California, in support of the inertial upper stage (IUS) booster vehicle. Over a five year period the system progressed from rapid prototype to operational system. ISTAR has been used to support five IUS missions and countless mission simulations. There were a significant number of lessons learned with respect to integrating an expert system capability into an existing ground system.

  2. Development of a RT-PCR ELISA for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants and its evaluation on clinical samples.

    PubMed

    Dubey, Pooja; Mishra, N; Rajukumar, K; Behera, S P; Kalaiyarasu, S; Nema, R K; Prakash, A

    2015-03-01

    The aim of this study was to develop a reverse transcription polymerase chain reaction ELISA (RT-PCR ELISA) for detection of ruminant pestiviruses and to evaluate its diagnostic performance on clinical samples obtained from cattle, sheep and goats. Optimization was carried out by serial dilution of home-made digoxygenin-labelled RT-PCR product standards obtained from pestivirus isolates and pestivirus infected animals. The detection limit of the assay was 10TCID50/ml, similar to virus isolation and real-time RT-PCR but 10-fold higher than RT-PCR. The assay had high analytical specificity along with a good reproducibility. When the assay was evaluated on the samples obtained from animals infected experimentally with BVDV and from the field using virus isolation as standard, it showed a high diagnostic sensitivity (95.9%) and specificity (98.6%) and there was strong agreement (97.5% concordance) between the two tests. However, it displayed an increased diagnostic specificity and sensitivity over RT-PCR. Additionally, when a few samples (n=26) were tested by RT-PCR ELISA and real-time RT-PCR, 100% concordance was obtained between them. Our results showed that RT-PCR ELISA can be a rapid, cost effective and alternative molecular diagnostic test for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants in ordinary laboratory settings. PMID:25486086

  3. Real-time Stability Analysis for Disruption Avoidance in ITER

    NASA Astrophysics Data System (ADS)

    Glasser, Alexander; Kolemen, Egemen; Glasser, Alan

    2015-11-01

    ITER is intended to operate at plasma parameters approaching the frontier of achievable stability limits. And yet, plasma disruptions at ITER must be kept to a bare minimum to avoid damage to its plasma-facing structures. These competing goals necessitate real-time plasma stability analysis and feedback control at ITER. This work aims to develop a mechanism for real-time analysis of a large and virulent class of disruptions driven by the rapid growth of ideal MHD unstable modes in tokamak equilibria. Such modes will be identified by a parallelized, low-latency implementation of A.H. Glasser's well-tested DCON (Direct Criterion of Newcomb) code, which measures the energetics of modes in the bulk plasma fluid, as well as M.S. Chance's VACUUM code, which measures the same in the vacuum between the plasma and tokamak chamber wall. Parallelization of these codes is intended to achieve a time-savings of 40x, thereby reducing latency to a timescale of order 100ms and making the codes viable for ideal MHD stability control at ITER. The hardware used to achieve this parallelization will be an Intel Xeon Phi server with 77 cores (308 threads). Supported by the US DOE under DE-AC02-09CH11466.

  4. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  5. Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a Newly Described RT-PCR Targeting the Pertactin Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently a real-time PCR (RT-PCT) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here we report that the B. pertussis pertactin gene sequence for the region encompassing the RT-PCR probe and primers is nearly identical to that of many B....

  6. Quantitative real-time single particle analysis of virions

    SciTech Connect

    Heider, Susanne; Metzner, Christoph

    2014-08-15

    Providing information about single virus particles has for a long time been mainly the domain of electron microscopy. More recently, technologies have been developed—or adapted from other fields, such as nanotechnology—to allow for the real-time quantification of physical virion particles, while supplying additional information such as particle diameter concomitantly. These technologies have progressed to the stage of commercialization increasing the speed of viral titer measurements from hours to minutes, thus providing a significant advantage for many aspects of virology research and biotechnology applications. Additional advantages lie in the broad spectrum of virus species that may be measured and the possibility to determine the ratio of infectious to total particles. A series of disadvantages remain associated with these technologies, such as a low specificity for viral particles. In this review we will discuss these technologies by comparing four systems for real-time single virus particle analysis and quantification. - Highlights: • We introduce four methods for virus particle-based quantification of viruses. • They allow for quantification of a wide range of samples in under an hour time. • The additional measurement of size and zeta potential is possible for some.

  7. Real-time Analysis of Lateral Root Organogenesis in Arabidopsis

    PubMed Central

    Marhavý, Peter; Benková, Eva

    2016-01-01

    Plants maintain capacity to form new organs such as leaves, flowers, lateral shoots and roots throughout their postembryonic lifetime. Lateral roots (LRs) originate from a few pericycle cells that acquire attributes of founder cells (FCs), undergo series of anticlinal divisions, and give rise to a few short initial cells. After initiation, coordinated cell division and differentiation occur, giving rise to lateral root primordia (LRP). Primordia continue to grow, emerge through the cortex and epidermal layers of the primary root, and finally a new apical meristem is established taking over the responsibility for growth of mature lateral roots [for detailed description of the individual stages of lateral root organogenesis see Malamy and Benfey (1997)]. To examine this highly dynamic developmental process and to investigate a role of various hormonal, genetic and environmental factors in the regulation of lateral root organogenesis, the real time imaging based analyses represent extremely powerful tools (Laskowski et al., 2008; De Smet et al., 2012; Marhavý et al., 2013 and 2014). Herein, we describe a protocol for real time lateral root primordia (LRP) analysis, which enables the monitoring of an onset of the specific gene expression and subcellular protein localization during primordia organogenesis, as well as the evaluation of the impact of genetic and environmental perturbations on LRP organogenesis. PMID:27331080

  8. Portable real time analysis system for regional cerebral blood flow

    SciTech Connect

    Tiernan, T.; Entine, G.; Stump, D.A.; Prough, D.S.

    1988-02-01

    A very portable, regional cerebral blood flow (rCBF) analysis instrument system suitable for use in the operating theater during surgery is under development. Cadmium telluride (CdTe) solid state radiation detectors, an 8086 based data acquisition and communications module and a DEC Microvax computer are used so that the instrument is very compact, yet has the computational power to provide real time data analysis in the clinical environment. The instrument is currently being used at Bowman Gray School of Medicine to study rCBF during cardiopulmonary bypass surgery (CPB). Preliminary studies indicate that monitoring rCBF during this surgical procedure may provide insights into the mechanism that causes a significant fraction of these patients to suffer post operative neuropsychological deficit.

  9. Cloud-ECG for real time ECG monitoring and analysis.

    PubMed

    Xia, Henian; Asif, Irfan; Zhao, Xiaopeng

    2013-06-01

    Recent advances in mobile technology and cloud computing have inspired numerous designs of cloud-based health care services and devices. Within the cloud system, medical data can be collected and transmitted automatically to medical professionals from anywhere and feedback can be returned to patients through the network. In this article, we developed a cloud-based system for clients with mobile devices or web browsers. Specially, we aim to address the issues regarding the usefulness of the ECG data collected from patients themselves. Algorithms for ECG enhancement, ECG quality evaluation and ECG parameters extraction were implemented in the system. The system was demonstrated by a use case, in which ECG data was uploaded to the web server from a mobile phone at a certain frequency and analysis was performed in real time using the server. The system has been proven to be functional, accurate and efficient. PMID:23261079

  10. Method of Real-Time Principal-Component Analysis

    NASA Technical Reports Server (NTRS)

    Duong, Tuan; Duong, Vu

    2005-01-01

    Dominant-element-based gradient descent and dynamic initial learning rate (DOGEDYN) is a method of sequential principal-component analysis (PCA) that is well suited for such applications as data compression and extraction of features from sets of data. In comparison with a prior method of gradient-descent-based sequential PCA, this method offers a greater rate of learning convergence. Like the prior method, DOGEDYN can be implemented in software. However, the main advantage of DOGEDYN over the prior method lies in the facts that it requires less computation and can be implemented in simpler hardware. It should be possible to implement DOGEDYN in compact, low-power, very-large-scale integrated (VLSI) circuitry that could process data in real time.

  11. VLBI real-time analysis by Kalman Filtering

    NASA Astrophysics Data System (ADS)

    Karbon, Maria; Soja, Benedikt; Nilson, Tobias; Heinkelmann, Robert; Liu, Li; Lu, Ciuxian; Xu, Minghui; Raposo-Pulido, Virginia; Mora-Diaz, Julian; Schuh, Harald

    2014-05-01

    Geodetic Very Long Baseline Interferometry (VLBI) is one of the primary space geodetic techniques. It provides the full set of Earth Orientation Parameter (EOP) and is unique for observing long term Universal Time (UT1) and precession/nutation. Currently the VLBI products are delivered with a delay of about two weeks from the moment of the observation. However, the need for near-real time estimates of the parameters is increasing, e.g. for satellite based navigation and positioning or for enabling precise tracking of interplanetary spacecraft. The goal is thus to reduce the time span between observation and the final result to less than one day. This can be archived by replacing the classical least squares method with an adaptive Kalman filter. We have developed a Kalman filter for VLBI data analysis. This method has the advantage that it is simultaneously possible to estimate stationary parameters, e.g. station positions, and to model the highly variable stochastic behavior of non-stationary parameters like clocks or atmospheric parameters. The filter is able to perform without any human interaction, making it a completely autonomous tool. In this work we describe the filter and discuss its application for EOP determination and prediction. We discuss the implementation of the stochastic models to statistically account for unpredictable changes in EOP. Furthermore, additional data like results from other techniques can be included to improve the performance. For example, atmospheric angular momentum calculated from numerical weather models can be introduced to supplement the short-term prediction of UT1 and polar motion. This Kalman filter will be extended and embedded in the newly developed Vienna VLBI Software (VieVS) as a completely autonomous tool enabling the VLBI analysis in near real-time and providing all the parameters of interest with the highest possible accuracy.

  12. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  13. Cloning of fox (Vulpes vulpes) Il2, Il6, Il10 and IFNgamma and analysis of their expression by quantitative RT-PCR in fox PBMC after in vitro stimulation by Concanavalin A.

    PubMed

    Rolland-Turner, Magali; Farré, Guillaume; Boué, Franck

    2006-04-15

    The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterised, and specific immunological tools are needed. A quantitative RT-PCR using SyBR Green to investigate fox cytokine expression after antigen PBMC in vitro re-stimulation is presented here. First, we cloned by homology with dog cytokine sequences the fox IL2, IL6, IL10, IFNgamma and a partial 18S sequence. Fox specific primers were then defined and used to set up a species-specific quantitative RT-PCR assay using SyBR Green and 18S housekeeping gene as internal standard. The technique was validated using total RNA from fox PBMC stimulated with a polyclonal activator, Concanavaline A. PMID:16321447

  14. Reference Gene Selection and Evaluation for Gene Expression Studies Using qRT-PCR in the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

    PubMed

    An, Xing-kui; Hou, Mao-lin; Liu, Yu-di

    2016-04-01

    The white-backed planthopper, Sogatella furcifera (Hemiptera, Delphacidae), is one of the most devastating rice pests. For a better control strategy, various genetic studies have been conducted using reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR). The appropriate application of qRT-PCR requires reliable endogenous controls; however, studies on this aspect of the white-backed planthopper are lacking. In the present study, nine commonly used reference genes, elongation factor 1-α (EF1-α), polyubiquitin (UB), ribosomal protein S18 (RPS18), actin 1 (ACT), α-1 tubulin (TUB), glyceraldehyde-3-phosphate (GAPDH), ribosomal protein L9 (RPL9), ribosomal protein L10 (RPL10), and 18S ribosomal RNA (18S), were evaluated by qRT-PCR for their expression stability under four different experimental conditions (different developmental stages, acquisition of Southern rice black-streaked dwarf virus (SRBSDV), different tissues, and different temperature stress). These results were analyzed using four software programs (geNorm, NormFinder, BestKeeper, and the delta Ct method) and a Web-based comprehensive tool RefFinder to compare and rank candidate reference genes. According to the results of RefFinder analysis, which integrates the abovementioned four software programs, TUB was ranked as the most suitable reference gene at different developmental stages and under different temperature stress, and GAPDH and RPL9 showed the highest stability for acquisition of SRBSDV and different tissues, respectively. These results will provide a solid foundation for future gene expression study on the white-backed planthopper, and also will give aids in establishing a standardized qRT-PCR procedure for other related insects. PMID:26612891

  15. Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

    PubMed

    Kundu, K; Rysánek, P

    2004-01-01

    Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected. PMID:15595212

  16. VLBI real-time analysis by Kalman Filtering

    NASA Astrophysics Data System (ADS)

    Karbon, M.; Nilsson, T.; Soja, B.; Heinkelmann, R.; Raposo-Pulido, V.; Schuh, H.

    2013-12-01

    Geodetic Very Long Baseline Interferometry (VLBI) is one of the primary space geodetic techniques providing the full set of Earth Orientation Parameter (EOP) and is unique for observing long term Universal Time (UT1) and precession/nutation. Accurate and continuous EOP obtained in near real-time are essential for satellite based navigation and positioning and for enabling the precise tracking of interplanetary spacecrafts. To meet this necessity the International VLBI Service for Geodesy and Astrometry (IVS) increased its efforts to reduce the time span between the VLBI observations and the availability of the final results. Currently the timeliness is about two weeks, but the goal is to reduce it to less than one day with the future VGOS (VLBI2010 Global Observing System) network. The FWF project VLBI-ART contributes to this new generation VLBI system by considerably accelerating the VLBI analysis procedure through the implementation of an elaborate Kalman filter. This true real-time Kalman filter will be embedded in the Vienna VLBI Software (VieVS) as a completely automated tool with no need of human interaction. This filter also allows the prediction and combination of EOP from various space geodetic techniques by implementing stochastic models to statistically account for unpredictable changes in EOP. Additionally, atmospheric angular momenta calculated from numerical weather prediction models are introduced to support the short-term EOP prediction. To optimize the performance of the new software various investigations with real as well as simulated data are foreseen. The results are compared to the ones obtained by conventional VLBI parameter estimation methods (e.g. least squares method) and to corresponding parameter series from other techniques, such as from the Global Navigation Satellite Systems (GNSS).

  17. Petroleomics by Direct Analysis in Real Time-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Romão, Wanderson; Tose, Lilian V.; Vaz, Boniek G.; Sama, Sara G.; Lobinski, Ryszard; Giusti, Pierre; Carrier, Hervé; Bouyssiere, Brice

    2016-01-01

    The analysis of crude oil and its fractions by applying ambient ionization techniques remains underexplored in mass spectrometry (MS). Direct analysis in real time (DART) in the positive-ion mode was coupled to a linear quadrupole ion trap Orbitrap mass spectrometer (LTQ Orbitrap) to analyze crude oil, paraffin samples, and porphyrin standard compounds. The ionization parameters of DART-MS were optimized for crude oil analysis. DART-MS rendered the optimum conditions of the operation using paper as the substrate, T = 400°C, helium as the carrier gas, and a sample concentration ≥6 mg mL-1. In the crude oils analysis, the DART(+)-Orbitrap mass spectra detected the typical N, NO, and O-containing compounds. In the paraffin samples, oxidized hydrocarbon species (Ox classes, where x = 1-4) with double-bond equivalent of 1-4 were detected, and their structures and connectivity were confirmed by collision-induced dissociation (CID) experiments. DART(+)-MS has identified the porphyrin standard compounds as [M + H]+ ions of m/ z 615.2502 and 680.1763, where M = C44H30N4 and C44H28N4OV, respectively, based on the formula assignment and by phenyl losses observed on CID experiments.

  18. Real-time chemical analysis of aerosol particles

    SciTech Connect

    Yang, M.; Whitten, W.B.; Ramsey, J.M.

    1995-04-01

    An important aspect of environmental atmospheric monitoring requires the characterization of airborne microparticles and aerosols. Unfortunately, traditional sample collection and handling techniques are prone to contamination and interference effects that can render an analysis invalid. These problems can be avoided by using real-time atmospheric sampling techniques followed by immediate mass spectrometric analysis. The former is achieved in these experiments via a two state differential pumping scheme that is attached directly to a commercially available quadruple ion trap mass spectrometer. Particles produced by an external particle generator enter the apparatus and immediately pass through two cw laser/fiberoptic based detectors positioned two centimeters apart. Timing electronics measure the time between detection events, estimate the particles arrival in the center of the ion trap and control the firing of a YAG laser. Ions produced when the UV laser light ablates the particle`s surface are stored by the ion trap for mass analysis. Ion trap mass spectrometers have several advantages over conventional time-of-flight instruments. First, they are capable of MS/MS analysis by the collisional dissociation of a stored species, This permits complete chemical characterization of airborne samples. Second, ion traps are small and lend themselves to portable, field oriented applications.

  19. Petroleomics by Direct Analysis in Real Time-Mass Spectrometry.

    PubMed

    Romão, Wanderson; Tose, Lilian V; Vaz, Boniek G; Sama, Sara G; Lobinski, Ryszard; Giusti, Pierre; Carrier, Hervé; Bouyssiere, Brice

    2016-01-01

    The analysis of crude oil and its fractions by applying ambient ionization techniques remains underexplored in mass spectrometry (MS). Direct analysis in real time (DART) in the positive-ion mode was coupled to a linear quadrupole ion trap Orbitrap mass spectrometer (LTQ Orbitrap) to analyze crude oil, paraffin samples, and porphyrin standard compounds. The ionization parameters of DART-MS were optimized for crude oil analysis. DART-MS rendered the optimum conditions of the operation using paper as the substrate, T = 400°C, helium as the carrier gas, and a sample concentration ≥6 mg mL(-1). In the crude oils analysis, the DART(+)-Orbitrap mass spectra detected the typical N, NO, and O-containing compounds. In the paraffin samples, oxidized hydrocarbon species (Ox classes, where x = 1-4) with double-bond equivalent of 1-4 were detected, and their structures and connectivity were confirmed by collision-induced dissociation (CID) experiments. DART(+)-MS has identified the porphyrin standard compounds as [M + H](+) ions of m/z 615.2502 and 680.1763, where M = C44H30N4 and C44H28N4OV, respectively, based on the formula assignment and by phenyl losses observed on CID experiments. PMID:26432579

  20. Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

  1. Cluster Computing For Real Time Seismic Array Analysis.

    NASA Astrophysics Data System (ADS)

    Martini, M.; Giudicepietro, F.

    A seismic array is an instrument composed by a dense distribution of seismic sen- sors that allow to measure the directional properties of the wavefield (slowness or wavenumber vector) radiated by a seismic source. Over the last years arrays have been widely used in different fields of seismological researches. In particular they are applied in the investigation of seismic sources on volcanoes where they can be suc- cessfully used for studying the volcanic microtremor and long period events which are critical for getting information on the volcanic systems evolution. For this reason arrays could be usefully employed for the volcanoes monitoring, however the huge amount of data produced by this type of instruments and the processing techniques which are quite time consuming limited their potentiality for this application. In order to favor a direct application of arrays techniques to continuous volcano monitoring we designed and built a small PC cluster able to near real time computing the kinematics properties of the wavefield (slowness or wavenumber vector) produced by local seis- mic source. The cluster is composed of 8 Intel Pentium-III bi-processors PC working at 550 MHz, and has 4 Gigabytes of RAM memory. It runs under Linux operating system. The developed analysis software package is based on the Multiple SIgnal Classification (MUSIC) algorithm and is written in Fortran. The message-passing part is based upon the LAM programming environment package, an open-source imple- mentation of the Message Passing Interface (MPI). The developed software system includes modules devote to receiving date by internet and graphical applications for the continuous displaying of the processing results. The system has been tested with a data set collected during a seismic experiment conducted on Etna in 1999 when two dense seismic arrays have been deployed on the northeast and the southeast flanks of this volcano. A real time continuous acquisition system has been simulated by

  2. Noncontact optical motion sensing for real-time analysis

    NASA Astrophysics Data System (ADS)

    Fetzer, Bradley R.; Imai, Hiromichi

    1990-08-01

    The adaptation of an image dissector tube (IDT) within the OPTFOLLOW system provides high resolution displacement measurement of a light discontinuity. Due to the high speed response of the IDT and the advanced servo loop circuitry, the system is capable of real time analysis of the object under test. The image of the discontinuity may be contoured by direct or reflected light and ranges spectrally within the field of visible light. The image is monitored to 500 kHz through a lens configuration which transposes the optical image upon the photocathode of the IDT. The photoelectric effect accelerates the resultant electrons through a photomultiplier and an enhanced current is emitted from the anode. A servo loop controls the electron beam, continually centering it within the IDT using magnetic focusing of deflection coils. The output analog voltage from the servo amplifier is thereby proportional to the displacement of the target. The system is controlled by a microprocessor with a 32kbyte memory and provides a digital display as well as instructional readout on a color monitor allowing for offset image tracking and automatic system calibration.

  3. Dimensional analysis of blood vessel images in real time

    NASA Astrophysics Data System (ADS)

    Smith, Peter R.; Eustaquio-Martin, Almudena; Thomason, Harry; Bennett, M.; Thurston, H.

    1996-01-01

    The physiology and pathology of dissected blood vessels are studied by perfusion myography combined with video microscopy. Images of the vessels are formed under diffuse white light illumination and contrast is achieved by differential absorption with respect to the vessel wall. To obtain the vessel dimensional information in quasi real time an edge-tracking algorithm is used, allowing the edges to be found by applying common image processing tools to a very small number of pixels rather than the whole image. Employing a low order optical model of the light transmission properties of vessels with circular cross section, a relationship between the positions of edges found by a typical image processing algorithm and actual dimensions is derived. The dimensional analysis is demonstrated on rat mesenteric resistance arteries (internal diameter less than 300 micrometer) mounted in a perfusion arteriograph. Segments of vessels are secured on two glass cannulae using single strands of a nylon braided suture. The artery is perfused with physiological salt solution and the perfusion pressure maintained at 60 mmHg before starting the experiment. Changes in vascular diameter to the vasoconstrictor noradrenaline and the endothelium-dependent vasodilator acetylcholine were then observed.

  4. Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time RT-PCR (RRT-PCR) is a high throughput molecular diagnostic test used for rapid detection of avian influenza virus (AIV) in clinical samples. However the performance of RRT-PCR can be adversely affected by RT-PCR inhibitors present in the sample. The tested commercial RNA extraction kits ...

  5. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  6. Infrared Signature Analysis: Real Time Monitoring Of Manufacturing Processes

    NASA Astrophysics Data System (ADS)

    Bangs, Edmund R.

    1988-01-01

    The ability to monitor manufacturing processes in an adaptive control mode and perform an inspection in real time is of interest to fabricators in the pressure vessel, aerospace, automotive, nuclear and shipbuilding industries. Results of a series of experiments using infrared thermography as the principal sensing mode are presented to show how artificial intelligence contained in infrared isotherm, contains vast critical process variables. Image processing computer software development has demonstrated in a spot welding application how the process can be monitored and controlled in real time. The IR vision sensor program is now under way. Research thus far has focused on fusion welding, resistance spot welding and metal removal.

  7. Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

    PubMed Central

    Wardhani, Puspa; Yohan, Benediktus; Trimarsanto, Hidayat; Fahri, Sukmal; Setianingsih, Tri Y.; Meutiawati, Febrina

    2014-01-01

    Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was increased to 76.6% (141 out of 184 samples). When tested in 40 sera that were confirmed by virus isolation as the gold standard, the conventional RT-PCR yielded 95% sensitivity while the sensitivity of SYBR Green real-time RT-PCR and Simplexa Dengue assay reached 97.5% and 100%, respectively. The specificities of all methods were 100% when tested in 43 non-dengue illness and 20 healthy human samples. Altogether, our data showed the higher detection rate of Simplexa Dengue compared to conventional and SYBR Green real-time RT-PCR in field/surveillance setting. In conclusion, Simplexa Dengue offers rapid and accurate detection and typing of dengue infection and is suitable for both routine diagnostic and surveillance. PMID:25102066

  8. Generic RT-PCR tests for detection and identification of tospoviruses.

    PubMed

    Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W

    2016-07-01

    A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories. PMID:27036502

  9. ANALYSIS OF REAL-TIME VEHICLE HYDROCARBON EMISSIONS DATA

    EPA Science Inventory

    The report gives results of analyses using real-time dynamometer test emissions data from 13 passenger cars to examine variations in emissions during different speeds or modes of travel. The resulting data provided a way to separately identify idle, cruise, acceleration, and dece...

  10. Real-Time Case Method: Analysis of a Second Implementation

    ERIC Educational Resources Information Center

    Theroux, James M.

    2009-01-01

    In 2005, M. Hopkins and J. Theroux implemented the second example of an experimental case study, at 11 business schools in the United States and Canada. The new type of case study, named the "real-time case (RTC) study," uses the Internet to bring business reality to business courses and to facilitate communication among faculty, students, and the…

  11. Real time in situ chemical characterization of submicrometer organic particles using direct analysis in real time-mass spectrometry.

    PubMed

    Nah, Theodora; Chan, ManNin; Leone, Stephen R; Wilson, Kevin R

    2013-02-19

    Direct analysis in real time mass spectrometry (DART-MS) is used to analyze the surface chemical composition of nanometer-sized organic aerosol particles in real time at atmospheric pressure. By introducing a stream of particles in between the DART ionization source and the atmospheric pressure inlet of the mass spectrometer, the aerosol is exposed to a thermal flow of helium or nitrogen gas containing some fraction of metastable helium atoms or nitrogen molecules. In this configuration, the molecular constituents of organic particles are desorbed, ionized, and detected with reduced molecular ion fragmentation, allowing for compositional identification. Aerosol particles detected include alkanes, alkenes, acids, esters, alcohols, aldehydes, and amino acids. The ion signal produced by DART-MS scales with the aerosol surface area rather than volume, suggesting that DART-MS is a viable technique to measure the chemical composition of the particle interface. For oleic acid, particle size measurements of the aerosol stream exiting the ionization region suggest that the probing depth depends upon the desorption temperature, and the probing depth is estimated to be on the order of 5 nm for a 185 nm diameter particle at a DART heater temperature of 500 °C with nitrogen as the DART gas. The reaction of ozone with submicrometer oleic acid particles is measured to demonstrate the ability of this technique to identify products and quantify reaction rates in a heterogeneous reaction. PMID:23330910

  12. Measurement of avian cytokines with real time RT-PCR following infection with avian influenza

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Both functional and molecular techniques have been employed to measure the production of cytokines following influenza infection. Historically, the use of functional or antibody based techniques were employed in mammalian immunology. In avian immunology, only a few commercial antibodies are availa...

  13. Development of a real-time RT-PCR assay for turkey cytokines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent pathogenesis studies with turkey-origin reoviruses (TRVs) in specific pathogen free (SPF) poults have revealed evidence of immuosuppression associated with TRV infection. The ability to quantitatively measure the abundance and/or relative amounts of cytokine mRNA in poult immune system tissu...

  14. Development of a real-time RT-PCR assay for the detection of marine caliciviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More than forty different marine caliciviruses (family Caliciviridae, genus Vesivirus) have been identified from marine and terrestrial host since their initial isolation in 1972. Marine vesiviruses have previously infected swine along the Western coast of the United States and produce a disease cl...

  15. Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

    PubMed Central

    Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

  16. Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

    PubMed

    Singh, Varinder; Kaul, Sunil C; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

  17. Analysis of real-time reservoir monitoring : reservoirs, strategies, & modeling.

    SciTech Connect

    Mani, Seethambal S.; van Bloemen Waanders, Bart Gustaaf; Cooper, Scott Patrick; Jakaboski, Blake Elaine; Normann, Randy Allen; Jennings, Jim; Gilbert, Bob; Lake, Larry W.; Weiss, Chester Joseph; Lorenz, John Clay; Elbring, Gregory Jay; Wheeler, Mary Fanett; Thomas, Sunil G.; Rightley, Michael J.; Rodriguez, Adolfo; Klie, Hector; Banchs, Rafael; Nunez, Emilio J.; Jablonowski, Chris

    2006-11-01

    The project objective was to detail better ways to assess and exploit intelligent oil and gas field information through improved modeling, sensor technology, and process control to increase ultimate recovery of domestic hydrocarbons. To meet this objective we investigated the use of permanent downhole sensors systems (Smart Wells) whose data is fed real-time into computational reservoir models that are integrated with optimized production control systems. The project utilized a three-pronged approach (1) a value of information analysis to address the economic advantages, (2) reservoir simulation modeling and control optimization to prove the capability, and (3) evaluation of new generation sensor packaging to survive the borehole environment for long periods of time. The Value of Information (VOI) decision tree method was developed and used to assess the economic advantage of using the proposed technology; the VOI demonstrated the increased subsurface resolution through additional sensor data. Our findings show that the VOI studies are a practical means of ascertaining the value associated with a technology, in this case application of sensors to production. The procedure acknowledges the uncertainty in predictions but nevertheless assigns monetary value to the predictions. The best aspect of the procedure is that it builds consensus within interdisciplinary teams The reservoir simulation and modeling aspect of the project was developed to show the capability of exploiting sensor information both for reservoir characterization and to optimize control of the production system. Our findings indicate history matching is improved as more information is added to the objective function, clearly indicating that sensor information can help in reducing the uncertainty associated with reservoir characterization. Additional findings and approaches used are described in detail within the report. The next generation sensors aspect of the project evaluated sensors and packaging

  18. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

    PubMed Central

    Varkonyi-Gasic, Erika; Wu, Rongmei; Wood, Marion; Walton, Eric F; Hellens, Roger P

    2007-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. PMID:17931426

  19. Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections

    PubMed Central

    Alm, Erik; Lesko, Birgitta; Lindegren, Gunnel; Ahlm, Clas; Söderholm, Sandra; Falk, Kerstin I.; Lagerqvist, Nina

    2014-01-01

    Background Dengue is a mosquito-borne viral disease that has become more prevalent in the last few decades. Most patients are viremic when they present with symptoms, and early diagnosis of dengue is important in preventing severe clinical complications associated with this disease and also represents a key factor in differential diagnosis. Here, we designed and validated a hydrolysis-probe-based one-step real-time RT-PCR assay that targets the genomes of dengue virus serotypes 1–4. Methodology/Principal Findings The primers and probe used in our RT-PCR assay were designed to target the 3′ untranslated region of all complete genome sequences of dengue virus available in GenBank (n = 3,305). Performance of the assay was evaluated using in vitro transcribed RNA, laboratory-adapted virus strains, external control panels, and clinical specimens. The linear dynamic range was found to be 104–1011 GCE/mL, and the detection limit was between 6.0×102 and 1.1×103 GCE/mL depending on target sequence. The assay did not cross-react with human RNA, nor did it produce false-positive results for other human pathogenic flaviviruses or clinically important etiological agents of febrile illnesses. We used clinical serum samples obtained from returning travelers with dengue-compatible symptomatology (n = 163) to evaluate the diagnostic relevance of our assay, and laboratory diagnosis performed by the RT-PCR assay had 100% positive agreement with diagnosis performed by NS1 antigen detection. In a retrospective evaluation including 60 archived serum samples collected from confirmed dengue cases 1–9 days after disease onset, the RT-PCR assay detected viral RNA up to 9 days after appearance of symptoms. Conclusions/Significance The validation of the RT-PCR assay presented here indicates that this technique can be a reliable diagnostic tool, and hence we suggest that it be introduced as the method of choice during the first 5 days of dengue symptoms. PMID:25522325

  20. SYBR(®) Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses.

    PubMed

    Poojari, Sudarsana; Alabi, Olufemi J; Okubara, Patricia A; Naidu, Rayapati A

    2016-09-01

    A SYBR(®) Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt-curve analysis (MCA) was optimized for the detection of nine grapevine viruses. The detection limits for simplex qRT-PCR for all nine grapevine viruses were estimated to be in the range of 214-1112 copies of the virus genome. Amplicons with melting temperatures (Tm) separated by at least 2°C in the MCA could differentiate two viruses in the same reaction. Therefore, eight of the nine viruses could be co-diagnosed in five different combinations of duplex assays. Of 305 grape leaf samples from the field or greenhouse, 162 were positive for at least one of the nine grapevine viruses using the duplex qRT-PCR assays. In contrast, only 127 samples were positive using endpoint RT-PCR and PCR assays, indicating the enhanced sensitivity of duplex real-time PCR. In addition, the duplex qRT-PCR assays were be used to detect Grapevine leafroll associated virus 3 (GLRaV-3) in its vector, the grape mealybug (Pseudococcus maritimus Ehrhorn), and Grapevine red blotch-associated virus (GRBaV) in Virginia creeper leafhopper (Erythroneura ziczac Walsh). The simplex and duplex real-time PCR assays developed in this study can be used to examine transmission of co-occruing viruses by insect vectors as well as for rapid and sensitive detection of viruses in infected grapevines. PMID:27246908

  1. Dopamine Receptors in Human Lymphocytes: Radioligand Binding and Quantitative RT-PCR Assays

    PubMed Central

    Kirillova, Galina P.; Hrutkay, Rebecca J.; Shurin, Michael R.; Shurin, Galina V.; Tourkova, Irina L.; Vanyukov, Michael M.

    2008-01-01

    Analysis of dopamine receptors (DR) in lymphocytes of the human peripheral blood mononuclear cell (PBMC) fraction is an attractive tool for evaluation of functional properties of dopaminergic function underlying variation in complex psychological/psychopathological traits. Receptor binding assays (RBA) with selective radioligands, which are widely used in CNS studies, have not produced consistent results when applied to isolated PBMC. We tested the assay conditions that could be essential for detection of DR in human PBMC and their membrane preparations. Using [3H]SCH23390, a dopamine D1-like receptor antagonist, we demonstrated the presence of two binding sites in PBMC-derived membrane fraction. One of them is characterized by the Kd value consistent with that reported for D5 dopamine receptors in human lymphocytes, whereas the other Kd value possibly corresponds to serotonin receptor(s). Although D5 receptor binding sites in PBMC membranes could be characterized by binding assays, the low protein expression and the large volume of blood needed for membrane preparation render the binding method impracticable for individual phenotyping. In contrast, real-time RT-PCR may be used for this purpose, contingent on the relationship between DR expression in the brain and in lymphocytes. The expression of the DRD2-DRD5 genes, as detected by this method, varied widely among samples, whereas the DRD1 expression was not detected. The expression levels were comparable with those in the brain for DRD3 and DRD4, and were significantly lower for DRD2 and DRD5. PMID:18721826

  2. Analysis of gene expression in emerald ash borer (Agrilus planipennis) using quantitative real time-PCR.

    PubMed

    Bhandary, Binny; Rajarapu, Swapna Priya; Rivera-Vega, Loren; Mittapalli, Omprakash

    2010-01-01

    Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1(st)-, 2(nd)-, 3(rd)-, 4(th)-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium. PMID:20445495

  3. Real Time Analysis and Display of Aircraft Approach Maneuvers

    NASA Technical Reports Server (NTRS)

    Lynch, Robert E. (Inventor); Chidester, Thomas R. (Inventor); Lawrence, Robert E. (Inventor)

    2007-01-01

    Method and system for monitoring and comparing, in real time, performance of an aircraft during an approach to touchdown along a conventional approach path and along a contemplated modified approach path to touchdown. In a first procedure, a flight parameter value at a selected location is compared and displayed, for the planned path and for the modified path. In a second procedure, flight parameter values FP(t(sub m)) at a sequence (t(sub n)}n, of measurement times is compared and displayed, for the planned path and for a contemplated or presently-executed modified path. If the flight parameter for the planned path and for the modified path differ too much from each other, the pilot in command has an option of terminating the approach along the modified path.

  4. Real-time analysis of incinerator emissions: The missing link

    SciTech Connect

    Manuel, J.

    1994-11-01

    Incineration has long been, and continues to be, one of the most cost-effective technologies for disposing of the world's growing volume of municipal and hazardous waste. Yet anyone who has been involved in an attempt to site an incinerator in recent years knows the political nightmare this process has become. The public has become extremely suspicious of the health and environmental impact of incinerators, and not without reason. Incinerators have been known to release unacceptably high levels of toxic substances into the air, including dioxins, furans, and other pollutants. Worse, there are no monitoring devices that can continuously measure trace gases in incinerator emissions to allow operators to know exactly what substances are being released and allow for quick corrective action. To address the problems, several teams of university scientists are developing techniques for real-time emissions monitoring that may simultaneously allow industry to operate incinerators in the most efficient manner and assure the public that their health is being protected.

  5. Sensitive, semi-nested RT-PCR amplification of fusion gene sequences for the rapid detection and differentiation of Newcastle disease virus.

    PubMed

    Zhang, Lei; Pan, Zhiming; Geng, Shizhong; Chen, Xiang; Hu, Shunlin; Liu, Huimo; Wu, Yantao; Jiao, Xinan; Liu, Xiufan

    2010-10-01

    A rapid, sensitive and specific semi-nested RT-PCR was developed to detect and differentiate virulent and avirulent strains of Newcastle disease virus (NDV). For a total of 67 NDV strains, the results obtained from the semi-nested RT-PCR were consistent with those from nucleotide sequence analysis, plaque forming assays, mean death time (MDT) measurements and intracerebral pathogenicity index (ICPI). Furthermore, 13 class I NDV strains can be characterized by the semi-nested RT-PCR approach, which was feasible by the conventional methods. The detection limit for the semi-nested RT-PCR was two plaque forming units (PFU) both for NDV strain F48E9 in allantoic fluid and for isolate APMV1/ch/ChinaND4031 in oral or cloacal swabs. In conclusion, this semi-nested RT-PCR method offers a new assay for the rapid detection and differentiation of NDVs. PMID:20219221

  6. Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes

    PubMed Central

    Deepak, SA; Kottapalli, KR; Rakwal, R; Oros, G; Rangappa, KS; Iwahashi, H; Masuo, Y; Agrawal, GK

    2007-01-01

    Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR — detection and expression analysis of gene(s) in real-time — has revolutionized the 21st century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant. PMID:18645596

  7. Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus.

    PubMed

    Delporte, Marianne; Legrand, Guillaume; Hilbert, Jean-Louis; Gagneul, David

    2015-01-01

    Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds, i.e., chlorogenic, isochlorogenic, caftaric, and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR) should be considered. Because cell cultures are a model of choice for specialized metabolism investigations, this study described for the first time the validation of reference genes for this system in chicory. Eighteen potential reference genes were obtained by mining expressed sequence tag databases of chicory for orthologs of Arabidopsis thaliana genes currently used as reference genes. Twelve genes passed the qRT-PCR standard requirements and their expression stability across different samples was tested using three distinct softwares: geNorm, NormFinder, and BestKeeper. In cell cultures grown under various conditions, TIP41 (TIP41 like protein) was shown to be the most stable gene. Further validation of the proposed reference genes was done by normalization of expression levels of a group of genes of interest. In order to assess the potentiality of the proposed list of candidate reference genes, theses genes were in parallel tested on another experimental design, i.e., chicory seedlings. In this case, the best reference gene identified was Clath (Clathrin adaptator complex subunit). The results highlight the importance of the use of properly validated reference genes to achieve relevant interpretation of qRT-PCR analyses. Here, we provide a list of reference genes suitable for future gene expression studies in chicory. PMID:26347767

  8. Combination of conventional immunohistochemistry and qRT-PCR to detect ALK rearrangement

    PubMed Central

    2014-01-01

    Background Compared with FISH and qRT-PCR analyses, immunohistochemistry (IHC) is the preferred screening test in most pathology practices for ALK-rearrangement detection. With 100% sensitivity and 98% specificity, the VENTANA ALK (D5F3) IHC assay has been approved in the EU and some Asian countries for ALK-rearrangement detection. However, an automated Ventana IHC platform is not available in most pathology labs. In this study, we evaluated the applicability of conventional IHC with D5F3 antibody in routine pathological practice and proposed detection methods and procedures that ensure that patients with ALK+ are not missed. Methods FISH and IHC analyses were performed on 297 lung adenocarcinoma cases. VENTANA IHC and qRT-PCR assay were applied to evaluate ALK-fusion status in the discordant cases of FISH and IHC. The association of ALK+ with clinicopathological characteristics was statistically analyzed. Results IHC had 100% sensitivity and 81.8% specificity for detecting ALK+. Eight ALK-expressed cases were ALK-, five of which had ALK fusion detected by qRT-PCR analysis. Three of these five cases showed ALK expression using VENTANA IHC assay. ALK+ was associated with younger age and lymph node metastasis in this Chinese lung adenocarcinoma patient cohort. Conclusions The advantages of low cost and 100% sensitivity allow conventional IHC to serve as a robust diagnostic tool for screening patients with ALK+, especially in pathology labs without a VENTANA IHC platform. For cases in which ALK is weakly expressed, qRT-PCR is necessary as a diagnostic test for ALK-fusion detection. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2269448351088278. PMID:24422905

  9. Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations.

    PubMed

    Carrasco, Adriano de Oliveira Torres; Rodrigues, Juliana Nogueira Martins; Seki, Meire Christina; de Moraes, Fabricio Edgar; Silva, Jaqueline Raymondi; Durigon, Edison Luis; Pinto, Aramis Augusto

    2013-02-01

    The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota--LS--(vaccine strain) and Sao Joao do Meriti--SJM--strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity. PMID:22983878

  10. Comparison and optimization of detection methods for noroviruses in frozen strawberries containing different amounts of RT-PCR inhibitors.

    PubMed

    Bartsch, Christina; Szabo, Kathrin; Dinh-Thanh, Mai; Schrader, Christina; Trojnar, Eva; Johne, Reimar

    2016-12-01

    Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl(®)-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors. PMID:27554153

  11. Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens.

    PubMed

    Cordey, Samuel; Thomas, Yves; Suter, Patricia; Kaiser, Laurent

    2012-01-01

    The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology.This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values ≥ 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques. PMID:22611461

  12. Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens

    PubMed Central

    Cordey, Samuel; Thomas, Yves; Suter, Patricia; Kaiser, Laurent

    2012-01-01

    The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology. This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values ≥ 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques. PMID:22611461

  13. DEVELOPMENT OF AN ASTROVIRUS RT-PCR DETECTION ASSAY FOR USE WITH CONVENTIONAL, REAL-TIME, AND INTEGRATED CELL CULTURE/RT-PCR

    EPA Science Inventory

    Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed an...

  14. Detection of Grapevine Leafroll-associated virus 7 using real-time qRT-PCR and conventional RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nine isolates of Grapevine Leafroll-associated Virus 7 (GLRaV-7) from California have been sequenced to design more sensitive molecular diagnostic tools. These sequences were from the coat protein (CP) and the homologous heat shock protein (hHSP70) genes. Sequence identity among these isolates rang...

  15. RSV Growth and Quantification by Microtitration and qRT-PCR Assays.

    PubMed

    Caidi, Hayat; Harcourt, Jennifer L; Haynes, Lia M

    2016-01-01

    Defective interfering viral particles have been reported as important determinants of the course of viral infection, and they can markedly temper the virulence of the infection. Here, we describe a simple method, based on limiting dilution, for the removal of defective interfering particles from RSV. This method results in a high-titer viral preparation from both HEp-2 and Vero cell lines. We evaluated two concentrations of sucrose to stabilize the virus preparation, and demonstrate that RSV is stable when prepared and stored in 25 % sucrose at -152 °C. In addition, this chapter describes some commonly used methods of RSV titration, detection using microtitration and quantitative real-time RT-PCR, and the use of immunostaining for antigenic characterization. PMID:27464684

  16. Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea

    PubMed Central

    Ma, Rui; Xu, Sheng; Zhao, Yucheng; Xia, Bing; Wang, Ren

    2016-01-01

    Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA

  17. Detection of bovine group a rotavirus using rapid antigen detection kits, rt-PCR and next-generation DNA sequencing.

    PubMed

    Minami-Fukuda, Fujiko; Nagai, Makoto; Takai, Hikaru; Murakami, Toshiaki; Ozawa, Tadashi; Tsuchiaka, Shinobu; Okazaki, Sachiko; Katayama, Yukie; Oba, Mami; Nishiura, Naomi; Sassa, Yukiko; Omatsu, Tsutomu; Furuya, Tetsuya; Koyama, Satoshi; Shirai, Junsuke; Tsunemitsu, Hiroshi; Fujii, Yoshiki; Katayama, Kazuhiko; Mizutani, Tetsuya

    2013-12-30

    We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick 'Eiken' Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 10⁰-10³-fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes. PMID:23912876

  18. Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

    PubMed Central

    Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A.; Brennan, Patrick J.

    2012-01-01

    Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations. PMID:22170923

  19. Structural analysis in real time using continuous monitoring

    NASA Astrophysics Data System (ADS)

    Braunstein, Juergen; Viano, Charles; Hodac, Bernard

    2005-05-01

    OSMOS developed a completely automatic monitoring-system, which is ideal for the determination and monitoring of the structural state of civil engineering structures. Static and dynamic data are recorded as needed and are available via internet for further analysis. In case of bridges, automatic calculation of the axle load of the flowing traffic is implemented, a weigh in motion system (WIMS). When configurable thresholds are exceeded alarms are sent by SMS, e-mail, SNMP-trap for facility-management-systems or by fax.

  20. Human movement analysis with image processing in real time

    NASA Astrophysics Data System (ADS)

    Fauvet, Eric; Paindavoine, Michel; Cannard, F.

    1991-04-01

    In the field of the human sciences, a lot of applications needs to know the kinematic characteristics of the human movements Psycology is associating the characteristics with the control mechanism, sport and biomechariics are associating them with the performance of the sportman or of the patient. So the trainers or the doctors can correct the gesture of the subject to obtain a better performance if he knows the motion properties. Roherton's studies show the children motion evolution2 . Several investigations methods are able to measure the human movement But now most of the studies are based on image processing. Often the systems are working at the T.V. standard (50 frame per secund ). they permit only to study very slow gesture. A human operator analyses the digitizing sequence of the film manually giving a very expensive, especially long and unprecise operation. On these different grounds many human movement analysis systems were implemented. They consist of: - markers which are fixed to the anatomical interesting points on the subject in motion, - Image compression which is the art to coding picture data. Generally the compression Is limited to the centroid coordinates calculation tor each marker. These systems differ from one other in image acquisition and markers detection.

  1. Real Time Intelligent Target Detection and Analysis with Machine Vision

    NASA Technical Reports Server (NTRS)

    Howard, Ayanna; Padgett, Curtis; Brown, Kenneth

    2000-01-01

    We present an algorithm for detecting a specified set of targets for an Automatic Target Recognition (ATR) application. ATR involves processing images for detecting, classifying, and tracking targets embedded in a background scene. We address the problem of discriminating between targets and nontarget objects in a scene by evaluating 40x40 image blocks belonging to an image. Each image block is first projected onto a set of templates specifically designed to separate images of targets embedded in a typical background scene from those background images without targets. These filters are found using directed principal component analysis which maximally separates the two groups. The projected images are then clustered into one of n classes based on a minimum distance to a set of n cluster prototypes. These cluster prototypes have previously been identified using a modified clustering algorithm based on prior sensed data. Each projected image pattern is then fed into the associated cluster's trained neural network for classification. A detailed description of our algorithm will be given in this paper. We outline our methodology for designing the templates, describe our modified clustering algorithm, and provide details on the neural network classifiers. Evaluation of the overall algorithm demonstrates that our detection rates approach 96% with a false positive rate of less than 0.03%.

  2. Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

    PubMed

    Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

    2016-05-01

    Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. PMID:26546824

  3. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses

    PubMed Central

    Müller, Oliver A.; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens. PMID:26313760

  4. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses.

    PubMed

    Müller, Oliver A; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens. PMID:26313760

  5. Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments.

    PubMed

    Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang

    2015-01-01

    To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions. PMID:26528312

  6. Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

    PubMed Central

    Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang

    2015-01-01

    To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions. PMID:26528312

  7. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

    PubMed Central

    2014-01-01

    Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study

  8. Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.)

    PubMed Central

    Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min

    2016-01-01

    Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR. PMID:27524995

  9. Does the length of specimen storage affect influenza testing results by real-time reverse transcription-polymerase chain reaction? an analysis of influenza surveillance specimens, 2008 to 2010.

    PubMed

    Caselton, Dl; Arunga, G; Emukule, G; Muthoka, P; Mayieka, L; Kosgey, A; Ochola, R; Waiboci, Lw; Feikin, Dr; Mott, Ja; Breiman, Rf; Katz, Ma

    2014-01-01

    In some influenza surveillance systems, timely transport to laboratories for reverse transcription-polymerase chain reaction (RT-PCR) testing is challenging.Guidelines suggest that samples can be stored at 4°Cfor up to 96 hours but the effect of longer storage times has not been systematically evaluated. We collected nasopharyngeal and oropharyngeal specimens from patients in Kenya and stored them in viral transport medium at 2 to 8°C before testing for influenza A and B using real-time RT-PCR. From April 2008 to November 2010, we collected 7,833 samples; 940 (12%) were positive for influenza. In multivariable analysis, specimens stored for six days were less likely to be influenza-positive compared to specimens stored between zero and one day (adjusted odds ratio (a OR): 0.49, 95%confidence interval (CI): 0.27–0.93). There was no statistically significant difference in influenza positivity of specimens stored for five days compared to zero to one day. There was no statistically significant relationship between days in refrigeration and cycle threshold(Ct) values for positive samples (p=0.31). We found that samples could remain in storage for at least five days without affecting the proportion-positive of samples,potentially increasing the feasibility of including influenza surveillance sites in remote areas. PMID:25232920

  10. Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.).

    PubMed

    Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min

    2016-01-01

    Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR. PMID:27524995

  11. Effective detection of human noroviruses in Hawaiian waters using enhanced RT-PCR methods.

    PubMed

    Tong, Hsin-I; Connell, Christina; Boehm, Alexandria B; Lu, Yuanan

    2011-11-15

    The current recreational water quality criteria using growth-based measurements of fecal indicator bacteria (FIB) concentration have their limitations for swimmer protection. To evaluate the possible use of enteric viruses as an improved indicator of human sewage contamination in recreational waters for enhanced health risk assessment, human norovirus (huNoV) was tested as a model in this study. To establish a highly sensitive protocol for effective huNoV detection in waters, 16 published and newly designed reverse transcription polymerase chain reaction (RT-PCR) primer pairs specific for huNoV genogroup I (GI) and genogroup II (GII) were comparatively evaluated side-by-side using single sources of huNoV RNA stock extracted from local clinical isolates. Under optimized conditions, these RT-PCR protocols shared a very different pattern of detection sensitivity for huNoV. The primer sets COG2F/COG2R and QNIF4/NV1LCR were determined to be the most sensitive ones for huNoV GII and GI, respectively, with up to 10(5)- and 10(6)-fold more sensitive as compared to other sets tested. These two sensitive protocols were validated by positive detection of huNoV in untreated and treated urban wastewater samples. In addition, these RT-PCR protocols enabled detection of the prevalence of huNoV in 5 (GI) and 10 (GII) of 16 recreational water samples collected around the island of O'ahu, which was confirmed by DNA sequencing and sequence analysis. Findings from this study support the possible use of enteric viral pathogens for environmental monitoring and argue the importance and essentiality for such monitoring activity to ensure a safe use of recreational waters. PMID:21945082

  12. Lung Cancer Lymph Node Micrometastasis Detection Using RT-PCR – Correlation with Vascular Endothelial Growth Factor (VEGF) expression

    PubMed Central

    Nwogu, Chukwumere E.; Yendamuri, Sai; Tan, Wei; Kannisto, Eric; Bogner, Paul; Morrison, Carl; Cheney, Richard; Dexter, Elisabeth; Picone, Anthony; Hennon, Mark; Hutson, Alan; Reid, Mary; Adjei, Alex; Demmy, Todd L.

    2013-01-01

    Objectives Lymph node (LN) staging provides critical information in non-small cell lung cancer (NSCLC) patients. Lymphangiogenesis may be an important contributor to the pathophysiology of lymphatic metastases. We hypothesized that the presence of lymph node micrometastases positively correlates with VEGF-A/C/D and VEGF-receptor-3 (lymphangiogenic factors) expression in lymph nodes. Methods Forty NSCLC patients had pre-operative PET-CT and mediastinoscopy. RT-PCR assays for mRNA expression of epithelial markers (CK-7, CEACAM-5 and PLUNC) were performed in selected fluorodeoxyglucose (FDG)-avid lymph nodes. VEGF-A/C/D and VEGF-receptor-3 expression levels were measured in primary tumors and lymph nodes. Wilcoxon rank sum test was run for the association between the RT-PCR epithelial marker levels and VEGF expression levels in the LNs. Results RT-PCR for CK-7, CEACAM5 or PLUNC indicated lymph node micrometastatic disease in 19 of 35 patients (54%). There was a high correlation between detection of micrometastases and VEGF-A/C/D or VEGF-receptor-3 expression levels in lymph nodes. Median follow-up was 12.6 months. Conclusions RT-PCR analysis of FDG-avid lymph nodes results in up-staging of patients. Micrometastases correlate with the expression of VEGF in lymph nodes in NSCLC patients. This may reflect the role of lymphangiogenesis in promoting metastases. PMID:23414988

  13. Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents.

    PubMed

    Takekawa, John Y; Iverson, Samuel A; Schultz, Annie K; Hill, Nichola J; Cardona, Carol J; Boyce, Walter M; Dudley, Joseph P

    2010-06-01

    Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID((R)), Idaho Technologies, Salt Lake City, UT) that employed lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies) was compared to virus isolation combined with real-time RT-PCR conducted in a laboratory. This study included both field- and experimental-based sampling. Field samples were collected from migratory shorebirds captured in northern California, while experimental samples were prepared by spiking fecal material with an H6N2 AIV isolate. Results indicated that the portable rRT-PCR unit had equivalent specificity to virus isolation with no false positives, but sensitivity was compromised at low viral titers. Use of portable rRT-PCR with lyophilized reagents may expedite surveillance results, paving the way to a better understanding of wild bird involvement in HPAIV H5N1 transmission. PMID:20206650

  14. Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents

    USGS Publications Warehouse

    Takekawa, John Y.; Iverson, Samuel A.; Schultz, Annie K.; Hill, Nichola J.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.

    2010-01-01

    Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID(Registered), Idaho Technologies, Salt Lake City, UT) that employed lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies) was compared to virus isolation combined with real-time RT-PCR conducted in a laboratory. This study included both field and experimental-based sampling. Field samples were collected from migratory shorebirds captured in northern California, while experimental samples were prepared by spiking fecal material with an H6N2 AIV isolate. Results indicated that the portable rRT-PCR unit had equivalent specificity to virus isolation with no false positives, but sensitivity was compromised at low viral titers. Use of portable rRT-PCR with lyophilized reagents may expedite surveillance results, paving the way to a better understanding of wild bird involvement in HPAIV H5N1 transmission.

  15. An integrated software solution for real-time PCR analysis based on microfluidic biochip

    NASA Astrophysics Data System (ADS)

    Wang, Qinghui; Gong, Haiqing

    2003-04-01

    In this paper, we present an integrated and automated prototype system which has been developed for real-time polymerase chain reaction (PCR) analysis based on microfluidic PCR array chips. The system integrates the PCR thermal cycling and optical detection capabilities to enable real-time fluorescence imaging and image processing for data analysis. The main advantage of the system is that it provides a solution that can rapidly perform and evaluate PCR experiment simultaneously on microfluidic PCR array chips. The system has demonstrated fast and efficient on-chip real-time PCR analysis using human genomic DNA samples. The implementation of the system integration is a multi-thread Windows software with component structure which is written in Visual C++.

  16. Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

    PubMed Central

    Zhang, Haili; Zhu, Guan

    2015-01-01

    Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds. PMID:26441920

  17. Detection of Avian bornavirus in multiple tissues of infected psittacine birds using real-time reverse transcription polymerase chain reaction.

    PubMed

    Delnatte, Pauline; Mak, Matthew; Ojkic, Davor; Raghav, Raj; DeLay, Josepha; Smith, Dale A

    2014-03-01

    Avian bornavirus (ABV), the cause of proventricular dilation disease in psittacine birds, has been detected in multiple tissues of infected birds using immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR). In the current study, real-time RT-PCR, using primers targeting the ABV matrix gene, was used to detect ABV in 146 tissues from 7 ABV-infected psittacine birds. Eighty-six percent of the samples tested positive, with crossing point values ranging from 13.82 to 37.82 and a mean of 22.3. These results were compared to the findings of a previous study using gel-based RT-PCR and IHC on the same samples. The agreement between the 2 RT-PCR techniques was 91%; when tests disagreed it was because samples were negative using gel-based RT-PCR but positive on real-time RT-PCR. Agreement with IHC was 77%; 16 out of 74 samples were negative using IHC but positive on real-time RT-PCR. The results suggest that real-time RT-PCR is a more sensitive technique than gel-based RT-PCR and IHC to detect ABV in tissues. The tissues that were ranked most frequently as having a high amount of viral RNA were proventriculus, kidney, colon, cerebrum, and cerebellum. Skeletal muscle, on the other hand, was found to have a consistently low amount of viral RNA. PMID:24518276

  18. An integrated, subsurface characterization system for real-time, in-situ field analysis

    SciTech Connect

    Baumgart, C.W.; Creager, J.; Mathes, J.; Pounds, T.; VanDeusen, A.; Warthen, B.

    1996-02-01

    This paper describes current efforts at AlliedSignal Federal Manufacturing and Technologies (FM and T) to develop and field an in-situ, data analysis platform to acquire, process, and display site survey data in near real-time. In past years, FM and T has performed a number of site survey tasks. Each of these surveys was unique in application as well as in the type of data processing and analysis that was required to extract and visualize useful site characterization information. However, common to each of these surveys were the following specific computational and operational requirements: (1) a capability to acquire, process, and visualize the site survey data in the field; (2) a capability to perform all processing in a timely fashion (ideally real-time); and (3) a technique for correlating (or fusing) data streams from multiple sensors. Two more general, but no less important, requirements include system architecture modularity and positioning capability. Potential applications include: survey, evaluation, and remediation of numerous Department of Defense and Department of Energy waste sites; real-time detection and characterization of unexploded ordnance and landmines; survey, evaluation, and remediation of industrial waste sites; location of underground utility lines; and providing law enforcement agencies with real-time surveys of crime scenes. The paper describes an integrated data acquisition, processing, and visualization platform that is capable of performing in-situ data processing, interpretation, and visualization in real-time.

  19. Implementation and application of real-time motion analysis based on passive markers.

    PubMed

    Baroni, G; Ferrigno, G; Pedotti, A

    1998-11-01

    A method for real-time motion analysis based on passive markers is presented. An opto-electronic automatic motion analyser was used as hardware platform and the real-time operation was based on the interfacing between two levels of the system architecture. True real-time acquisition, processing and representation of two-dimensional and three-dimensional kinematics data were implemented through a newly conceived data acquisition procedure and high speed optimisation of the kinematics data processing. The method allows one to operate the motion analysis system in real-time; even when the data elaboration unit is required to perform other processing functions, the only consequence is a decrease in system sampling rate. The maximum number of processed and plotted markers in three dimensions at the highest system sampling rate (100 Hz) turned out to be suitable for the implementation of analytical and visual kinematics biofeedback. An example of the achievable level of complexity in terms of marker disposition model and graphic representation is reported by describing a demonstration of the real-time representation of human face movements. A clinical application of the method for patient position definition and control at radiotherapy units is presented. PMID:10367459

  20. Analysis of liver connexin expression using reverse transcription quantitative real-time polymerase chain reaction

    PubMed Central

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283

  1. Analysis of Liver Connexin Expression Using Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction.

    PubMed

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin RNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction, and data analysis. PMID:27207283

  2. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation

    PubMed Central

    Goodell, Christa K.; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O’Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J.

    2016-01-01

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10−1 to 10−8). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated. PMID:26733728

  3. Modular Sampling and Analysis Techniques for the Real-Time Analysis of Human Breath

    SciTech Connect

    Frank, M; Farquar, G; Adams, K; Bogan, M; Martin, A; Benner, H; Spadaccini, C; Steele, P; Davis, C; Loyola, B; Morgan, J; Sankaran, S

    2007-07-09

    At LLNL and UC Davis, we are developing several techniques for the real-time sampling and analysis of trace gases, aerosols and exhaled breath that could be useful for a modular, integrated system for breath analysis. Those techniques include single-particle bioaerosol mass spectrometry (BAMS) for the analysis of exhaled aerosol particles or droplets as well as breath samplers integrated with gas chromatography mass spectrometry (GC-MS) or MEMS-based differential mobility spectrometry (DMS). We describe these techniques and present recent data obtained from human breath or breath condensate, in particular, addressing the question of how environmental exposure influences the composition of breath.

  4. Multi-channel holographic birfurcative neural network system for real-time adaptive EOS data analysis

    NASA Technical Reports Server (NTRS)

    Liu, Hua-Kuang; Diep, J.; Huang, K.

    1991-01-01

    Viewgraphs on multi-channel holographic bifurcative neural network system for real-time adaptive Earth Observing System (EOS) data analysis are presented. The objective is to research and develop an optical bifurcating neuromorphic pattern recognition system for making optical data array comparisons and to evaluate the use of the system for EOS data classification, reduction, analysis, and other applications.

  5. Novel Laser-Based Technique is Ideal for Real-Time Environmental Analysis

    ERIC Educational Resources Information Center

    Journal of Chemical Education, 2005

    2005-01-01

    Ocean Optics offers laser-induced breakdown spectrometer systems (LIBS) that can be used to identify light to heavy metals in a variety of sample types and geometries in environmental analysis applications. LIBS are versatile, real-time, high-resolution analyzers for qualitative analysis, in less than one second, of every element in solids,…

  6. Evaluation of oxidative stress via total antioxidant status, sialic acid, malondialdehyde and RT-PCR findings in sheep affected with bluetongue

    PubMed Central

    Aytekin, I.; Aksit, H.; Sait, A.; Kaya, F.; Aksit, D.; Gokmen, M.; Baca, A. Unsal

    2015-01-01

    Introduction Bluetongue (BT) is a non-contagious infectious disease of ruminants. The disease agent bluetongue virus (BTV) is classified in the Reoviridae family Orbivirus. Aims and objectives The aim of this study was to determine serum malondialdehyde (MDA), total antioxidative stres (TAS), total sialic acid (TSA), ceruloplasmin, triglyceride, alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), cholesterol, creatinine, albumin, and total protein levels in sheep with and without bluetongue (BT). Materials and Methods The study included 13 Sakiz crossbreed sheep, aged 1–4 years and usually in the last stage of pregnancy, as the BT group and a control group consisting of 10 healthy sheep. All sheep were clinically examined before collecting blood samples. Serum ALT, AST, cholesterol, triglyceride, albumin, GGT, total protein, creatinine and TAS levels were measured using commercially available kits as per manufacturer's recommendations using a Biochemistry Auto Analyzer (Sinnowa D280, China). Serum lipid peroxidation was estimated through a previously described method in which MDA reacts with thiobarbituric acid (TBA) to form a coloured complex at a maximum absorbance of 535 nm. The TSA value was measured at 549 nm using the method described by Warren (1959): sialic acid was oxidised to formyl-pyruvic acid, which reacts with TBA to form a pink product. The ceruloplasmin concentration was measured according to Sunderman and Nomoto (1970): ceruloplasmin and p-phenylenediamine formed a coloured oxidation product that was proportional to the concentration of serum ceruloplasmin. Real time RT-PCR and conventional RT-PCR were performed as described by Shaw and others (2007). Results Biochemistry analysis of serum showed that in the BT group, TSA, MDA, triglyceride and ALT and AST were higher and that ceruloplasmin and TAS were lower than in the control group. Serum albumin, cholesterol, creatinine, total protein and GGT did

  7. Evaluation of Four Different Systems for Extraction of RNA from Stool Suspensions Using MS-2 Coliphage as an Exogenous Control for RT-PCR Inhibition

    PubMed Central

    Shulman, Lester M.; Hindiyeh, Musa; Muhsen, Khitam; Cohen, Dani; Mendelson, Ella; Sofer, Danit

    2012-01-01

    Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A); MagNA Pure LC2.0 Automatic extractor (B); KingFisher (C); and NucliSENS EasyMag (D) RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) of MS-2 coliphage spiked into each system’s lysis buffer served as an external control for both. Cycle thresholds (Cts) of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93) or varying amounts of inhibitors (n = 92). Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) of the first 93 RNA extracts, and 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples. PMID:22815706

  8. Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool

    PubMed Central

    Vongsouvath, Manivanh; Phommasone, Koukeo; Sengvilaipaseuth, Onanong; Kosoltanapiwat, Nathamon; Chantratita, Narisara; Blacksell, Stuart D.; Lee, Sue J.; de Lamballerie, Xavier; Mayxay, Mayfong; Keomany, Sommay; Newton, Paul N.; Dubot-Pérès, Audrey

    2016-01-01

    Background Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. Methodology/Principal Findings We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. Conclusions/Significance Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions. PMID:27159058

  9. A simplified holographic-interferometry technique for real-time flow visualization and analysis

    NASA Technical Reports Server (NTRS)

    Long, S. A.; Spencer, R. C.

    1974-01-01

    A holographic-interferometry technique for flow visualization and analysis that produces real-time moire fringes is described from both experimental and application considerations. It has three chief advantages: real-time data for continuous observation and photography, ease of optical adjustment, and capability of using ordinary-glass test-section windows without affecting the results. A theoretical discussion is presented describing the formation of the fringes in holographic terms and then comparing this result to that which is obtained from a conventional moire approach. A discussion on obtaining density information from the fringe pattern is also included.

  10. An optical real-time 3D measurement for analysis of facial shape and movement

    NASA Astrophysics Data System (ADS)

    Zhang, Qican; Su, Xianyu; Chen, Wenjing; Cao, Yiping; Xiang, Liqun

    2003-12-01

    Optical non-contact 3-D shape measurement provides a novel and useful tool for analysis of facial shape and movement in presurgical and postsurgical regular check. In this article we present a system, which allows a precise 3-D visualization of the patient's facial before and after craniofacial surgery. We discussed, in this paper, the real time 3-D image capture, processing and the 3-D phase unwrapping method to recover complex shape deformation when the movement of the mouth. The result of real-time measurement for facial shape and movement will be helpful for the more ideal effect in plastic surgery.

  11. A new method for robust quantitative and qualitative analysis of real-time PCR

    PubMed Central

    Shain, Eric B.; Clemens, John M.

    2008-01-01

    An automated data analysis method for real-time PCR needs to exhibit robustness to the factors that routinely impact the measurement and analysis of real-time PCR data. Robust analysis is paramount to providing the same interpretation for results regardless of the skill of the operator performing or reviewing the work. We present a new method for analysis of real-time PCR data, the maxRatio method, which identifies a consistent point within or very near the exponential region of the PCR signal without requiring user intervention. Compared to other analytical techniques that generate only a cycle number, maxRatio generates several measurements of amplification including cycle numbers and relative measures of amplification efficiency and curve shape. By using these values, the maxRatio method can make highly reliable reactive/nonreactive determination along with quantitative evaluation. Application of the maxRatio method to the analysis of quantitative and qualitative real-time PCR assays is shown along with examples of method robustness to, and detection of, amplification response anomalies. PMID:18603594

  12. Protein Analysis Using Real-Time PCR Instrumentation: Incorporation in an Integrated, Inquiry-Based Project

    ERIC Educational Resources Information Center

    Southard, Jonathan N.

    2014-01-01

    Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein…

  13. Real-time fMRI-based activation analysis and stimulus control

    NASA Astrophysics Data System (ADS)

    Moench, Tobias; Hollmann, Maurice; Bernarding, Johannes

    2007-03-01

    The real-time analysis of brain activation using functional MRI data offers a wide range of new experiments such as investigating self-regulation or learning strategies. However, besides special data acquisition and real-time data analysing techniques such examination requires dynamic and adaptive stimulus paradigms and self-optimising MRI-sequences. This paper presents an approach that enables the unified handling of parameters influencing the different software systems involved in the acquisition and analysing process. By developing a custom-made Experiment Description Language (EDL) this concept is used for a fast and flexible software environment which treats aspects like extraction and analysis of activation as well as the modification of the stimulus presentation. We describe how extracted real-time activation is subsequently evaluated by comparing activation patterns to previous acquired templates representing activated regions of interest for different predefined conditions. According to those results the stimulus presentation is adapted. The results showed that the developed system in combination with EDL is able to reliably detect and evaluate activation patterns in real-time. With a processing time for data analysis of about one second the approach is only limited by the natural time course of the hemodynamic response function of the brain activation.

  14. A rule-based system for real-time analysis of control systems

    NASA Technical Reports Server (NTRS)

    Larson, Richard R.; Millard, D. Edward

    1992-01-01

    An approach to automate the real-time analysis of flight critical health monitoring and system status is being developed and evaluated at the NASA Dryden Flight Research Facility. A software package was developed in-house and installed as part of the extended aircraft interrogation and display system. This design features a knowledge-base structure in the form of rules to formulate interpretation and decision logic of real-time data. This technique has been applied for ground verification and validation testing and flight testing monitoring where quick, real-time, safety-of-flight decisions can be very critical. In many cases post processing and manual analysis of flight system data are not required. The processing is described of real-time data for analysis along with the output format which features a message stack display. The development, construction, and testing of the rule-driven knowledge base, along with an application using the X-31A flight test program, are presented.

  15. In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

    PubMed

    Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H

    2016-01-01

    Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions. PMID:27525844

  16. An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended cons...

  17. Methodology for object-oriented real-time systems analysis and design: Software engineering

    NASA Technical Reports Server (NTRS)

    Schoeffler, James D.

    1991-01-01

    Successful application of software engineering methodologies requires an integrated analysis and design life-cycle in which the various phases flow smoothly 'seamlessly' from analysis through design to implementation. Furthermore, different analysis methodologies often lead to different structuring of the system so that the transition from analysis to design may be awkward depending on the design methodology to be used. This is especially important when object-oriented programming is to be used for implementation when the original specification and perhaps high-level design is non-object oriented. Two approaches to real-time systems analysis which can lead to an object-oriented design are contrasted: (1) modeling the system using structured analysis with real-time extensions which emphasizes data and control flows followed by the abstraction of objects where the operations or methods of the objects correspond to processes in the data flow diagrams and then design in terms of these objects; and (2) modeling the system from the beginning as a set of naturally occurring concurrent entities (objects) each having its own time-behavior defined by a set of states and state-transition rules and seamlessly transforming the analysis models into high-level design models. A new concept of a 'real-time systems-analysis object' is introduced and becomes the basic building block of a series of seamlessly-connected models which progress from the object-oriented real-time systems analysis and design system analysis logical models through the physical architectural models and the high-level design stages. The methodology is appropriate to the overall specification including hardware and software modules. In software modules, the systems analysis objects are transformed into software objects.

  18. Multiplex titration RT-PCR: rapid determination of gene expression patterns for a large number of genes

    NASA Technical Reports Server (NTRS)

    Nebenfuhr, A.; Lomax, T. L.

    1998-01-01

    We have developed an improved method for determination of gene expression levels with RT-PCR. The procedure is rapid and does not require extensive optimization or densitometric analysis. Since the detection of individual transcripts is PCR-based, small amounts of tissue samples are sufficient for the analysis of expression patterns in large gene families. Using this method, we were able to rapidly screen nine members of the Aux/IAA family of auxin-responsive genes and identify those genes which vary in message abundance in a tissue- and light-specific manner. While not offering the accuracy of conventional semi-quantitative or competitive RT-PCR, our method allows quick screening of large numbers of genes in a wide range of RNA samples with just a thermal cycler and standard gel analysis equipment.

  19. Novel, In-House, SYBR Green Based One-Step rRT-PCR: Rapid and Accurate Diagnosis of Crimean-Congo Hemorrhagic Fever Virus in Suspected Patients From Iran

    PubMed Central

    Zahraei, Bentolhoda; Hashemzadeh, Mohammad Sadegh; Najarasl, Mohammad; Zahiriyeganeh, Samaneh; Tat, Mahdi; Metanat, Maliheh; Sepehri Rad, Nahid; Khansari-nejad, Behzad; Zafari, Ehsan; Sharti, Mojtaba; Dorostkar, Ruhollah

    2016-01-01

    Background The Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans, with a high mortality rate. Since, there is no approved vaccine or specific treatment for CCHF, an early and accurate diagnosis, as well as reliable surveillance, is essential for case management and patient improvement. Objectives For this research, our aim was to evaluate the application of a novel SYBR Green based one-step real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay for the in-house diagnosis of the CCHF virus. Patients and Methods In this experimental study, the highly conserved S-region sequence of the CCHF viral genome was first adapted from GenBank, and the specific primers targeting this region were designed. Then, the viral RNA was extracted from 75 serum samples from different patients in eastern Iran. The sensitivity and specificity of the primers were also evaluated in positive serum samples previously confirmed to have the CCHF virus, by this one-step rRT-PCR assay, as well as a DNA sequencing analysis. Results From a total of 75 suspected serum samples, 42 were confirmed to be positive for CCHF virus, with no false-positives detected by the sequencing results. After 40 amplification cycles, the melting curve analysis revealed a mean melting temperature (Tm) of 86.5 ± 0.6°C (quite different from those of the primer-dimers), and the positive samples showed only a small variation in the parameters. In all of the positive samples, the predicted length of 420 bp was confirmed by electrophoresis. Moreover, the sensitivity test showed that this assay can detect less than 20 copies of viral RNA per reaction. Conclusions This study showed that this novel one-step rRT-PCR assay is a rapid, reliable, repeatable, specific, sensitive, and simple tool for the detection of the CCHF virus. PMID:27099688

  20. Real-time Sample Analysis using Sampling Probe and Miniature Mass Spectrometer

    PubMed Central

    Chen, Chien-Hsun; Lin, Ziqing; Tian, Ran; Shi, Riyi; Cooks, R. Graham; Ouyang, Zheng

    2016-01-01

    A miniature mass spectrometry system with a sampling probe has been developed for real-time analysis of chemicals from sample surfaces. The sampling probe is 1.5m in length and is comprised of one channel for introducing the spray and the other channel for transferring the charged species back to the Mini MS. This system provides a solution to the problem of real-time mass spectrometry analysis of a three-dimensional object in the field and is successful with compounds including those in inks, agrochemicals, explosives, and animal tissues. This system can be implemented in the form of a backpack MS with a sampling probe for forensic analysis or in the form of a compact MS with an intra-surgical probe for tissue analysis. PMID:26237577

  1. Real-time sample analysis using a sampling probe and miniature mass spectrometer.

    PubMed

    Chen, Chien-Hsun; Lin, Ziqing; Tian, Ran; Shi, Riyi; Cooks, R Graham; Ouyang, Zheng

    2015-09-01

    A miniature mass spectrometry system with a sampling probe has been developed for real-time analysis of chemicals from sample surfaces. The sampling probe is 1.5 m in length and is comprised of one channel for introducing the spray and the other channel for transferring the charged species back to the Mini MS. This system provides a solution to the problem of real-time mass spectrometry analysis of a three-dimensional object in the field and is successful with compounds including those in inks, agrochemicals, explosives, and animal tissues. This system can be implemented in the form of a backpack MS with a sampling probe for forensic analysis or in the form of a compact MS with an intrasurgical probe for tissue analysis. PMID:26237577

  2. Biofilm reactor based real-time analysis of biochemical oxygen demand.

    PubMed

    Liu, Changyu; Jia, Jianbo; Dong, Shaojun

    2013-04-15

    We reported a biofilm reactor (BFR) based analytical system for real-time biochemical oxygen demand (BOD) monitoring. It does not need a blank solution and other chemical reagents to operate. The initial dissolved oxygen (DO) in sample solution was measured as blank, while DO in the BFR effluent was measured as response. The DO difference obtained before and after the sample solution flowed through the BFR was regarded as an indicator of real-time BOD. The analytical performance of this reagent-free BFR system was equal to the previous BFR system operated using phosphate buffer saline (PBS) and high purity deionized water in reproducibility, accuracy and long-term stability. Besides, this method embraces many notable advantages, such as no secondary pollution. Additionally, the sample solutions are free from temperature controlling and air-saturation before injection. Significantly, this is a real-time BOD analysis method. This method was successfully carried out in a simulated emergency, and the obtained results agreed well with conventional BOD₅. These advantages, coupled with simplicity in device, convenience in operation and minimal maintenance, make such a reagent-free BFR analytical system promising for practical BOD real-time warning. PMID:23228491

  3. Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti

    PubMed Central

    2012-01-01

    Background Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. Results We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). Conclusions The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods. PMID:22264275

  4. Real-time GPS seismology using a single receiver: method comparison, error analysis and precision validation

    NASA Astrophysics Data System (ADS)

    Li, Xingxing

    2014-05-01

    Earthquake monitoring and early warning system for hazard assessment and mitigation has traditional been based on seismic instruments. However, for large seismic events, it is difficult for traditional seismic instruments to produce accurate and reliable displacements because of the saturation of broadband seismometers and problematic integration of strong-motion data. Compared with the traditional seismic instruments, GPS can measure arbitrarily large dynamic displacements without saturation, making them particularly valuable in case of large earthquakes and tsunamis. GPS relative positioning approach is usually adopted to estimate seismic displacements since centimeter-level accuracy can be achieved in real-time by processing double-differenced carrier-phase observables. However, relative positioning method requires a local reference station, which might itself be displaced during a large seismic event, resulting in misleading GPS analysis results. Meanwhile, the relative/network approach is time-consuming, particularly difficult for the simultaneous and real-time analysis of GPS data from hundreds or thousands of ground stations. In recent years, several single-receiver approaches for real-time GPS seismology, which can overcome the reference station problem of the relative positioning approach, have been successfully developed and applied to GPS seismology. One available method is real-time precise point positioning (PPP) relied on precise satellite orbit and clock products. However, real-time PPP needs a long (re)convergence period, of about thirty minutes, to resolve integer phase ambiguities and achieve centimeter-level accuracy. In comparison with PPP, Colosimo et al. (2011) proposed a variometric approach to determine the change of position between two adjacent epochs, and then displacements are obtained by a single integration of the delta positions. This approach does not suffer from convergence process, but the single integration from delta positions to

  5. The hsp 16 gene of the probiotic Lactobacillus acidophilus is differently regulated by salt, high temperature and acidic stresses, as revealed by reverse transcription quantitative PCR (qRT-PCR) analysis.

    PubMed

    Capozzi, Vittorio; Arena, Mattia Pia; Crisetti, Elisabetta; Spano, Giuseppe; Fiocco, Daniela

    2011-01-01

    Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group. PMID:21954366

  6. One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

    PubMed

    De Paula, Sérgio Oliveira; de Melo Lima, Cristiane; Torres, Maria Paula; Pereira, Márcia Rodrigues Garbin; Lopes da Fonseca, Benedito Antônio

    2004-08-01

    Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis. PMID:15163417

  7. Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    PubMed

    Oguchi, Taichi; Onishi, Mari; Minegishi, Yasutaka; Kurosawa, Yasunori; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2009-06-01

    A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this. PMID:19602858

  8. Application of real time systems to the analysis of neuronal dynamics

    NASA Astrophysics Data System (ADS)

    Cymbalyuk, Gennady; Shilnikov, Andrey

    2008-03-01

    Neurons exhibit various activity regimes and transitions in between. The central pattern generator controlling the leech's heartbeat contains identified pairs of mutually inhibitory neurons (Calabrese et al. 1995). We describe real time systems approaches to the analysis of their activity. The hybrid system consists of a living neuron and a model neuron (or an artificial silicon neuron) interacting in the real time. Dynamic clamp is used to implement artificial ionic currents and synapses in the system (Sharp et al. 1993). Our study determines the mechanisms underlying and regulating bursting activity, based on intrinsic membrane dynamics and network interactions. The complexity of endogenous dynamics originates from the diversity of ionic currents operating on different time scales. Hybrid system analysis and slow-fast dynamical systems analysis have been combined in our studies of bursting, its origin and transformations in heart interneurons both as single cells and in the mutually inhibitory configuration.

  9. Real time automatic detection of bearing fault in induction machine using kurtogram analysis.

    PubMed

    Tafinine, Farid; Mokrani, Karim

    2012-11-01

    A proposed signal processing technique for incipient real time bearing fault detection based on kurtogram analysis is presented in this paper. The kurtogram is a fourth-order spectral analysis tool introduced for detecting and characterizing non-stationarities in a signal. This technique starts from investigating the resonance signatures over selected frequency bands to extract the representative features. The traditional spectral analysis is not appropriate for non-stationary vibration signal and for real time diagnosis. The performance of the proposed technique is examined by a series of experimental tests corresponding to different bearing conditions. Test results show that this signal processing technique is an effective bearing fault automatic detection method and gives a good basis for an integrated induction machine condition monitor. PMID:23145702

  10. Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR.

    PubMed

    Apaza, Sonia; Espetia, Susan; Gilman, Robert H; Montenegro, Sonia; Pineda, Susana; Herhold, Fanny; Pomari, Romeo; Kosek, Margaret; Vu, Nancy; Saito, Mayuko

    2012-01-01

    consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups. PMID:22898754

  11. Evaluation of a multiplex real-time polymerase chain reaction assay for the detection of influenza and respiratory syncytial viruses.

    PubMed

    Esposito, Susanna; Scala, Alessia; Tagliabue, Claudia; Zampiero, Alberto; Bianchini, Sonia; Principi, Nicola

    2016-01-01

    Nasopharyngeal swabs from 424 children were used to compare the performances of the new multiplex real-time polymerase chain reaction (RT-PCR) RIDA®GENE Flu & RSV kit and monospecific RT-PCR assays in detecting respiratory syncytial and influenza viruses. The easy-to-use kit was highly sensitive and specific and is recommended for routine practice. PMID:26458277

  12. Real-time open-loop frequency response analysis of flight test data

    NASA Technical Reports Server (NTRS)

    Bosworth, J. T.; West, J. C.

    1986-01-01

    A technique has been developed to compare the open-loop frequency response of a flight test aircraft real time with linear analysis predictions. The result is direct feedback to the flight control systems engineer on the validity of predictions and adds confidence for proceeding with envelope expansion. Further, gain and phase margins can be tracked for trends in a manner similar to the techniques used by structural dynamics engineers in tracking structural modal damping.

  13. A Real-Time Analysis and Feedback System for Quality Control of Dam Foundation Grouting Engineering

    NASA Astrophysics Data System (ADS)

    Zhong, D. H.; Yan, F. G.; Li, M. C.; Huang, C. X.; Fan, K.; Tang, J. F.

    2015-09-01

    Real-time analysis and feedback systems play a vital role in obtaining good results from grouting processes. However, when there are intense construction schedules and complex geological structures, it is difficult for existing systems to provide to site engineers, prior to the borehole construction, prompt and accurate feedback, such as detailed geological information about grouting boreholes, which limits the use of such systems in practical applications. This paper proposes combining a three-dimensional (3D) geological model with real-time data collection technology in a system for both monitoring grouting, and providing analysis and feedback. This integrated grouting model, which comprises the geological model, the grouting borehole model and the grouting parameter database set, can be coupled and associated dynamically with grouting data. Additionally, the following methods are applied in this system: real-time grouting data processing and a monitoring alarm, prediction and visualization of geological conditions, forecasting of rock uplift, and visualization analysis of grouting parameters. The application of this system in Hydropower Project A, China is used as a case study. The predictions of geological conditions are closely matched with the actual situation, and this system can be used to monitor construction processes remotely and to help site engineers to design reasonable construction plans, optimize layouts for grouting boreholes and adjust construction measures.

  14. Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

    PubMed Central

    Yan, Zhaoping; Gao, Jinhang; Lv, Xiuhe; Yang, Wenjuan; Wen, Shilei; Tong, Huan; Tang, Chengwei

    2016-01-01

    The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis. PMID:27069927

  15. Multi-layer holographic bifurcative neural network system for real-time adaptive EOS data analysis

    NASA Technical Reports Server (NTRS)

    Liu, Hua-Kuang; Huang, K.; Diep, J.

    1992-01-01

    Optical data processing techniques have the inherent advantage of high data throughout, low weight and low power requirements. These features are particularly desirable for onboard spacecraft in-situ real-time data analysis and data compression applications. The proposed multi-layer optical holographic neural net pattern recognition technique will utilize the nonlinear photorefractive devices for real-time adaptive learning to classify input data content and recognize unexpected features. Information can be stored either in analog or digital form in a nonlinear photorefractive device. The recording can be accomplished in time scales ranging from milliseconds to microseconds. When a system consisting of these devices is organized in a multi-layer structure, a feed forward neural net with bifurcating data classification capability is formed. The interdisciplinary research will involve the collaboration with top digital computer architecture experts at the University of Southern California.

  16. Multi-layer holographic bifurcative neural network system for real-time adaptive EOS data analysis

    NASA Technical Reports Server (NTRS)

    Liu, Hua-Kuang; Huang, K. S.; Diep, J.

    1993-01-01

    Optical data processing techniques have the inherent advantage of high data throughout, low weight and low power requirements. These features are particularly desirable for onboard spacecraft in-situ real-time data analysis and data compression applications. the proposed multi-layer optical holographic neural net pattern recognition technique will utilize the nonlinear photorefractive devices for real-time adaptive learning to classify input data content and recognize unexpected features. Information can be stored either in analog or digital form in a nonlinear photofractive device. The recording can be accomplished in time scales ranging from milliseconds to microseconds. When a system consisting of these devices is organized in a multi-layer structure, a feedforward neural net with bifurcating data classification capability is formed. The interdisciplinary research will involve the collaboration with top digital computer architecture experts at the University of Southern California.

  17. A CAMAC based real-time noise analysis system for nuclear reactors

    NASA Astrophysics Data System (ADS)

    Ciftcioglu, Özer

    1987-05-01

    A CAMAC based real-time noise analysis system was designed for the TRIGA MARK II nuclear reactor at the Institute for Nuclear Energy, Istanbul. The input analog signals obtained from the radiation detectors are introduced to the system through CAMAC interface. The signals converted into digital form are processed by a PDP-11 computer. The fast data processing based on auto/cross power spectral density computations is carried out by means of assembly written FFT algorithms in real-time and the spectra obtained are displayed on a CAMAC driven display system as an additional monitoring device. The system has the advantage of being software programmable and controlled by a CAMAC system so that it is operated under program control for reactor surveillance, anomaly detection and diagnosis. The system can also be used for the identification of nonstationary operational characteristics of the reactor in long term by comparing the noise power spectra with the corresponding reference noise patterns prepared in advance.

  18. Single-channel multiplexing without melting curve analysis in real-time PCR.

    PubMed

    Lee, Young-Jo; Kim, Daeyoung; Lee, Kihoon; Chun, Jong-Yoon

    2014-01-01

    Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics. PMID:25501038

  19. Real-Time Volumetric Phase Monitoring: Advancing Chemical Analysis by Countercurrent Separation.

    PubMed

    Pauli, Guido F; Pro, Samuel M; Chadwick, Lucas R; Burdick, Thomas; Pro, Luke; Friedl, Warren; Novak, Nick; Maltby, John; Qiu, Feng; Friesen, J Brent

    2015-07-21

    Countercurrent separation (CCS) utilizes the differential partitioning behavior of analytes between two immiscible liquid phases. We introduce the first platform ("CherryOne") capable of real-time monitoring, metering, and control of the dynamic liquid-liquid CCS process. Automated phase monitoring and volumetrics are made possible with an array of sensors, including the new permittivity-based phase metering apparatus (PMA). Volumetric data for each liquid phase are converted into a dynamic real-time display of stationary phase retention (Sf) and eluent partition coefficients (K), which represent critical parameters of CCS reproducibility. When coupled with the elution-extrusion operational mode (EECCC), automated Sf and K determination empowers untargeted and targeted applications ranging from metabolomic analysis to preparative purifications. PMID:26152934

  20. Single-channel multiplexing without melting curve analysis in real-time PCR

    PubMed Central

    Lee, Young-Jo; Kim, Daeyoung; Lee, Kihoon; Chun, Jong-Yoon

    2014-01-01

    Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics. PMID:25501038

  1. Real-time analysis of mechanical and electrical resonances with open-source sound card software

    NASA Astrophysics Data System (ADS)

    Makan, G.; Kopasz, K.; Gingl, Z.

    2014-01-01

    We present an easily reproducible, open-source, sound card based experimental set-up to support transfer function measurement. Our system is able to visualize the signals of mechanical and electrical resonances and their spectra in real time. We give a brief description of the system, and show some examples of electrical and mechanical resonance experiments that are supported by the system. The theoretical background, experimental set-up, component selection and digital signal processing are all discussed, and more detailed information (building instructions, software download) is provided on a dedicated web page (www.noise.inf.u-szeged.hu/edudev/RealTimeAnalysisOfResonances/). The experimental set-up can support the undergraduate and graduate education of students of physics, physics education and engineering by means of experimental demonstrations and laboratory exercises. The very low cost, high efficiency and transparent system provides a scalable experimental environment that can be easily built in several instances.

  2. Real-time lossy compression of hyperspectral images using iterative error analysis on graphics processing units

    NASA Astrophysics Data System (ADS)

    Sánchez, Sergio; Plaza, Antonio

    2012-06-01

    Hyperspectral image compression is an important task in remotely sensed Earth Observation as the dimensionality of this kind of image data is ever increasing. This requires on-board compression in order to optimize the donwlink connection when sending the data to Earth. A successful algorithm to perform lossy compression of remotely sensed hyperspectral data is the iterative error analysis (IEA) algorithm, which applies an iterative process which allows controlling the amount of information loss and compression ratio depending on the number of iterations. This algorithm, which is based on spectral unmixing concepts, can be computationally expensive for hyperspectral images with high dimensionality. In this paper, we develop a new parallel implementation of the IEA algorithm for hyperspectral image compression on graphics processing units (GPUs). The proposed implementation is tested on several different GPUs from NVidia, and is shown to exhibit real-time performance in the analysis of an Airborne Visible Infra-Red Imaging Spectrometer (AVIRIS) data sets collected over different locations. The proposed algorithm and its parallel GPU implementation represent a significant advance towards real-time onboard (lossy) compression of hyperspectral data where the quality of the compression can be also adjusted in real-time.

  3. Multiplex RT-PCR detection of H3N2 influenza A virus in dogs.

    PubMed

    Lee, EunJung; Kim, Eun-Ju; Kim, Bo-Hye; Song, Jae-Young; Cho, In-Soo; Shin, Yeun-Kyung

    2016-02-01

    A multiplex RT-PCR (mRT-PCR) assay to detect H3N2 CIV genomic segments was developed as a rapid and cost-effective method. Its performance was evaluated with forty-six influenza A viruses from different hosts using three primer sets which amplify four segments of H3N2 CIV simultaneously. The mRT-PCR has been successful in detecting the viral segments, indicating that it can improve the speed of diagnosis for H3N2 CIV and its reassortants. PMID:26738688

  4. Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis

    PubMed Central

    Ma, Jun-Xin; Wang, Lin-Nong; Zhou, Ru-Xia; Yu, Yang; Du, Tong-Xin

    2016-01-01

    AIM To design, optimize and validate a rapid, internally controlled real-time polymerase chain reaction (RT-PCR) test for herpes simplex virus (HSV) in the diagnosis of necrotizing herpes stromal keratitis. METHODS Tears alone or together with corneal epithelium scrapings from 30 patients (30 eyes) suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores. RESULTS The positive rate (46.4%) in the corneal epithelium group before the therapy was significantly higher than that (13.3%) in the tears group (P=0.006). There were 13 positive HSV patients before the therapy, the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group (paired t-test, P=0.0397). Multilevel mixed-effects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant (P=0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment (r=0.844, P<0.0001). CONCLUSION RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis. PMID:27275421

  5. Quantitative RT-PCR assessment of melanoma cells in peripheral blood during immunotherapy for metastatic melanoma.

    PubMed

    Schmidt, H; Sørensen, B S; von der Maase, H; Bang, C; Agger, R; Hokland, M; Nexo, E

    2002-12-01

    Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival. PMID:12459648

  6. Molecular characterization and clinical impact of TMPRSS2-ERG rearrangement on prostate cancer: comparison between FISH and RT-PCR.

    PubMed

    Fernández-Serra, A; Rubio, L; Calatrava, A; Rubio-Briones, J; Salgado, R; Gil-Benso, R; Espinet, B; García-Casado, Z; López-Guerrero, J A

    2013-01-01

    Prostate cancer (PCa) is a very heterogeneous disease, and there are constraints in its current diagnosis. Serum PSA levels, digital rectal examination (DRE), and histopathologic analysis often drive to overdiagnosis and overtreatment. Since 2005, the presence of the genetic rearrangement between transmembrane-serine protease gene (TMPRSS2) and the erythroblast transformation-specific (ETS) member ERG (v-ets erythroblastosis virus E26 oncogene homolog avian) has been demonstrated in almost half of PCa cases. Both FISH and RT-PCR are useful tools for detecting these rearrangements, but very few comparatives between both techniques have been published. In this study, we included FFPE tumors from 294 PCa patients treated with radical prostatectomy with more than 5 years of followup. We constructed a total of 20 tissue microarrays in order to perform break-apart and tricolor probe FISH approaches that were compared with RT-PCR, showing a concordance of 80.6% (P < 0.001). The presence of TMPRSS2-ERG rearrangement was observed in 56.6% of cases. No association between TMPRSS2-ERG status and clinicopathological parameters nor biochemical progression and clinical progression free survival was found. In conclusion, this study demonstrates that both FISH and RT-PCR are useful tools in the assessment of the TMPRSS2-ERG fusion gene status in PCa patients and that this genetic feature per se lacks prognostic value. PMID:23781502

  7. Portable Dew Point Mass Spectrometry System for Real-Time Gas and Moisture Analysis

    NASA Technical Reports Server (NTRS)

    Arkin, C.; Gillespie, Stacey; Ratzel, Christopher

    2010-01-01

    A portable instrument incorporates both mass spectrometry and dew point measurement to provide real-time, quantitative gas measurements of helium, nitrogen, oxygen, argon, and carbon dioxide, along with real-time, quantitative moisture analysis. The Portable Dew Point Mass Spectrometry (PDP-MS) system comprises a single quadrupole mass spectrometer and a high vacuum system consisting of a turbopump and a diaphragm-backing pump. A capacitive membrane dew point sensor was placed upstream of the MS, but still within the pressure-flow control pneumatic region. Pressure-flow control was achieved with an upstream precision metering valve, a capacitance diaphragm gauge, and a downstream mass flow controller. User configurable LabVIEW software was developed to provide real-time concentration data for the MS, dew point monitor, and sample delivery system pressure control, pressure and flow monitoring, and recording. The system has been designed to include in situ, NIST-traceable calibration. Certain sample tubing retains sufficient water that even if the sample is dry, the sample tube will desorb water to an amount resulting in moisture concentration errors up to 500 ppm for as long as 10 minutes. It was determined that Bev-A-Line IV was the best sample line to use. As a result of this issue, it is prudent to add a high-level humidity sensor to PDP-MS so such events can be prevented in the future.

  8. Real-time analysis of breath for carbon tetrachloride and its metabolites

    SciTech Connect

    Kenny, D.V.; Callahan, P.J.; Thrall, K.D.

    1995-12-31

    Real-time breath analysis offers a non-invasive method to detect exposure to toxic air pollutants. Breath measurements are useful in environmental exposure studies, and may provide evidence about previous long-term or repeated exposures in environments that are not easy to monitor. If breath samples are collected during or immediately following a short term exposure, breath measurements can be used to predict the peak exposure. Previous breath sampling methodologies have been to collect repeated 1-minute breath samples at 5 minute intervals. Although this method can aid in describing the rapid exponential emptying of the blood compartment that occurs following peak exposure, sampling 5 minute intervals still forces an approximation of the true shape of the clearance kinetics. The authors have developed a methodology to quantitively measure the concentration of exhaled volatile compounds in real-time using laboratory rats. Continuously monitoring breath for a parent toxicant and its metabolite(s) provides input into physiologically based pharmacokinetic (PBPK) models to describe the bio-kinetics of a chemical in the body. Real-time analyses are better than batch sampling methods because of the rapidly changing kinetics of elimination of some volatile chemicals from the body.

  9. Circadian Variation of the Human Metabolome Captured by Real-Time Breath Analysis

    PubMed Central

    Martinez-Lozano Sinues, Pablo; Tarokh, Leila; Li, Xue; Kohler, Malcolm; Brown, Steven A.; Zenobi, Renato; Dallmann, Robert

    2014-01-01

    Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36–49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings. PMID:25545545

  10. Near Real Time Review of Instrument Performance using the Airborne Data Processing and Analysis Software Package

    NASA Astrophysics Data System (ADS)

    Delene, D. J.

    2014-12-01

    Research aircraft that conduct atmospheric measurements carry an increasing array of instrumentation. While on-board personnel constantly review instrument parameters and time series plots, there are an overwhelming number of items. Furthermore, directing the aircraft flight takes up much of the flight scientist time. Typically, a flight engineer is given the responsibility of reviewing the status of on-board instruments. While major issues like not receiving data are quickly identified during a flight, subtle issues like low but believable concentration measurements may go unnoticed. Therefore, it is critical to review data after a flight in near real time. The Airborne Data Processing and Analysis (ADPAA) software package used by the University of North Dakota automates the post-processing of aircraft flight data. Utilizing scripts to process the measurements recorded by data acquisition systems enables the generation of data files within an hour of flight completion. The ADPAA Cplot visualization program enables plots to be quickly generated that enable timely review of all recorded and processed parameters. Near real time review of aircraft flight data enables instrument problems to be identified, investigated and fixed before conducting another flight. On one flight, near real time data review resulted in the identification of unusually low measurements of cloud condensation nuclei, and rapid data visualization enabled the timely investigation of the cause. As a result, a leak was found and fixed before the next flight. Hence, with the high cost of aircraft flights, it is critical to find and fix instrument problems in a timely matter. The use of a automated processing scripts and quick visualization software enables scientists to review aircraft flight data in near real time to identify potential problems.

  11. Real-time whole slide mosaicing for non-automated microscopes in histopathology analysis

    PubMed Central

    Gherardi, Alessandro; Bevilacqua, Alessandro

    2013-01-01

    Context: Mosaics of Whole Slides (WS) are a valuable resource for pathologists to have the whole sample available at high resolution. The WS mosaic provides pathologists with an overview of the whole sample at a glance, helping them to make a reliable diagnosis. Despite recent solutions exist for creating WS mosaics based, for instance, on automated microscopes with motorized stages or WS scanner, most of the histopathology analysis are still performed in laboratories endowed with standard manual stage microscopes. Nowadays, there are lots of dedicated devices and hardware to achieve WS automatically and in batch, but only few of them are conceived to work tightly connected with a microscope and none of them is capable of working in real-time with common light microscopes. However, there is a need of having low-cost yet effective mosaicing applications even in small laboratories to improve routine histopathological analyses or to perform remote diagnoses. Aims: The purpose of this work is to study and develop a real-time mosaicing algorithm working even using non-automated microscopes, to enable pathologists to achieve WS while moving the holder manually, without exploiting any dedicated device. This choice enables pathologists to build WS in real-time, while browsing the sample as they are accustomed to, helping them to identify, locate, and digitally annotate lesions fast. Materials and Methods: Our method exploits fast feature tracker and frame to frame registration that we implemented on common graphics processing unit cards. The system work with common light microscopes endowed with a digital camera and connected to a commodity personal computer. Result and Conclusion: The system has been tested on several histological samples to test the effectiveness of the algorithm to work with mosaicing having different appearances as far as brightness, contrast, texture, and detail levels are concerned, attaining sub-pixel registration accuracy at real-time interactive

  12. Development of a Rift Valley fever real-time RT-PCR assay that can detect all three genome segments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Outbreaks of Rift Valley fever in Kenya, Madagascar, Mauritania, and South Africa had devastating effects on livestock and human health. In addition, this disease is a food security issue for endemic countries. There is growing concern for the potential introduction of RVF into non-endemic countries...

  13. Factors associated with real-time RT-PCR cycle threshold values among medically attended influenza episodes.

    PubMed

    Spencer, Sarah; Chung, Jessie; Thompson, Mark; Piedra, Pedro A; Jewell, Alan; Avadhanula, Vasanthi; Mei, Minghua; Jackson, Michael L; Meece, Jennifer; Sundaram, Maria; Belongia, Edward A; Cross, Rachel; Johnson, Emileigh; Bullotta, Arlene; Rinaldo, Charles; Gaglani, Manjusha; Murthy, Kempapura; Clipper, Lydia; Berman, LaShondra; Flannery, Brendan

    2016-04-01

    We evaluated the cycle threshold (CT) values of 1,160 influenza A positive and 806 influenza B positive specimens from two seasons of the US Flu VE Network to identify factors associated with CT values. Low CT values (high genomic load) were associated with shorter intervals between illness onset and specimen collection, young age (ages 3-8 years old), and self-rated illness severity for both influenza A and B. Low CT values were also associated with reported fever/feverishness and age ≥65 years for influenza A. PMID:26334765

  14. Detection of Grapevine leafroll-associated virus 7 using real-time PCR and conventional RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grapevine leafroll-associated virus 7 (GLRaV-7) is an unassigned member in the Closteroviridae family that was first recorded in an asymptomatic white-berried grapevine cultivar from Albania. In California, the virus has been detected in several cultivars including Chardonnay, Merlot, Pinot Noir, Em...

  15. Design of a multiplex real time RT-PCR assay to detect Newcastle disease viruses from classes I and II

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease (ND) is a major concern for poultry producers around the world and the rapid diagnosis of an outbreak is crucial to any control program. Prompt detection of the causative agent of ND, virulent strains of Newcastle disease virus (vNDV), and differentiation of these viruses from tho...

  16. Feasibility of real-time satisfaction surveys through automated analysis of patients' unstructured comments and sentiments.

    PubMed

    Alemi, Farrokh; Torii, Manabu; Clementz, Laura; Aron, David C

    2012-01-01

    This article shows how sentiment analysis (an artificial intelligence procedure that classifies opinions expressed within the text) can be used to design real-time satisfaction surveys. To improve participation, real-time surveys must be radically short. The shortest possible survey is a comment card. Patients' comments can be found online at sites organized for rating clinical care, within e-mails, in hospital complaint registries, or through simplified satisfaction surveys such as "Minute Survey." Sentiment analysis uses patterns among words to classify a comment into a complaint, or praise. It further classifies complaints into specific reasons for dissatisfaction, similar to broad categories found in longer surveys such as Consumer Assessment of Healthcare Providers and Systems. In this manner, sentiment analysis allows one to re-create responses to longer satisfaction surveys from a list of comments. To demonstrate, this article provides an analysis of sentiments expressed in 995 online comments made at the RateMDs.com Web site. We focused on pediatrician and obstetrician/gynecologist physicians in District of Columbia, Maryland, and Virginia. We were able to classify patients' reasons for dissatisfaction and the analysis provided information on how practices can improve their care. This article reports the accuracy of classifications of comments. Accuracy will improve as the number of comments received increases. In addition, we ranked physicians using the concept of time-to-next complaint. A time-between control chart was used to assess whether time-to-next complaint exceeded historical patterns and therefore suggested a departure from norms. These findings suggest that (1) patients' comments are easily available, (2) sentiment analysis can classify these comments into complaints/praise, and (3) time-to-next complaint can turn these classifications into numerical benchmarks that can trace impact of improvements over time. The procedures described in the

  17. DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES

    EPA Science Inventory

    Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...

  18. High altitude aircraft direction using real-time scientific analysis of telemetered data

    NASA Technical Reports Server (NTRS)

    Wegener, Steven; Chan, K. Roland; Pfister, Leonhard; Arvesen, John

    1991-01-01

    Microscale and some mesoscale scientific targets are often too small and fast moving in a dynamic atmosphere. In this environment the ER-2 may spend only a small time sampling the phenomena of interest. The ER-2 scientists currently rely on post-flight analysis to determine the success of the mission and to provide input for planning the next flight. Real time analysis, combined with realistic modeling, can provide important clues to significantly enhance the probability of success in the investigation of specific scientific targets.

  19. Combining real-time monitoring and knowledge-based analysis in MARVEL

    NASA Technical Reports Server (NTRS)

    Schwuttke, Ursula M.; Quan, A. G.; Angelino, R.; Veregge, J. R.

    1993-01-01

    Real-time artificial intelligence is gaining increasing attention for applications in which conventional software methods are unable to meet technology needs. One such application area is the monitoring and analysis of complex systems. MARVEL, a distributed monitoring and analysis tool with multiple expert systems, was developed and successfully applied to the automation of interplanetary spacecraft operations at NASA's Jet Propulsion Laboratory. MARVEL implementation and verification approaches, the MARVEL architecture, and the specific benefits that were realized by using MARVEL in operations are described.

  20. Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

    PubMed

    Brault, Aaron C; Fang, Ying; Reisen, William K

    2015-05-01

    Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. PMID:26334826

  1. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption.

    PubMed

    Yi, Jianzhong; Liu, Chengqian

    2011-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses. PMID:21535888

  2. Overview of real-time computer systems technical analysis of the Modcomp implementation of a proprietary system MAX IV'' and real-time UNIX system REAL/IX''

    SciTech Connect

    Cummings, J.

    1990-10-01

    There many applications throughout industry and government requiring real-time computing. Any application that monitors and/or controls a process would fit into this category. Some examples are: Nuclear power plants, Steel mills, Space program, etc. General Atomics uses eight real-time computer systems for control and high speed data acquisition required to run the nuclear fusion experiments. Real-Time computing can be defined as the ability to respond to asynchronous external events in a predictable (preferably fast) time frame. Real-Time computer systems are similar to other computers in many ways and may by used for general computing requirements such as Time-Sharing. However special hardware, operating systems and software had to be developed to meet the requirement for real-time computing. Traditionally, real-time computing has been a realm of proprietary operating systems with real-time applications written in FORTRAN and assembly language. In the past, these systems adequately served the needs of the real-time world. Many of these systems that were developed 15 years ago are still being used today. However the real-time world is now changing, demanding new systems to be developed. This paper gives a description of general real-time computer systems and how they differ from other systems. However, the main purpose of this paper is to give a detailed technical description of the hardware and operating systems of an existing proprietary system and a real-time UNIX system. The two real-time computer systems described in detail are Modcomp Classic III/95 with the MAX IV operating system and Modcomp TRI-D 9750 with the REAL/IX.2 operating system.

  3. Real time video processing software for the analysis of endoscopic guided-biopsies

    NASA Astrophysics Data System (ADS)

    Ordoñez, C.; Bouchet, A.; Pastore, J.; Blotta, E.

    2011-12-01

    The severity in Barrett esophagus disease is, undoubtedly, the possibility of its malignization. To make an early diagnosis in order to avoid possible complications, it is absolutely necessary collect biopsies to make a histological analysis. This should be done under endoscopic control to avoid mucus areas that may co-exist within the columnar epithelial, which could lead to a false diagnosis. This paper presents a video processing software in real-time in order to delineate and enhance areas of interest to facilitate the work of the expert.

  4. Real-time Visualisation and Analysis of Tera-scale Datasets

    NASA Astrophysics Data System (ADS)

    Fluke, Christopher J.

    2015-03-01

    As we move ever closer to the Square Kilometre Array era, support for real-time, interactive visualisation and analysis of tera-scale (and beyond) data cubes will be crucial for on-going knowledge discovery. However, the data-on-the-desktop approach to analysis and visualisation that most astronomers are comfortable with will no longer be feasible: tera-scale data volumes exceed the memory and processing capabilities of standard desktop computing environments. Instead, there will be an increasing need for astronomers to utilise remote high performance computing (HPC) resources. In recent years, the graphics processing unit (GPU) has emerged as a credible, low cost option for HPC. A growing number of supercomputing centres are now investing heavily in GPU technologies to provide O(100) Teraflop/s processing. I describe how a GPU-powered computing cluster allows us to overcome the analysis and visualisation challenges of tera-scale data. With a GPU-based architecture, we have moved the bottleneck from processing-limited to bandwidth-limited, achieving exceptional real-time performance for common visualisation and data analysis tasks.

  5. A NEAR REAL-TIME BERYLLIUM MONITOR WITH CAM AND WIPE ANALYSIS CAPABILITIES

    SciTech Connect

    D.T. Kendrick; Steven Saggese

    2002-12-01

    Science & Engineering Associates, Inc. (SEA), under contract No. DE-AC26-00NT40768, was tasked by the US Department of Energy--National Energy Technology Laboratory to develop and test a near real-time beryllium monitor for airborne and surface measurements. Recent public awareness of the health risks associated with exposure to beryllium has underscored the need for better, faster beryllium monitoring capabilities within the DOE. A near real-time beryllium monitor will offer significant improvements over the baseline monitoring technology currently in use. Whereas the baseline technology relies upon collecting an air sample on a filter and the subsequent analysis of the filter by an analytical laboratory, this effort developed a monitor that offers near real-time measurement results while work is in progress. Since the baseline typically only offers after-the-fact documentation of exposure levels, the near real-time capability provides a significant increase in worker protection. The beryllium monitor developed utilizes laser induced breakdown spectroscopy, or LIBS as the fundamental measurement technology. LIBS has been used in a variety of laboratory and field based instrumentation to provide real-time, and near-real-time elemental analysis capabilities. LIBS is an analytical technique where a pulsed high energy laser beam is focused to a point on the sample to be interrogated. The high energy density produces a small high temperature plasma plume, sometimes called a spark. The conditions within this plasma plume result in the constituent atoms becoming excited and emitting their characteristic optical emissions. The emission light is collected and routed to an optical spectrometer for quantitative spectral analysis. Each element has optical emissions, or lines, of a specific wavelength that can be used to uniquely identify that element. In this application, the intensity of the beryllium emission is used to provide a quantitative measure of the abundance of the

  6. Real-time Functional Analysis of Inertial Microfluidic Devices via Spectral Domain Optical Coherence Tomography.

    PubMed

    Dong, Biqin; Chen, Siyu; Zhou, Fan; Chan, Christina H Y; Yi, Ji; Zhang, Hao F; Sun, Cheng

    2016-01-01

    We report the application of spectral-domain optical coherence tomography (SD-OCT) technology that enables real-time functional analysis of sorting microparticles and cells in an inertial microfluidic device. We demonstrated high-speed, high-resolution acquisition of cross-sectional images at a frame rate of 350 Hz, with a lateral resolution of 3 μm and an axial resolution of 1 μm within the microfluidic channel filled with water. We analyzed the temporal sequence of cross-sectional SD-OCT images to determine the position and diameter of microspheres in a spiral microfluidic channel under various flow rates. We used microspheres with known diameters to validate the sub-micrometer precision of the particle size analysis based on a scattering model of spherical microparticles. An additional investigation of sorting live HT-29 cells in the spiral microfluidic channel indicated that the distribution of cells within in the microchannel has a close correspondence with the cells' size distribution. The label-free real-time imaging and analysis of microscale particles in flow offers robustness for practical applications with live cells and allows us to better understand the mechanisms of particle separations in microfluidic sorting systems. PMID:27619202

  7. Real-time flight test analysis and display techniques for the X-29A aircraft

    NASA Technical Reports Server (NTRS)

    Hicks, John W.; Petersen, Kevin L.

    1988-01-01

    The X-29A advanced technology demonstrator flight envelope expansion program and the subsequent flight research phase gave impetus to the development of several innovative real-time analysis and display techniques. These new techniques produced significant improvements in flight test productivity, flight research capabilities, and flight safety. These techniques include real-time measurement and display of in-flight structural loads, dynamic structural mode frequency and damping, flight control system dynamic stability and control response, aeroperformance drag polars, and aircraft specific excess power. Several of these analysis techniques also provided for direct comparisons of flight-measured results with analytical predictions. The aeroperformance technique was made possible by the concurrent development of a new simplified in-flight net thrust computation method. To achieve these levels of on-line flight test analysis, integration of ground and airborne systems was required. The capability of NASA Ames Research Center, Dryden Flight Research Facility's Western Aeronautical Test Range was a key factor in enabling implementation of these methods.

  8. [Research of Left Ventricle Function Analysis Using Real-time Cardiac Magnetic Resonance Imaging].

    PubMed

    Yang, Fan; He, Yan; Zhang, Jie; Wu, Yin

    2015-12-01

    Real-time free breathing cardiac cine imaging is a reproducible method with shorter acquisition time and without breath-hold for cardiac magnetic resonance imaging. However, the detection of end-diastole and end-systole frames of real-time free breathing cardiac cine imaging for left ventricle function analysis is commonly completed by visual identification, which is time-consuming and laborious. In order to save processing time, we propose a method for semi-automatic identification of end-diastole and end-systole frames. The method fits respiratory motion signal and acquires the expiration phase, end-diastole and end-systole frames by cross correlation coefficient. The procedure successfully worked on ten healthy volunteers and validated by the analysis of left ventricle function compared to the standard breath-hold steady-state free precession cardiac cine imaging without any significant statistical differences. The results demonstrated that the present method could correctly detect end-diastole and end-systole frames. In the future, this technique may be used for rapid left ventricle function analysis in clinic. PMID:27079101

  9. Identification and evaluation of endogenous control genes for use in quantitative RT-PCR during wheat (Triticum aestivum L.) grain filling.

    PubMed

    Wu, D; Dong, J; Yao, Y J; Zhao, W C; Gao, X

    2015-01-01

    The use of appropriate reference genes is essential for the generation of accurate and biologically meaningful results from quantitative real-time PCR (qRT-PCR) analysis. However, studies have found that the expression of most commonly used reference genes is not always independent of the tissues, treatments, or developmental stages studied. geNormPlus, NormFinder, and BestKeeper, were applied and the expression stability of nine candidate genes was evaluated in different data sets during wheat grain development. Varying degrees of diversity in either single or multiple reference genes were observed among the results generated from the different computer programs, parameters, and data sets. Therefore, the reliability of identified reference genes in the flag leaf and the complete set of samples was estimated by monitoring the expression dynamics of three NAM genes (TaNAM-A1, TaNAM-B1, and TaNAM-B2). The results suggest that a single control gene identified by geNormPlus for use with the complete set of samples, and multiple reference genes selected by geNormPlus and NormFinder exclusively for the flag leaf outperformed others owing to the consistent results with previous analyses of these genes, which were normalized against a verified single control gene. Given the limit of NormFinder in gene numbers of multiple reference genes, robust quantification can be achieved by normalizing against Ta27922 or multiple reference genes chosen by geNormPlus for individual tissues. PMID:26400285

  10. Surrogate analysis and index developer (SAID) tool and real-time data dissemination utilities

    USGS Publications Warehouse

    Domanski, Marian M.; Straub, Timothy D.; Wood, Molly S.; Landers, Mark N.; Wall, Gary R.; Brady, Steven J.

    2015-01-01

    The use of acoustic and other parameters as surrogates for suspended-sediment concentrations (SSC) in rivers has been successful in multiple applications across the Nation. Critical to advancing the operational use of surrogates are tools to process and evaluate the data along with the subsequent development of regression models from which real-time sediment concentrations can be made available to the public. Recent developments in both areas are having an immediate impact on surrogate research, and on surrogate monitoring sites currently in operation. The Surrogate Analysis and Index Developer (SAID) standalone tool, under development by the U.S. Geological Survey (USGS), assists in the creation of regression models that relate response and explanatory variables by providing visual and quantitative diagnostics to the user. SAID also processes acoustic parameters to be used as explanatory variables for suspended-sediment concentrations. The sediment acoustic method utilizes acoustic parameters from fixed-mount stationary equipment. The background theory and method used by the tool have been described in recent publications, and the tool also serves to support sediment-acoustic-index methods being drafted by the multi-agency Sediment Acoustic Leadership Team (SALT), and other surrogate guidelines like USGS Techniques and Methods 3-C4 for turbidity and SSC. The regression models in SAID can be used in utilities that have been developed to work with the USGS National Water Information System (NWIS) and for the USGS National Real-Time Water Quality (NRTWQ) Web site. The real-time dissemination of predicted SSC and prediction intervals for each time step has substantial potential to improve understanding of sediment-related water-quality and associated engineering and ecological management decisions.

  11. Real-Time Drug Release Analysis of Enzyme and pH Responsive Polysaccharide Nanovesicles.

    PubMed

    Pramod, Poothayil Subash; Deshpande, Nilesh Umakant; Jayakannan, Manickam

    2015-08-20

    The accurate estimation of drug release kinetics of polymeric vehicles is an indispensable prerequisite for the developments of successful drug carriers for cancer therapy. The present investigation reports the development of time-resolved fluorescence spectroscopic approach for the real-time release kinetics of fluorophore loaded polysaccharide vesicles that are potential vectors in cancer treatment. The polysaccharide vesicles were custom designed with appropriate enzyme and pH responsiveness and loaded with water-soluble biocompatible fluorophore Rhodamine B (Rh-B). The semipermeable membrane dialysis method along with steady state absorbance spectroscopic technique was found to be inaccurate for the estimation of drug release. Time correlated single photon counting (TCSPC) technique was found to exhibit significant difference in excited state decay profiles and fluorescent lifetime of Rh-B in the free and polymer bound states. This enabled the establishment of real-time drug release protocols by TCSPC method for polysaccharide vesicles that are responsible to pH and enzyme with respect to intracellular compartments. Real-time analysis predicted the release kinetics 20-25% higher accuracy when compared to the dialysis method under in vitro conditions. Moreover, the ability of enzyme to cleave the polysaccharide vesicles was further validated by docking studies. The positioning of the molecules in active site of enzyme and the binding energy data were generated using AUTODOCK program to study the rupture of polysaccharide vesicles. This new TCSPC technique could be very useful for studying the drug release pattern of synthetic polymer vesicles loaded with Rh-B fluorophore. PMID:26237375

  12. Real-Time Analysis of Raman Spectra for Temperature Field Characterization in Aircraft Exhaust Noise Studies

    NASA Astrophysics Data System (ADS)

    Wormhoudt, J.; Nelson, D. D.; Annen, K.; Locke, R. J.; Wernet, M.

    2009-06-01

    Raman scattering has long been used as a non-intrusive diagnostic of temperatures in combustion exhaust flows, using a variety of spectral analysis techniques. As part of their ongoing program of experiments to support development of computer codes that calculate exhaust flow fields and predict jet noise, NASA Glenn Research Center is developing a laser Raman diagnostic system that will measure mean temperatures and temperature fluctuations in hot and cold jet flows. We describe a software package, ART (Analysis for Raman Temperatures), that analyzes Raman spectra of air for temperature and density using vibrational or resolved or unresolved rotational bands, presenting results in a variety of real-time displays. Each analysis technique presents its own challenges in obtaining the most precise and accurate values, and we will comment on these issues by exhibiting example spectra of each type. The ART program is closely related to another Aerodyne software package (TDLWintel) which automates the acquisition and analysis of tunable laser absorption spectra.

  13. Independent component analysis algorithm FPGA design to perform real-time blind source separation

    NASA Astrophysics Data System (ADS)

    Meyer-Baese, Uwe; Odom, Crispin; Botella, Guillermo; Meyer-Baese, Anke

    2015-05-01

    The conditions that arise in the Cocktail Party Problem prevail across many fields creating a need for of Blind Source Separation. The need for BSS has become prevalent in several fields of work. These fields include array processing, communications, medical signal processing, and speech processing, wireless communication, audio, acoustics and biomedical engineering. The concept of the cocktail party problem and BSS led to the development of Independent Component Analysis (ICA) algorithms. ICA proves useful for applications needing real time signal processing. The goal of this research was to perform an extensive study on ability and efficiency of Independent Component Analysis algorithms to perform blind source separation on mixed signals in software and implementation in hardware with a Field Programmable Gate Array (FPGA). The Algebraic ICA (A-ICA), Fast ICA, and Equivariant Adaptive Separation via Independence (EASI) ICA were examined and compared. The best algorithm required the least complexity and fewest resources while effectively separating mixed sources. The best algorithm was the EASI algorithm. The EASI ICA was implemented on hardware with Field Programmable Gate Arrays (FPGA) to perform and analyze its performance in real time.

  14. FPGA Based Real-time Network Traffic Analysis using Traffic Dispersion Patterns

    SciTech Connect

    Khan, F; Gokhale, M; Chuah, C N

    2010-03-26

    The problem of Network Traffic Classification (NTC) has attracted significant amount of interest in the research community, offering a wide range of solutions at various levels. The core challenge is in addressing high amounts of traffic diversity found in today's networks. The problem becomes more challenging if a quick detection is required as in the case of identifying malicious network behavior or new applications like peer-to-peer traffic that have potential to quickly throttle the network bandwidth or cause significant damage. Recently, Traffic Dispersion Graphs (TDGs) have been introduced as a viable candidate for NTC. The TDGs work by forming a network wide communication graphs that embed characteristic patterns of underlying network applications. However, these patterns need to be quickly evaluated for mounting real-time response against them. This paper addresses these concerns and presents a novel solution for real-time analysis of Traffic Dispersion Metrics (TDMs) in the TDGs. We evaluate the dispersion metrics of interest and present a dedicated solution on an FPGA for their analysis. We also present analytical measures and empirically evaluate operating effectiveness of our design. The mapped design on Virtex-5 device can process 7.4 million packets/second for a TDG comprising of 10k flows at very high accuracies of over 96%.

  15. Development of Non-proximate Probe Electrospray Ionization for Real-Time Analysis of Living Animal

    PubMed Central

    Yoshimura, Kentaro; Chen, Lee Chuin; Johno, Hisashi; Nakajima, Mayutaka; Hiraoka, Kenzo; Takeda, Sen

    2014-01-01

    Ambient ionization mass spectrometry is one of the most challenging analytical tools in the field of biomedical research. We previously demonstrated that probe electrospray ionization mass spectrometry (PESI-MS) could potentially be used in the rapid diagnosis of cancer. Although this technique does not require a tedious sample pretreatment process, it was not possible for our previously reported setup to be applied to cases involving the direct sampling of tissues from living animal and large animal subjects, because there would not be enough room to accommodate the larger bodies juxtaposed to the ion inlet. To make PESI-MS more applicable for the real-time analysis of living animals, a long auxiliary ion sampling tube has been connected to the ion inlet of the mass spectrometer to allow for the collection of ions and charged droplets from the PESI source (hereafter, referred to as non-proximate PESI). Furthermore, an additional ion sampling tube was connected to a small diaphragm pump to increase the uptake rate of air carrying the ions and charged droplets to the ion inlet. This modification allows for the extended ion sampling orifice to be positioned closer to the specimens, even when they are too large to be placed inside the ionization chamber. In this study, we have demonstrated the use of non-proximate PESI-MS for the real-time analysis for biological molecules and pharmacokinetic parameters from living animals. PMID:26819892

  16. Raman Based Process Monitor For Continuous Real-Time Analysis Of High Level Radioactive Waste Components

    SciTech Connect

    Bryan, Samuel A.; Levitskaia, Tatiana G.; Schlahta, Stephan N.

    2008-05-27

    ABSTRACT A new monitoring system was developed at Pacific Northwest National Laboratory (PNNL) to quickly generate real-time data/analysis to facilitate a timely response to the dynamic characteristics of a radioactive high level waste stream. The developed process monitor features Raman and Coriolis/conductivity instrumentation configured for the remote monitoring, MatLab-based chemometric data processing, and comprehensive software for data acquisition/storage/archiving/display. The monitoring system is capable of simultaneously and continuously quantifying the levels of all the chemically significant anions within the waste stream including nitrate, nitrite, phosphate, carbonate, chromate, hydroxide, sulfate, and aluminate. The total sodium ion concentration was also determined independently by modeling inputs from on-line conductivity and density meters. In addition to the chemical information, this monitoring system provides immediate real-time data on the flow parameters, such as flow rate and temperature, and cumulative mass/volume of the retrieved waste stream. The components and analytical tools of the new process monitor can be tailored for a variety of complex mixtures in chemically harsh environments, such as pulp and paper processing liquids, electroplating solutions, and radioactive tank wastes. The developed monitoring system was tested for acceptability before it was deployed for use in Hanford Tank S-109 retrieval activities. The acceptance tests included performance inspection of hardware, software, and chemometric data analysis to determine the expected measurement accuracy for the different chemical species that are encountered during S-109 retrieval.

  17. Raman Based Process Monitor for Continuous Real-Time Analysis Of High Level Radioactive Waste Components

    SciTech Connect

    Bryan, S.; Levitskaia, T.; Schlahta, St.

    2008-07-01

    A new monitoring system was developed at Pacific Northwest National Laboratory (PNNL) to quickly generate real-time data/analysis to facilitate a timely response to the dynamic characteristics of a radioactive high level waste stream. The developed process monitor features Raman and Coriolis/conductivity instrumentation configured for the remote monitoring, MatLab-based chemometric data processing, and comprehensive software for data acquisition/storage/archiving/display. The monitoring system is capable of simultaneously and continuously quantifying the levels of all the chemically significant anions within the waste stream including nitrate, nitrite, phosphate, carbonate, chromate, hydroxide, sulfate, and aluminate. The total sodium ion concentration was also determined independently by modeling inputs from on-line conductivity and density meters. In addition to the chemical information, this monitoring system provides immediate real-time data on the flow parameters, such as flow rate and temperature, and cumulative mass/volume of the retrieved waste stream. The components and analytical tools of the new process monitor can be tailored for a variety of complex mixtures in chemically harsh environments, such as pulp and paper processing liquids, electroplating solutions, and radioactive tank wastes. The developed monitoring system was tested for acceptability before it was deployed for use in Hanford Tank S-109 retrieval activities. The acceptance tests included performance inspection of hardware, software, and chemometric data analysis to determine the expected measurement accuracy for the different chemical species that are encountered during S-109 retrieval. (authors)

  18. Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

    PubMed Central

    Hao, Xinyuan; Horvath, David P.; Chao, Wun S.; Yang, Yajun; Wang, Xinchao; Xiao, Bin

    2014-01-01

    Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions. PMID:25474086

  19. Tendency for interlaboratory precision in the GMO analysis method based on real-time PCR.

    PubMed

    Kodama, Takashi; Kurosawa, Yasunori; Kitta, Kazumi; Naito, Shigehiro

    2010-01-01

    The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio. PMID:20480922

  20. Real-time Image Analysis of Living Cellular-Biology Measurements of Intelligent Chemistry

    SciTech Connect

    Solinsky, James C.; Budge, Scott E.; Majors, Paul D.; Rex, Bruce B.

    2003-08-01

    This paper reports on the Pacific Northwest National Laboratory (PNNL) DOE Initiative in Image Science and Technology (ISAT) research, which is developing algorithms and software tool sets for remote sensing and biological applications. In particular, the PNNL ISAT work is applying these research results to the automated analysis of real-time cellular biology imagery to assist the biologist in determining the correct data collection region for the current state of a conglomerate of living cells in three-dimensional motion. The real-time computation of the typical 120 MB/sec multi-spectral data sets is executed in a Field Programmable Gate Array (FPGA) technology, which has very high processing rates due to large-scale parallelism. The outcome of this artificial vision work will allow the biologist to work with imagery as a creditable set of dye-tagged chemistry measurements in formats for individual cell tracking through regional feature extraction, and animation visualization through individual object isolation/characterization of the microscopy imagery.

  1. Real-time breath analysis in type 2 diabetes patients during cognitive effort.

    PubMed

    Mazzatenta, Andrea; Pokorski, Mieczyslaw; Di Giulio, Camillo

    2013-01-01

    The understanding the functional expression of exhaled volatile organic compounds (VOCs) has gradually expanded from the initial identification of breath pathological markers to direct expression of physiological activity. In the present study we investigated the potential application of breath analysis in real-time monitoring of type 2 diabetes mellitus (T2DM) patients versus control subjects while performing a cognitive task. T2DM is associated with cognitive impairment and neural deficits, because of insulin resistance and high expression of insulin receptors in the hippocampus. We set out to seek the evidence for mutual associations among breath exhale, on the one side, and T2DM and cognitive effort, on the other side. The recording system consisted of a metal oxide semiconductor (MOS) which is able to detect a broad range of volatile organic compounds. The sensor provides a measure of VOCs as ppm CO2 equivalents. The MOS is suitable for a non-invasive real-time monitoring of the breath exhale in humans. The study demonstrates that, apart from the T2DM metabolic derangement, performing a cognitive task, taken as an index of central neural effort, evoked distinct alterations in exhaled breath content. We conclude that exhaled breath content measurement might offer a novel diagnostic and therapeutic non-invasive approach in metabolic and neurodegenerative derangements. PMID:23835985

  2. Real time culture and analysis of embryo metabolism using a microfluidic device with deformation based actuation.

    PubMed

    Heo, Yun Seok; Cabrera, Lourdes M; Bormann, Charles L; Smith, Gary D; Takayama, Shuichi

    2012-06-21

    We report a computerized microfluidic real time embryo culture and assay device that can perform automated periodic analyses of embryo metabolism. This automated program uses a modified "gated injection" scheme (sample injection, reagent mixing, enzyme reaction of 15 min incubation, and sample detection) to sequentially measure fluorescence from sample, reference, and background (without any analyte) every hour. Measurements assessed with reference solutions demonstrated the stability of these microfluidic measurements over a 24 h period. Furthermore, this system was able to measure time dependent nutrient consumption by single or multiple (10) live mouse blastocyst-stage embryos with pmol h(-1) sensitivity. Mechanical deformation-based microfluidic actuation created by computerized movement of Braille pins enables automated fluid pumping and valving sequences without unwanted gravity-driven backflow or exposure to electrical fields as would be required in electrokinetic schemes. The convenient, non-invasive, and automated nature of these assays open the way for the development of integrated microfluidic platforms for practical single embryo culture and real time biochemical analysis to assess embryo viability and select embryos with the greatest implantation potential, thus improving success in clinical assisted reproductive technology laboratories. PMID:22402469

  3. Real-Time Adaptive EEG Source Separation Using Online Recursive Independent Component Analysis.

    PubMed

    Hsu, Sheng-Hsiou; Mullen, Tim R; Jung, Tzyy-Ping; Cauwenberghs, Gert

    2016-03-01

    Independent component analysis (ICA) has been widely applied to electroencephalographic (EEG) biosignal processing and brain-computer interfaces. The practical use of ICA, however, is limited by its computational complexity, data requirements for convergence, and assumption of data stationarity, especially for high-density data. Here we study and validate an optimized online recursive ICA algorithm (ORICA) with online recursive least squares (RLS) whitening for blind source separation of high-density EEG data, which offers instantaneous incremental convergence upon presentation of new data. Empirical results of this study demonstrate the algorithm's: 1) suitability for accurate and efficient source identification in high-density (64-channel) realistically-simulated EEG data; 2) capability to detect and adapt to nonstationarity in 64-ch simulated EEG data; and 3) utility for rapidly extracting principal brain and artifact sources in real 61-channel EEG data recorded by a dry and wearable EEG system in a cognitive experiment. ORICA was implemented as functions in BCILAB and EEGLAB and was integrated in an open-source Real-time EEG Source-mapping Toolbox (REST), supporting applications in ICA-based online artifact rejection, feature extraction for real-time biosignal monitoring in clinical environments, and adaptable classifications in brain-computer interfaces. PMID:26685257

  4. Collaborative real-time motion video analysis by human observer and image exploitation algorithms

    NASA Astrophysics Data System (ADS)

    Hild, Jutta; Krüger, Wolfgang; Brüstle, Stefan; Trantelle, Patrick; Unmüßig, Gabriel; Heinze, Norbert; Peinsipp-Byma, Elisabeth; Beyerer, Jürgen

    2015-05-01

    Motion video analysis is a challenging task, especially in real-time applications. In most safety and security critical applications, a human observer is an obligatory part of the overall analysis system. Over the last years, substantial progress has been made in the development of automated image exploitation algorithms. Hence, we investigate how the benefits of automated video analysis can be integrated suitably into the current video exploitation systems. In this paper, a system design is introduced which strives to combine both the qualities of the human observer's perception and the automated algorithms, thus aiming to improve the overall performance of a real-time video analysis system. The system design builds on prior work where we showed the benefits for the human observer by means of a user interface which utilizes the human visual focus of attention revealed by the eye gaze direction for interaction with the image exploitation system; eye tracker-based interaction allows much faster, more convenient, and equally precise moving target acquisition in video images than traditional computer mouse selection. The system design also builds on prior work we did on automated target detection, segmentation, and tracking algorithms. Beside the system design, a first pilot study is presented, where we investigated how the participants (all non-experts in video analysis) performed in initializing an object tracking subsystem by selecting a target for tracking. Preliminary results show that the gaze + key press technique is an effective, efficient, and easy to use interaction technique when performing selection operations on moving targets in videos in order to initialize an object tracking function.

  5. Rapid Detection of Hepatitis B Virus Variants Associated with Lamivudine and Adefovir Resistance by Multiplex Ligation-Dependent Probe Amplification Combined with Real-Time PCR

    PubMed Central

    Jia, Shuangrong; Wang, Feng; Li, Fake; Chang, Kai; Yang, Shaojun; Zhang, Kejun; Jiang, Wenbin; Shang, Ya

    2014-01-01

    Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice. PMID:24478474

  6. Information Gap Analysis: near real-time evaluation of disaster response

    NASA Astrophysics Data System (ADS)

    Girard, Trevor

    2014-05-01

    Disasters, such as major storm events or earthquakes, trigger an immediate response by the disaster management system of the nation in question. The quality of this response is a large factor in its ability to limit the impacts on the local population. Improving the quality of disaster response therefore reduces disaster impacts. Studying past disasters is a valuable exercise to understand what went wrong, identify measures which could have mitigated these issues, and make recommendations to improve future disaster planning and response. While such ex post evaluations can lead to improvements in the disaster management system, there are limitations. The main limitation that has influenced this research is that ex post evaluations do not have the ability to inform the disaster response being assessed for the obvious reason that they are carried out long after the response phase is over. The result is that lessons learned can only be applied to future disasters. In the field of humanitarian relief, this limitation has led to the development of real time evaluations. The key aspect of real time humanitarian evaluations is that they are completed while the operation is still underway. This results in findings being delivered at a time when they can still make a difference to the humanitarian response. Applying such an approach to the immediate disaster response phase requires an even shorter time-frame, as well as a shift in focus from international actors to the nation in question's government. As such, a pilot study was started and methodology developed, to analyze disaster response in near real-time. The analysis uses the information provided by the disaster management system within the first 0 - 5 days of the response. The data is collected from publicly available sources such as ReliefWeb and sorted under various categories which represent each aspect of disaster response. This process was carried out for 12 disasters. The quantity and timeliness of information

  7. Real-time areal precipitation determination from radar by means of statistical objective analysis

    NASA Astrophysics Data System (ADS)

    Gerstner, E.-M.; Heinemann, G.

    2008-05-01

    SummaryPrecipitation measurement by radar allows for areal rainfall determination with a high spatial and temporal resolution. However, hydrological applications require an accuracy of the precipitation quantification which cannot be obtained by today's weather radar devices. The quality of the radar-derived precipitation can be significantly improved with the aid of ground measurements. In this paper, a complete processing pipeline for real-time radar precipitation determination using a modified statistical objective analysis method is presented. Thereby, several additional algorithms, such as a dynamical use of Z-R relationships, a bias correction and an advection correction scheme are employed. The performance of the algorithms is tested for several case studies. For an error analysis, an eight months data set of X-band radar scans and rain gauge precipitation measurements is used. We show a reduction in the radar-rain gauge RMS difference of up to 59% for the optimal combination of the different algorithms.

  8. Real-time automated failure analysis for on-orbit operations

    NASA Technical Reports Server (NTRS)

    Kirby, Sarah; Lauritsen, Janet; Pack, Ginger; Ha, Anhhoang; Jowers, Steven; Mcnenny, Robert; Truong, The; Dell, James

    1993-01-01

    A system which is to provide real-time failure analysis support to controllers at the NASA Johnson Space Center Control Center Complex (CCC) for both Space Station and Space Shuttle on-orbit operations is described. The system employs monitored systems' models of failure behavior and model evaluation algorithms which are domain-independent. These failure models are viewed as a stepping stone to more robust algorithms operating over models of intended function. The described system is designed to meet two sets of requirements. It must provide a useful failure analysis capability enhancement to the mission controller. It must satisfy CCC operational environment constraints such as cost, computer resource requirements, verification, and validation. The underlying technology and how it may be used to support operations is also discussed.

  9. Load Balancing Using Time Series Analysis for Soft Real Time Systems with Statistically Periodic Loads

    NASA Technical Reports Server (NTRS)

    Hailperin, Max

    1993-01-01

    This thesis provides design and analysis of techniques for global load balancing on ensemble architectures running soft-real-time object-oriented applications with statistically periodic loads. It focuses on estimating the instantaneous average load over all the processing elements. The major contribution is the use of explicit stochastic process models for both the loading and the averaging itself. These models are exploited via statistical time-series analysis and Bayesian inference to provide improved average load estimates, and thus to facilitate global load balancing. This thesis explains the distributed algorithms used and provides some optimality results. It also describes the algorithms' implementation and gives performance results from simulation. These results show that our techniques allow more accurate estimation of the global system load ing, resulting in fewer object migration than local methods. Our method is shown to provide superior performance, relative not only to static load-balancing schemes but also to many adaptive methods.

  10. Real Time, On Line Crop Monitoring and Analysis with Near Global Landsat-class Mosaics

    NASA Astrophysics Data System (ADS)

    Varlyguin, D.; Hulina, S.; Crutchfield, J.; Reynolds, C. A.; Frantz, R.

    2015-12-01

    The presentation will discuss the current status of GDA technology for operational, automated generation of 10-30 meter near global mosaics of Landsat-class data for visualization, monitoring, and analysis. Current version of the mosaic combines Landsat 8 and Landsat 7. Sentinel-2A imagery will be added once it is operationally available. The mosaics are surface reflectance calibrated and are analysis ready. They offer full spatial resolution and all multi-spectral bands of the source imagery. Each mosaic covers all major agricultural regions of the world and 16 day time window. 2014-most current dates are supported. The mosaics are updated in real-time, as soon as GDA downloads Landsat imagery, calibrates it to the surface reflectances, and generates data gap masks (all typically under 10 minutes for a Landsat scene). The technology eliminates the complex, multi-step, hands-on process of data preparation and provides imagery ready for repetitive, field-to-country analysis of crop conditions, progress, acreages, yield, and production. The mosaics can be used for real-time, on-line interactive mapping and time series drilling via GeoSynergy webGIS platform. The imagery is of great value for improved, persistent monitoring of global croplands and for the operational in-season analysis and mapping of crops across the globe in USDA FAS purview as mandated by the US government. The presentation will overview operational processing of Landsat-class mosaics in support of USDA FAS efforts and will look into 2015 and beyond.

  11. An rRT-PCR assay to detect the matrix gene of a broad range of avian paramyxovirus serotype-1 strains.

    PubMed

    Hines, Nichole L; Killian, Mary Lea; Pedersen, Janice C; Reising, Monica M; Mosos, Nestor A; Mathieu-Benson, Christian; Miller, Cathy L

    2012-06-01

    The current U.S. Department of Agriculture (USDA)-validated real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay designed to detect the matrix gene of avian paramyxovirus serotype-1 (APMV-1) is the primary screening assay used in the United States. It has previously been shown to be unable to consistently detect all members of class I APMV-1. Diagnostic testing relies on rRT-PCR to quickly detect APMV-1 in wild birds, backyard flocks, live bird markets, commercial poultry, and for export testing. Limitations of the current USDA assay have raised concerns about the potential for some strains of APMV-1 to remain undetected by the primary screening assay. Mismatches in the probe were shown to cause a loss in template binding efficiency, resulting in lack of detection by the assay. Here, we describe the development and analytical validation of a new rRT-PCR assay designed to target a highly conserved region of the matrix gene across a wide range of APMV-1 strains. Limit of detection testing revealed a 3 log10 decrease in sensitivity for one low-virulence strain when compared to the USDA validated assay. Conversely, the assay showed increased sensitivity for a class I isolate and two virulent strains of APMV-1 that were not detected by the USDA-validated assay. The new assay also demonstrated a high degree of specificity by the lack of detection of 43 non-APMV-1 viruses. PMID:22856199

  12. Quantitative analysis of real-time tissue elastography for evaluation of liver fibrosis

    PubMed Central

    Shi, Ying; Wang, Xing-Hua; Zhang, Huan-Hu; Zhang, Hai-Qing; Tu, Ji-Zheng; Wei, Kun; Li, Juan; Liu, Xiao-Li

    2014-01-01

    The present study aimed to investigate the feasibility of quantitative analysis of liver fibrosis using real-time tissue elastography (RTE) and its pathological and molecule biological basis. Methods: Fifty-four New Zealand rabbits were subcutaneously injected with thioacetamide (TAA) to induce liver fibrosis as the model group, and another eight New Zealand rabbits served as the normal control group. Four rabbits were randomly taken every two weeks for real-time tissue elastography (RTE) and quantitative analysis of tissue diffusion. The obtained twelve characteristic quantities included relative mean value (MEAN), standard deviation (SD), blue area % (% AREA), complexity (COMP), kurtosis (KURT), skewness (SKEW), contrast (CONT), entropy (ENT), inverse different moment (IDM), angular secon moment (ASM), correlation (CORR) and liver fibrosis index (LF Index). Rabbits were executed and liver tissues were taken for pathological staging of liver fibrosis (grouped by pathological stage into S0 group, S1 group, S2 group, S3 group and S4 group). In addition, the collagen I (Col I) and collagen III (Col III) expression levels in liver tissue were detected by Western blot. Results: Except for KURT, there were significant differences among the other eleven characteristic quantities (P < 0.05). LF Index, Col I and Col III expression levels showed a rising trend with increased pathological staging of liver fibrosis, presenting a positive correlation with the pathological staging of liver fibrosis (r = 0.718, r = 0.693, r = 0.611, P < 0.05). Conclusion: RTE quantitative analysis is expected for noninvasive evaluation of the pathological staging of liver fibrosis. PMID:24955175

  13. Automated Detection and Analysis of Interplanetary Shocks Running Real-Time on the Web

    NASA Astrophysics Data System (ADS)

    Vorotnikov, V.; Smith, C. W.; Hu, Q.; Szabo, A.; Skoug, R. M.; Cohen, C. M.; Davis, A. J.

    2008-05-01

    The ACE real-time data stream provides web-based now-casting capabilities for solar wind conditions upstream of Earth. We have built a fully automated code that finds and analyzes interplanetary shocks as they occur and posts their solutions on the Web for possible real-time application to space weather nowcasting. Shock analysis algorithms based on the Rankine-Hugoniot jump conditions exist and are in wide-spread use today for the interactive analysis of interplanetary shocks yielding parameters such as shock speed and propagation direction and shock strength in the form of compression ratios. At a previous meeting we reported on efforts to develop a fully automated code that used ACE Level-2 (science quality) data to prove the applicability and correctness of the code and the associated shock-finder. We have since adapted the code to run ACE RTSW data provided by NOAA. This data lacks the full 3-dimensional velocity vector for the solar wind and contains only a single component wind speed. We show that by assuming the wind velocity to be radial strong shock solutions remain essentially unchanged and the analysis performs as well as it would if 3-D velocity components were available. This is due, at least in part, to the fact that strong shocks tend to have nearly radial shock normals and it is the strong shocks that are most effective in space weather applications. Strong shocks are the only shocks that concern us in this application. The code is now running on the Web and the results are available to all.

  14. Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage

    PubMed Central

    Hu, Yu; Xie, Shuying; Yao, Jihua

    2016-01-01

    Reference genes used in normalizing qRT-PCR data are critical for the accuracy of gene expression analysis. However, many traditional reference genes used in zebrafish early development are not appropriate because of their variable expression levels during embryogenesis. In the present study, we used our previous RNA-Seq dataset to identify novel reference genes suitable for gene expression analysis during zebrafish early developmental stages. We first selected 197 most stably expressed genes from an RNA-Seq dataset (29,291 genes in total), according to the ratio of their maximum to minimum RPKM values. Among the 197 genes, 4 genes with moderate expression levels and the least variation throughout 9 developmental stages were identified as candidate reference genes. Using four independent statistical algorithms (delta-CT, geNorm, BestKeeper and NormFinder), the stability of qRT-PCR expression of these candidates was then evaluated and compared to that of actb1 and actb2, two commonly used zebrafish reference genes. Stability rankings showed that two genes, namely mobk13 (mob4) and lsm12b, were more stable than actb1 and actb2 in most cases. To further test the suitability of mobk13 and lsm12b as novel reference genes, they were used to normalize three well-studied target genes. The results showed that mobk13 and lsm12b were more suitable than actb1 and actb2 with respect to zebrafish early development. We recommend mobk13 and lsm12b as new optimal reference genes for zebrafish qRT-PCR analysis during embryogenesis and early larval stages. PMID:26891128

  15. Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

    PubMed

    Hu, Yu; Xie, Shuying; Yao, Jihua

    2016-01-01

    Reference genes used in normalizing qRT-PCR data are critical for the accuracy of gene expression analysis. However, many traditional reference genes used in zebrafish early development are not appropriate because of their variable expression levels during embryogenesis. In the present study, we used our previous RNA-Seq dataset to identify novel reference genes suitable for gene expression analysis during zebrafish early developmental stages. We first selected 197 most stably expressed genes from an RNA-Seq dataset (29,291 genes in total), according to the ratio of their maximum to minimum RPKM values. Among the 197 genes, 4 genes with moderate expression levels and the least variation throughout 9 developmental stages were identified as candidate reference genes. Using four independent statistical algorithms (delta-CT, geNorm, BestKeeper and NormFinder), the stability of qRT-PCR expression of these candidates was then evaluated and compared to that of actb1 and actb2, two commonly used zebrafish reference genes. Stability rankings showed that two genes, namely mobk13 (mob4) and lsm12b, were more stable than actb1 and actb2 in most cases. To further test the suitability of mobk13 and lsm12b as novel reference genes, they were used to normalize three well-studied target genes. The results showed that mobk13 and lsm12b were more suitable than actb1 and actb2 with respect to zebrafish early development. We recommend mobk13 and lsm12b as new optimal reference genes for zebrafish qRT-PCR analysis during embryogenesis and early larval stages. PMID:26891128

  16. QPCR: Application for real-time PCR data management and analysis

    PubMed Central

    Pabinger, Stephan; Thallinger, Gerhard G; Snajder, René; Eichhorn, Heiko; Rader, Robert; Trajanoski, Zlatko

    2009-01-01

    Background Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. Results QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. Conclusion We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available at PMID:19712446

  17. AEP Ohio gridSMART Demonstration Project Real-Time Pricing Demonstration Analysis

    SciTech Connect

    Widergren, Steven E.; Subbarao, Krishnappa; Fuller, Jason C.; Chassin, David P.; Somani, Abhishek; Marinovici, Maria C.; Hammerstrom, Janelle L.

    2014-02-01

    This report contributes initial findings from an analysis of significant aspects of the gridSMART® Real-Time Pricing (RTP) – Double Auction demonstration project. Over the course of four years, Pacific Northwest National Laboratory (PNNL) worked with American Electric Power (AEP), Ohio and Battelle Memorial Institute to design, build, and operate an innovative system to engage residential consumers and their end-use resources in a participatory approach to electric system operations, an incentive-based approach that has the promise of providing greater efficiency under normal operating conditions and greater flexibility to react under situations of system stress. The material contained in this report supplements the findings documented by AEP Ohio in the main body of the gridSMART report. It delves into three main areas: impacts on system operations, impacts on households, and observations about the sensitivity of load to price changes.

  18. Real-time cellular analysis for quantitative detection of functional Clostridium difficile toxin in stool.

    PubMed

    Huang, Bin; Li, Haijing; Jin, Dazhi; Stratton, Charles W; Tang, Yi-Wei

    2014-04-01

    Rapid and accurate diagnosis and monitoring of Clostridium difficile infection (CDI) is critical for patient care and infection control. We will briefly review current laboratory techniques for the diagnosis of CDI and identify aspects needing improvement. We will also introduce a real-time cellular analysis (RTCA) assay developed for the diagnosis and monitoring of CDI using electronic impedance to assess the cell status. The RTCA assay uses impedance measurement to detect minute physiological changes in cells cultured on gold microelectrodes embedded in glass substrates in the bottom of microtiter wells. This assay has been adapted for quantitative detection of C. difficile functional toxin directly from stool specimens. Compared to conventional techniques and molecular assays, the RTCA assay provides a valuable tool for the diagnosis of CDI as well as for the assessment of clinical severity and for monitoring therapeutic efficacies. PMID:24649817

  19. Compton rejection for HPGe detectors via real-time pulse shape analysis

    SciTech Connect

    Beckedahl, D; Blair, J J; Friensehner, A; Kammeraad, J E; Kreek, S A; Payne, B; Schmid, G J

    1998-07-31

    A Lawrence Livermore National Laboratory-developed pulse shape analysis (PSA) technique which performs real-time Compton suppression in High Purity Germanium (HPGe) detectors without the use of anti-coincidence detectors is described. Some preliminary measurements of a variety of sources with a standard HPGe detector system and our prototype PSA algorithm have been made and indicate that a reduction in Compton continuum can be achieved via PSA. These measurements represent an initial assessment of the effectiveness of the prototype PSA system for the improvement of spectral quality and future improvements are expected. Additional work is progressing to optimize the effectiveness of the algorithm for Compton rejection in standard HPGe detectors. Work is also progressing to extend the methodology to segmented HPGe detectors which could potentially yield significantly better Compton rejection and gamma-ray ima

  20. Real time thermal imaging for analysis and control of crystal growth by the Czochralski technique

    NASA Technical Reports Server (NTRS)

    Wargo, M. J.; Witt, A. F.

    1992-01-01

    A real time thermal imaging system with temperature resolution better than +/- 0.5 C and spatial resolution of better than 0.5 mm has been developed. It has been applied to the analysis of melt surface thermal field distributions in both Czochralski and liquid encapsulated Czochralski growth configurations. The sensor can provide single/multiple point thermal information; a multi-pixel averaging algorithm has been developed which permits localized, low noise sensing and display of optical intensity variations at any location in the hot zone as a function of time. Temperature distributions are measured by extraction of data along a user selectable linear pixel array and are simultaneously displayed, as a graphic overlay, on the thermal image.

  1. Near Real-time Data Analysis of Core-Collapse Supernova Simulations With Bellerophon

    SciTech Connect

    Lingerfelt, Eric J; Messer, Bronson; Desai, Sharvari S; Holt, Chastity A; Lentz, Eric J

    2014-01-01

    We present an overview of a software system, Bellerophon, built to support a production-level HPC application called CHIMERA, which simulates core-collapse supernova events at the petascale. Developed over the last four years, Bellerophon enables CHIMERA s geographically dispersed team of collaborators to perform data analysis in near real-time. Its n-tier architecture provides an encapsulated, end-to-end software solution that enables the CHIMERA team to quickly and easily access highly customizable animated and static views of results from anywhere in the world via a web-deliverable, cross-platform desktop application. In addition, Bellerophon addresses software engineering tasks for the CHIMERA team by providing an automated mechanism for performing regression testing on a variety of supercomputing platforms. Elements of the team s workflow management needs are met with software tools that dynamically generate code repository statistics, access important online resources, and monitor the current status of several supercomputing resources.

  2. A specialized motion capture system for real-time analysis of mandibular movements using infrared cameras

    PubMed Central

    2013-01-01

    Background In the last years, several methods and devices have been proposed to record the human mandibular movements, since they provide quantitative parameters that support the diagnosis and treatment of temporomandibular disorders. The techniques currently employed suffer from a number of drawbacks including high price, unnatural to use, lack of support for real-time analysis and mandibular movements recording as a pure rotation. In this paper, we propose a specialized optical motion capture system, which causes a minimum obstruction and can support 3D mandibular movement analysis in real-time. Methods We used three infrared cameras together with nine reflective markers that were placed at key points of the face. Some classical techniques are suggested to conduct the camera calibration and three-dimensional reconstruction and we propose some specialized algorithms to automatically recognize our set of markers and track them along a motion capture session. Results To test the system, we developed a prototype software and performed a clinical experiment in a group of 22 subjects. They were instructed to execute several movements for the functional evaluation of the mandible while the system was employed to record them. The acquired parameters and the reconstructed trajectories were used to confirm the typical function of temporomandibular joint in some subjects and to highlight its abnormal behavior in others. Conclusions The proposed system is an alternative to the existing optical, mechanical, electromagnetic and ultrasonic-based methods, and intends to address some drawbacks of currently available solutions. Its main goal is to assist specialists in diagnostic and treatment of temporomandibular disorders, since simple visual inspection may not be sufficient for a precise assessment of temporomandibular joint and associated muscles. PMID:23433470

  3. Noninvasive Real-Time Automated Skin Lesion Analysis System for Melanoma Early Detection and Prevention

    PubMed Central

    Abuzaghleh, Omar; Barkana, Buket D.

    2015-01-01

    Melanoma spreads through metastasis, and therefore, it has been proved to be very fatal. Statistical evidence has revealed that the majority of deaths resulting from skin cancer are as a result of melanoma. Further investigations have shown that the survival rates in patients depend on the stage of the cancer; early detection and intervention of melanoma implicate higher chances of cure. Clinical diagnosis and prognosis of melanoma are challenging, since the processes are prone to misdiagnosis and inaccuracies due to doctors’ subjectivity. Malignant melanomas are asymmetrical, have irregular borders, notched edges, and color variations, so analyzing the shape, color, and texture of the skin lesion is important for the early detection and prevention of melanoma. This paper proposes the two major components of a noninvasive real-time automated skin lesion analysis system for the early detection and prevention of melanoma. The first component is a real-time alert to help users prevent skinburn caused by sunlight; a novel equation to compute the time for skin to burn is thereby introduced. The second component is an automated image analysis module, which contains image acquisition, hair detection and exclusion, lesion segmentation, feature extraction, and classification. The proposed system uses PH2 Dermoscopy image database from Pedro Hispano Hospital for the development and testing purposes. The image database contains a total of 200 dermoscopy images of lesions, including benign, atypical, and melanoma cases. The experimental results show that the proposed system is efficient, achieving classification of the benign, atypical, and melanoma images with accuracy of 96.3%, 95.7%, and 97.5%, respectively. PMID:27170906

  4. A prototype for the real-time analysis of the Cherenkov Telescope Array

    NASA Astrophysics Data System (ADS)

    Bulgarelli, Andrea; Fioretti, Valentina; Zoli, Andrea; Aboudan, Alessio; Rodríguez-Vázquez, Juan José; Maier, Gernot; Lyard, Etienne; Bastieri, Denis; Lombardi, Saverio; Tosti, Gino; De Rosa, Adriano; Bergamaschi, Sonia; Interlandi, Matteo; Beneventano, Domenico; Lamanna, Giovanni; Jacquemier, Jean; Kosack, Karl; Antonelli, Lucio Angelo; Boisson, Catherine; Burkowski, Jerzy; Buson, Sara; Carosi, Alessandro; Conforti, Vito; Contreras, Jose Luis; De Cesare, Giovanni; de los Reyes, Raquel; Dumm, Jon; Evans, Phil; Fortson, Lucy; Fuessling, Matthias; Graciani, Ricardo; Gianotti, Fulvio; Grandi, Paola; Hinton, Jim; Humensky, Brian; Knödlseder, Jürgen; Malaguti, Giuseppe; Marisaldi, Martino; Neyroud, Nadine; Nicastro, Luciano; Ohm, Stefan; Osborne, Julian; Rosen, Simon; Tacchini, Alessandro; Torresi, Eleonora; Testa, Vincenzo; Trifoglio, Massimo; Weinstein, Amanda

    2014-07-01

    The Cherenkov Telescope Array (CTA) observatory will be one of the biggest ground-based very-high-energy (VHE) γ- ray observatory. CTA will achieve a factor of 10 improvement in sensitivity from some tens of GeV to beyond 100 TeV with respect to existing telescopes. The CTA observatory will be capable of issuing alerts on variable and transient sources to maximize the scientific return. To capture these phenomena during their evolution and for effective communication to the astrophysical community, speed is crucial. This requires a system with a reliable automated trigger that can issue alerts immediately upon detection of γ-ray flares. This will be accomplished by means of a Real-Time Analysis (RTA) pipeline, a key system of the CTA observatory. The latency and sensitivity requirements of the alarm system impose a challenge because of the anticipated large data rate, between 0.5 and 8 GB/s. As a consequence, substantial efforts toward the optimization of highthroughput computing service are envisioned. For these reasons our working group has started the development of a prototype of the Real-Time Analysis pipeline. The main goals of this prototype are to test: (i) a set of frameworks and design patterns useful for the inter-process communication between software processes running on memory; (ii) the sustainability of the foreseen CTA data rate in terms of data throughput with different hardware (e.g. accelerators) and software configurations, (iii) the reuse of nonreal- time algorithms or how much we need to simplify algorithms to be compliant with CTA requirements, (iv) interface issues between the different CTA systems. In this work we focus on goals (i) and (ii).

  5. Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

    PubMed Central

    Jiang, Xiaomei; Yin, Guohua; Zhang, Xinquan; Qi, Xiao; Zhang, Yu; Yan, Yanhong; Ma, Xiao; Peng, Yan

    2014-01-01

    Background Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied. Methodology/Principal Findings In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments. Conclusions/Significance These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies. PMID:24699822

  6. Endothelial cell adhesion in real time. Measurements in vitro by tandem scanning confocal image analysis.

    PubMed

    Davies, P F; Robotewskyj, A; Griem, M L

    1993-06-01

    Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-substratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time. The topography of focal contacts was estimated by calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm. Subtraction of consecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhesions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance of new ones often occurred within minutes while adjacent sites underwent minimal remodelling. Tandem scanning confocal microscopy image analysis of

  7. Quantitative Analysis of Copy Number Variants Based on Real-Time LightCycler PCR

    PubMed Central

    Ma, Lijiang; Chung, Wendy K.

    2014-01-01

    Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples. PMID:24510682

  8. Writing/Thinking in Real Time: Digital Video and Corpus Query Analysis

    ERIC Educational Resources Information Center

    Park, Kwanghyun; Kinginger, Celeste

    2010-01-01

    The advance of digital video technology in the past two decades facilitates empirical investigation of learning in real time. The focus of this paper is the combined use of real-time digital video and a networked linguistic corpus for exploring the ways in which these technologies enhance our capability to investigate the cognitive process of…

  9. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    PubMed

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks. PMID:27010714

  10. Evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In April 2013 a porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time RT-PCR assays to detect PEDV were developed by several Veterinary Diagnostic Laboratories. This study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS...

  11. Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

    PubMed Central

    Edouard, Sophie; Prudent, Elsa; Gautret, Philippe; Memish, Ziad A.

    2014-01-01

    We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed. PMID:25552360

  12. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    PubMed

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya. PMID:26666186

  13. Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine.

    PubMed

    Crossland, Rachel E; Norden, Jean; Bibby, Louis A; Davis, Joanna; Dickinson, Anne M

    2016-02-01

    MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the

  14. Real-Time Application of Multi-Satellite Precipitation Analysis for Floods and Landslides

    NASA Technical Reports Server (NTRS)

    Adler, Robert; Hong, Yang; Huffman, George

    2007-01-01

    Satellite data acquired and processed in real time now have the potential to provide the spacetime information on rainfall needed to monitor flood and landslide events around the world. This can be achieved by integrating the satellite-derived forcing data with hydrological models and landslide algorithms. Progress in using the TRMM Multi-satellite Precipitation Analysis (TMPA) as input to flood and landslide forecasts is outlined, with a focus on understanding limitations of the rainfall data and impacts of those limitations on flood/landslide analyses. Case studies of both successes and failures will be shown, as well as comparison with ground comparison data sets-- both in terms of rainfall and in terms of flood/landslide events. In addition to potential uses in real-time, the nearly ten years of TMPA data allow retrospective running of the models to examine variations in extreme events. The flood determination algorithm consists of four major components: 1) multi-satellite precipitation estimation; 2) characterization of land surface including digital elevation from NASA SRTM (Shuttle Radar Terrain Mission), topography-derived hydrologic parameters such as flow direction, flow accumulation, basin, and river network etc.; 3) a hydrological model to infiltrate rainfall and route overland runoff; and 4) an implementation interface to relay the input data to the models and display the flood inundation results to potential users and decision-makers, In terms of landslides, the satellite rainfall information is combined with a global landslide susceptibility map, derived from a combination of global surface characteristics (digital elevation topography, slope, soil types, soil texture, and land cover classification etc.) using a weighted linear combination approach. In those areas identified as "susceptible" (based on the surface characteristics), landslides are forecast where and when a rainfall intensity/duration threshold is exceeded. Results are described

  15. Real-time cell analysis for monitoring cholera toxin-induced human intestinal epithelial cell response.

    PubMed

    Ye, Julian; Luo, Yun; Fang, Weijia; Pan, Junhang; Zhang, Zheng; Zhang, Yanjun; Chen, Zhiping; Jin, Dazhi

    2015-04-01

    The pathogenic mechanism of Vibrio cholerae manifests as diarrhea and causes life-threatening dehydration. Here, we observe the human intestinal epithelial cells (HIEC) response to Cholera toxin (CT) by a real-time cell analysis (RTCA) platform, and disclose the difference from CT-induced cytotoxicity and others in HIEC. An HIEC cell of 1.0 × 10(5) cells/mL was characterized as the suitable concentration for each well. For experimentation, the assay requires an inoculation of CT dissolved in Dulbecco's phosphate-buffered saline with 0.1 % gelatin for a period of 18-25 h. The dimensionless impedance cell index curve presented characteristic dose- and time-dependent drop responses at the first stage, and the CT-induced cytotoxicity was the most remarkable following exposure for 18-25 h (P = 0.0002). Following the obvious cytotoxic reaction, the CI curve gradually increased over time until the original CI value, indicating that self-recovery occurred. The CT-induced CI curve for HIEC was different from that induced by other toxins, including diphtheria and Clostridium difficile toxin. Collectively, these results suggest that the CT-induced cytotoxicity in HIEC was absolutely different from that induced by C. difficile and other toxins because of the different pathogeneses that were correlated with the specific CI curve generated by the RTCA system. In summary, our data show that the assay described here is a convenient and rapid high-throughput tool for real-time monitoring of host cellular responses to CT on the basis of the characteristic CI curve. PMID:25510171

  16. Real-time analysis application for identifying bursty local areas related to emergency topics.

    PubMed

    Sakai, Tatsuhiro; Tamura, Keiichi

    2015-01-01

    Since social media started getting more attention from users on the Internet, social media has been one of the most important information source in the world. Especially, with the increasing popularity of social media, data posted on social media sites are rapidly becoming collective intelligence, which is a term used to refer to new media that is displacing traditional media. In this paper, we focus on geotagged tweets on the Twitter site. These geotagged tweets are referred to as georeferenced documents because they include not only a short text message, but also the documents' posting time and location. Many researchers have been tackling the development of new data mining techniques for georeferenced documents to identify and analyze emergency topics, such as natural disasters, weather, diseases, and other incidents. In particular, the utilization of geotagged tweets to identify and analyze natural disasters has received much attention from administrative agencies recently because some case studies have achieved compelling results. In this paper, we propose a novel real-time analysis application for identifying bursty local areas related to emergency topics. The aim of our new application is to provide new platforms that can identify and analyze the localities of emergency topics. The proposed application is composed of three core computational intelligence techniques: the Naive Bayes classifier technique, the spatiotemporal clustering technique, and the burst detection technique. Moreover, we have implemented two types of application interface: a Web application interface and an android application interface. To evaluate the proposed application, we have implemented a real-time weather observation system embedded the proposed application. we used actual crawling geotagged tweets posted on the Twitter site. The weather observation system successfully detected bursty local areas related to observed emergency weather topics. PMID:25918679

  17. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug

    PubMed Central

    Bansal, Raman; Mittapelly, Priyanka; Chen, Yuting; Mamidala, Praveen; Zhao, Chaoyang; Michel, Andy

    2016-01-01

    The brown marmorated stink bug (Halyomorpha halys) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9) for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed. PMID:27144586

  18. Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata

    PubMed Central

    Deng, Tian; Chen, Letian; Wu, Hong; Zhang, Shouzhou

    2016-01-01

    Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species. PMID:27124298

  19. An Application for Near Real-time Analysis of XPS Data

    SciTech Connect

    Lea, Alan S.; Swanson, Kenneth R.; Haack, Jereme N.; Castle, James E.; Tougaard, Sven M.; Baer, Donald R.

    2010-04-22

    Real-time analysis of x-ray photoelectron spectroscopy (XPS) data has significant potential advantages to scientists and analysts in that it has the potential to qualitatively alter the way an experiment is carried forward. As an example, immediate information about the magnitude of contamination and the layering of the surface could allow the scientist to immediately ask the next level of question and quickly re-direct the next experiment or test, even before the sample has been removed from the spectrometer. In addition to changing the nature of possible experiments, the immediate automated analysis of relatively simple procedures that an operator would normally conduct manually after the data files are saved, the report generation summarizing these analyses, and saving of this report to files with the critical metadata attached, has the potential to improve the turn-around time for data analysis, increase the sophistication of data analysis reportable to the scientist and reduce the labor involved in data analysis, resulting in significant time and cost savings.

  20. High-Performance Computing for Real-Time Grid Analysis and Operation

    SciTech Connect

    Huang, Zhenyu; Chen, Yousu; Chavarría-Miranda, Daniel

    2013-10-31

    Power grids worldwide are undergoing an unprecedented transition as a result of grid evolution meeting information revolution. The grid evolution is largely driven by the desire for green energy. Emerging grid technologies such as renewable generation, smart loads, plug-in hybrid vehicles, and distributed generation provide opportunities to generate energy from green sources and to manage energy use for better system efficiency. With utility companies actively deploying these technologies, a high level of penetration of these new technologies is expected in the next 5-10 years, bringing in a level of intermittency, uncertainties, and complexity that the grid did not see nor design for. On the other hand, the information infrastructure in the power grid is being revolutionized with large-scale deployment of sensors and meters in both the transmission and distribution networks. The future grid will have two-way flows of both electrons and information. The challenge is how to take advantage of the information revolution: pull the large amount of data in, process it in real time, and put information out to manage grid evolution. Without addressing this challenge, the opportunities in grid evolution will remain unfulfilled. This transition poses grand challenges in grid modeling, simulation, and information presentation. The computational complexity of underlying power grid modeling and simulation will significantly increase in the next decade due to an increased model size and a decreased time window allowed to compute model solutions. High-performance computing is essential to enable this transition. The essential technical barrier is to vastly increase the computational speed so operation response time can be reduced from minutes to seconds and sub-seconds. The speed at which key functions such as state estimation and contingency analysis are conducted (typically every 3-5 minutes) needs to be dramatically increased so that the analysis of contingencies is both

  1. Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry.

    PubMed

    Wang, Chunyan; Zhu, Hongbin; Cai, Zongwei; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

    2013-04-01

    Phenylketonuria (PKU) is commonly included in the newborn screening panel of most countries, with various techniques being used for quantification of L-phenylalanine (Phe). To diagnose PKU as early as possible in newborn screening, a rapid and simple method of analysis was developed. Using direct analysis in real time (DART) ionization coupled with triple-quadrupole tandem mass spectrometry (TQ-MS/MS) and with use of a 12 DIP-it tip scanner autosampler in positive ion mode, we analyzed dried blood spot (DBS) samples from PKU newborns. The concentration of Phe was determined using multiple reaction monitoring mode with the nondeuterated internal standard N,N-dimethylphenylalanine. The results of the analysis of DBS samples from newborns indicated that the DART-TQ-MS/MS method is fast, accurate, and reproducible. The results prove that this assay as a newborn screen for PKU can be performed in 18 s per sample for the quantification of Phe in DBS samples. DART-TQ-MS/MS analysis of the Phe concentration in DBS samples allowed us to screen newborns for PKU. This innovative protocol is rapid and can be effectively applied on a routine basis to analyze a large number of samples in PKU newborn screening and PKU patient monitoring. PMID:23397086

  2. Advanced Automation for Ion Trap Mass Spectrometry-New Opportunities for Real-Time Autonomous Analysis

    NASA Technical Reports Server (NTRS)

    Palmer, Peter T.; Wong, C. M.; Salmonson, J. D.; Yost, R. A.; Griffin, T. P.; Yates, N. A.; Lawless, James G. (Technical Monitor)

    1994-01-01

    The utility of MS/MS for both target compound analysis and the structure elucidation of unknowns has been described in a number of references. A broader acceptance of this technique has not yet been realized as it requires large, complex, and costly instrumentation which has not been competitive with more conventional techniques. Recent advancements in ion trap mass spectrometry promise to change this situation. Although the ion trap's small size, sensitivity, and ability to perform multiple stages of mass spectrometry have made it eminently suitable for on-line, real-time monitoring applications, advance automation techniques are required to make these capabilities more accessible to non-experts. Towards this end we have developed custom software for the design and implementation of MS/MS experiments. This software allows the user to take full advantage of the ion trap's versatility with respect to ionization techniques, scan proxies, and ion accumulation/ejection methods. Additionally, expert system software has been developed for autonomous target compound analysis. This software has been linked to ion trap control software and a commercial data system to bring all of the steps in the analysis cycle under control of the expert system. These software development efforts and their utilization for a number of trace analysis applications will be described.

  3. Quality by Design Study of the Direct Analysis in Real Time Mass Spectrometry Response

    NASA Astrophysics Data System (ADS)

    Wang, Lu; Chen, Teng; Zeng, Shanshan; Qu, Haibin

    2013-12-01

    A mass spectrometry method has been developed using the Quality by Design (QbD) principle. Direct analysis in real time mass spectrometry (DART-MS) was adopted to analyze a pharmaceutical preparation. A fishbone diagram for DART-MS and the Plackett-Burman design were utilized to evaluate the impact of a number of factors on the method performance. Multivariate regression and Pareto ranking analysis indicated that the temperature, determined distance, and sampler speed were statistically significant (P < 0.05). Furthermore, the Box-Behnken design combined with response surface analysis was then employed to study the relationships between these three factors and the quality of the DART-MS analysis. The analytical design space of DART-MS was thus constructed and its robustness was validated. In this presented approach, method performance was mathematically described as a composite desirability function of the critical quality attributes (CQAs). Two terms of method validation, including analytical repeatability and method robustness, were carried out at an operating work point. Finally, the validated method was successfully applied to the pharmaceutical quality assurance in different manufacturing batches. These results revealed that the QbD concept was practical in DART-MS method development. Meanwhile, the determined quality was controlled by the analytical design space. This presented strategy provided a tutorial to the development of a robust QbD-compliant mass spectrometry method for industrial quality control.

  4. Antimicrobial efficacy of different root canal sealers by using real-time polymerase chain reaction: An ex vivo study

    PubMed Central

    Seelan, R Gnana; Kumar, A Arvind; Emil Sam, R Jonathan; Maheswari, S Uma

    2015-01-01

    Background: Root canal sealers help to minimize leakage, provides antimicrobial activity to reduce the possibility of residual bacteria, and to resolve periapical lesion. Aim: To compare five different root canal sealers against Enterococcus faecalis in an infected root canal model by using real-time polymerase chain reaction (RT-PCR). Settings and Design: Sixty human mandibular premolars were sectioned to standardize a uniform length of 14 mm. Fifty microliters of the inoculum containing E. faecalis were transferred into each microcentrifuge tube (n = 60). The samples were divided into six groups Tubli-Seal, Apexit Plus, Fillapex, AH Plus, RoekoSeal, and Positive control, respectively. Materials and Methods: Five groups after the incubation with the microorganism E. faecalis were coated with different root canal sealers and obturated using F3 ProTaper Gutta-percha point. The dentinal shavings were collected and analyzed for RT-PCR. Statistical Analysis: The mean difference between six groups was calculated using analysis of variance and post-hoc test. Results: The highest antibacterial activity was achieved with Tubli-Seal (1938.13 DNA in pictogram [pg]) and least by RoekoSeal (3034.54 DNA in pg). Conclusion: The maximum antimicrobial activity was achieved AH Plus and Tubli-Seal. RT-PCR can be used as a valuable and accurate tool for testing antimicrobial activity. PMID:26752843

  5. A real-time digital control, data acquisition and analysis system for the DIII-D multipulse Thomson scattering diagnostic

    NASA Astrophysics Data System (ADS)

    Greenfield, C. M.; Campbell, G. L.; Carlstrom, T. N.; Deboo, J. C.; Hsieh, C.-L.; Snider, R. T.; Trost, P. K.

    1990-10-01

    A VME-based real time computer systems for laser control, data acquisition and analysis for the DIII-D multipulse Thomson scattering diagnostic is described. The laser control task requires precise timing of up to 8 Nd:YAG lasers, each with an average firing rate of 20 Hz. A cpu module in real time multiprocessing computer system will operate the lasers with evenly staggered laser pulses or in a 'burst mode', where all available (fully charged) lasers can be fired at 50 to 100 msec intervals upon receipt of an external event trigger signal. One of more cpu modules, along with a LeCroy FERA (Fast Encoding and Readout ADC) system, will perform real time data acquisition and analysis. Partial electron temperature and density profiles will be available for plasma feedback control within 1 msec following each laser pulse. The VME-based computer system consists of 2 or more target processor modules (25 MHz Motorola 68030) running the VMEexec real time operating system connected to a Unix based Host system (also a 68030). All real time software is fully interrupt driven to maximize system efficiency. Operator interaction and (non real-time) data analysis takes place on a MicroVAX 3400 connected via DECnet.

  6. A real-time digital control, data acquisition and analysis system for the DIII-D multipulse Thomson scattering diagnostic

    SciTech Connect

    Greenfield, C.M.; Campbell, G.L.; Carlstrom, T.N.; DeBoo, J.C.; Hsieh, C.-L.; Snider, R.T.; Trost, P.K.

    1990-10-01

    A VME-based real-time computer systems for laser control, data acquisition and analysis for the DIII-D multipulse Thomson scattering diagnostic is described. The laser control task requires precise timing of up to 8 Nd:YAG lasers, each with an average firing rate of 20 Hz. A cpu module in real-time multiprocessing computer system will operate the lasers with evenly staggered laser pulses or in a burst mode'', where all available (fully charged) lasers can be fired at 50--100 {mu}sec intervals upon receipt of an external event trigger signal. One of more cpu modules, along with a LeCroy FERA (Fast Encoding and Readout ADC) system, will perform real-time data acquisition and analysis. Partial electron temperature and density profiles will be available for plasma feedback control within 1 msec following each laser pulse. The VME-based computer system consists of 2 or more target processor modules (25 MHz Motorola 68030) running the VMEexec real-time operating system connected to a Unix based host system (also a 68030). All real-time software is fully interrupt driven to maximize system efficiency. Operator interaction and (non real-time) data analysis takes place on a MicroVAX 3400 connected via DECnet. 17 refs., 1 fig.

  7. Real-time digital control, data acquisition, and analysis system for the DIII-D multipulse Thomson scattering diagnostic

    NASA Astrophysics Data System (ADS)

    Greenfield, C. M.; Campbell, G. L.; Carlstrom, T. N.; DeBoo, J. C.; Hsieh, C.-L.; Snider, R. T.; Trost, P. K.

    1990-10-01

    A VME-based real-time computer system for laser control, data acquisition, and analysis for the DIII-D multipulse Thomson scattering diagnostic is described. The laser control task requires precise timing of up to eight Nd:YAG lasers, each with an average firing rate of 20 Hz. A cpu module in a real-time multiprocessing computer system will operate the lasers with evenly staggered laser pulses or in a ``burst mode,'' where all available (fully charged) lasers can be fired at 50-100 μs intervals upon receipt of an external event trigger signal. One or more cpu modules, along with a LeCroy FERA (fast encoding and readout ADC) system, will perform real-time data acquisition and analysis. Partial electron temperature and density profiles will be available for plasma feedback control within 1 ms following each laser pulse. The VME-based computer system consists of two or more target processor modules (25 MHz Motorola 68030) running the VMEexec real-time operating system connected to a Unix-based host system (also a 68030). All real-time software is fully interrupt driven to maximize system efficiency. Operator interaction and (non-real-time) data analysis takes place on a MicroVAX 3400 connected via DECnet.

  8. Real-time digital control, data acquisition, and analysis system for the DIII-D multipulse Thomson scattering diagnostic

    SciTech Connect

    Greenfield, C.M.; Campbell, G.L.; Carlstrom, T.N.; DeBoo, J.C.; Hsieh, C.; Snider, R.T.; Trost, P.K. )

    1990-10-01

    A VME-based real-time computer system for laser control, data acquisition, and analysis for the DIII-D multipulse Thomson scattering diagnostic is described. The laser control task requires precise timing of up to eight Nd:YAG lasers, each with an average firing rate of 20 Hz. A cpu module in a real-time multiprocessing computer system will operate the lasers with evenly staggered laser pulses or in a burst mode,'' where all available (fully charged) lasers can be fired at 50--100 {mu}s intervals upon receipt of an external event trigger signal. One or more cpu modules, along with a LeCroy FERA (fast encoding and readout ADC) system, will perform real-time data acquisition and analysis. Partial electron temperature and density profiles will be available for plasma feedback control within 1 ms following each laser pulse. The VME-based computer system consists of two or more target processor modules (25 MHz Motorola 68030) running the VMEexec real-time operating system connected to a Unix-based host system (also a 68030). All real-time software is fully interrupt driven to maximize system efficiency. Operator interaction and (non-real-time) data analysis takes place on a MicroVAX 3400 connected via DECnet.

  9. Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis.

    PubMed

    Andréasson, Hanna; Gyllensten, Ulf; Allen, Marie

    2002-08-01

    The rapid development of molecular genetic analysis tools has made it possible to analyze most biological materialfound at the scene of a crime. Evidence materials containing DNA quantities too low to be analyzed using nuclear markers can be analyzed using the highly abundant mtDNA. However, there is a shortage of sensitive nDNA and mtDNA quantification assays. In this study, an assay for the quantification of very small amounts of DNA, based on the real-time Taq-Man assay, has been developed. This analysis will provide an estimate of the total number of nDNA copies and the total number of mtDNA molecules in a particular evidence material. The quantification is easy to perform, fast, and requires a minimum of the valuable DNA extracted from the evidence materiaL The results will aid in the evaluation of whether the specific sample is suitable for nDNA or mtDNA analysis. Furthermore, the optimal amount of DNA to be used in further analysis can be estimated ensuring that the analysis is successful and that the DNA is retained for future independent analysis. This assay has significant advantages over existing techniques because of its high sensitivity, accuracy, and the combined analysis of nDNA and mtDNA. Moreover, it has the potential to provide additional information about the presence of inhibitors in forensic samples. Subsequent mitochondrial and nuclear analysis of quantified samples illustrated the potential to predict the number of DNA copies required for a successful analysis in a certain typing assay. PMID:12188193

  10. Real-time visualization and analysis of airflow field by use of digital holography

    NASA Astrophysics Data System (ADS)

    Di, Jianglei; Wu, Bingjing; Chen, Xin; Liu, Junjiang; Wang, Jun; Zhao, Jianlin

    2013-04-01

    The measurement and analysis of airflow field is very important in fluid dynamics. For airflow, smoke particles can be added to visually observe the turbulence phenomena by particle tracking technology, but the effect of smoke particles to follow the high speed airflow will reduce the measurement accuracy. In recent years, with the advantage of non-contact, nondestructive, fast and full-field measurement, digital holography has been widely applied in many fields, such as deformation and vibration analysis, particle characterization, refractive index measurement, and so on. In this paper, we present a method to measure the airflow field by use of digital holography. A small wind tunnel model made of acrylic glass is built to control the velocity and direction of airflow. Different shapes of samples such as aircraft wing and cylinder are placed in the wind tunnel model to produce different forms of flow field. With a Mach-Zehnder interferometer setup, a series of digital holograms carrying the information of airflow filed distributions in different states are recorded by CCD camera and corresponding holographic images are numerically reconstructed from the holograms by computer. Then we can conveniently obtain the velocity or pressure information of the airflow deduced from the quantitative phase information of holographic images and visually display the airflow filed and its evolution in the form of a movie. The theory and experiment results show that digital holography is a robust and feasible approach for real-time visualization and analysis of airflow field.

  11. Real-Time Respiratory Motion Analysis Using 4-D Shape Priors.

    PubMed

    Wasza, Jakob; Fischer, Peter; Leutheuser, Heike; Oefner, Tobias; Bert, Christoph; Maier, Andreas; Hornegger, Joachim

    2016-03-01

    Respiratory motion analysis based on range imaging (RI) has emerged as a popular means of generating respiration surrogates to guide motion management strategies in computer-assisted interventions. However, existing approaches employ heuristics, require substantial manual interaction, or yield highly redundant information. In this paper, we propose a framework that uses preprocedurally obtained 4-D shape priors from patient-specific breathing patterns to drive intraprocedural RI-based real-time respiratory motion analysis. As the first contribution, we present a shape motion model enabling an unsupervised decomposition of respiration induced high-dimensional body surface displacement fields into a low-dimensional representation encoding thoracic and abdominal breathing. Second, we propose a method designed for GPU architectures to quickly and robustly align our models to high-coverage multiview RI body surface data. With our fully automatic method, we obtain respiration surrogates yielding a Pearson correlation coefficient (PCC) of 0.98 with conventional surrogates based on manually selected regions on RI body surface data. Compared to impedance pneumography as a respiration signal that measures the change of lung volume, we obtain a PCC of 0.96. Using off-the-shelf hardware, our framework enables high temporal resolution respiration analysis at 50 Hz. PMID:26258934

  12. Quantitative real-time analysis of collective cancer invasion and dissemination

    NASA Astrophysics Data System (ADS)

    Ewald, Andrew J.

    2015-05-01

    A grand challenge in biology is to understand the cellular and molecular basis of tissue and organ level function in mammals. The ultimate goals of such efforts are to explain how organs arise in development from the coordinated actions of their constituent cells and to determine how molecularly regulated changes in cell behavior alter the structure and function of organs during disease processes. Two major barriers stand in the way of achieving these goals: the relative inaccessibility of cellular processes in mammals and the daunting complexity of the signaling environment inside an intact organ in vivo. To overcome these barriers, we have developed a suite of tissue isolation, three dimensional (3D) culture, genetic manipulation, nanobiomaterials, imaging, and molecular analysis techniques to enable the real-time study of cell biology within intact tissues in physiologically relevant 3D environments. This manuscript introduces the rationale for 3D culture, reviews challenges to optical imaging in these cultures, and identifies current limitations in the analysis of complex experimental designs that could be overcome with improved imaging, imaging analysis, and automated classification of the results of experimental interventions.

  13. Real-time nonlinear finite element analysis for surgical simulation using graphics processing units.

    PubMed

    Taylor, Zeike A; Cheng, Mario; Ourselin, Sébastien

    2007-01-01

    Clinical employment of biomechanical modelling techniques in areas of medical image analysis and surgical simulation is often hindered by conflicting requirements for high fidelity in the modelling approach and high solution speeds. We report the development of techniques for high-speed nonlinear finite element (FE) analysis for surgical simulation. We employ a previously developed nonlinear total Lagrangian explicit FE formulation which offers significant computational advantages for soft tissue simulation. However, the key contribution of the work is the presentation of a fast graphics processing unit (GPU) solution scheme for the FE equations. To the best of our knowledge this represents the first GPU implementation of a nonlinear FE solver. We show that the present explicit FE scheme is well-suited to solution via highly parallel graphics hardware, and that even a midrange GPU allows significant solution speed gains (up to 16.4x) compared with equivalent CPU implementations. For the models tested the scheme allows real-time solution of models with up to 16000 tetrahedral elements. The use of GPUs for such purposes offers a cost-effective high-performance alternative to expensive multi-CPU machines, and may have important applications in medical image analysis and surgical simulation. PMID:18051120

  14. Quantitative Analysis Of Sperm Motion Kinematics From Real-Time Video-Edge Images

    NASA Astrophysics Data System (ADS)

    Davis, Russell O...; Katz, David F.

    1988-02-01

    A new model of sperm swimming kinematics, which uses signal processing methods and multivariate statistical techniques to identify individual cell-motion parameters and unique cell populations, is presented. Swimming paths of individual cells are obtained using real-time, video-edge digitization. Raw paths are adaptively filtered to identify average paths, and measurements of space-time oscillations about average paths are made. Time-dependent frequency information is extracted from spatial variations about average paths using harmonic analysis. Raw-path and average-path measures such as curvature, curve length, and straight-line length, and measures of oscillations about average paths such as time-dependent amplitude and frequency variations, are used in a multivariate, cluster analysis to identify unique cell populations. The entire process, including digitization of sperm video images, is computer-automated. Preliminary results indicate that this method of tracking, digitization, and kinematic analysis accurately identifies unique cell subpopulations, including: the relative numbers of cells in each subpopulation, how subpopulations differ, and the extent and significance of such differences. With appropriate work, this approach may be useful for clinical discrimination between normal and abnormal semen specimens.

  15. Load Balancing Using Time Series Analysis for Soft Real Time Systems with Statistically Periodic Loads

    NASA Technical Reports Server (NTRS)

    Hailperin, M.

    1993-01-01

    This thesis provides design and analysis of techniques for global load balancing on ensemble architectures running soft-real-time object-oriented applications with statistically periodic loads. It focuses on estimating the instantaneous average load over all the processing elements. The major contribution is the use of explicit stochastic process models for both the loading and the averaging itself. These models are exploited via statistical time-series analysis and Bayesian inference to provide improved average load estimates, and thus to facilitate global load balancing. This thesis explains the distributed algorithms used and provides some optimality results. It also describes the algorithms' implementation and gives performance results from simulation. These results show that the authors' techniques allow more accurate estimation of the global system loading, resulting in fewer object migrations than local methods. The authors' method is shown to provide superior performance, relative not only to static load-balancing schemes but also to many adaptive load-balancing methods. Results from a preliminary analysis of another system and from simulation with a synthetic load provide some evidence of more general applicability.

  16. A framework for analysis of the upper airway from real-time MRI sequences

    NASA Astrophysics Data System (ADS)

    Silva, Samuel; Teixeira, António

    2013-12-01

    In recent years, real-time Magnetic Resonance Imaging (RT-MRI) has been used to acquire vocal tract data to support articulatory studies. The large amount of images resulting from these acquisitions needs to be processed and the resulting data analysed to extract articulatory features. This analysis is often performed by linguists and phoneticists and requires not only tools providing a high level exploration of the data, to gather insight over the different aspects of speech, but also a set of features to compare different vocal tract configurations in static and dynamic scenarios. In order to make the data available in a faster and systematic fashion, without the continuous direct involvement of image processing specialists, a framework is being developed to bridge the gap between the more technical aspects of raw data and the higher level analysis required by speech researchers. In its current state it already includes segmentation of the vocal tract, allows users to explore the different aspects of the acquired data using coordinated views, and provides support for vocal tract configuration comparison. Beyond the traditional method of visual comparison of vocal tract profiles, a quantitative method is proposed, considering relevant anatomical features, supported by an abstract representation of the data both for static and dynamic analysis.

  17. Rapid detection of sacbrood virus in honeybee using ultra-rapid real-time polymerase chain reaction.

    PubMed

    Yoo, Mi-Sun; Thi, Kim Cuc Nguyen; Van Nguyen, Phu; Han, Sang-Hoon; Kwon, Soon-Hwan; Yoon, Byoung-Su

    2012-01-01

    A real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for the fast and highly sensitive detection of the sacbrood virus (SBV) genome and applied to honeybee samples. Using plasmid DNA containing a partial SBV genome and diluted serially, as few as 1×10(2)copies/μl (correlation co-efficiency >0.99) were detected by the qRT-PCR assay, whereas 1×10(3)copies/μl were detected by the conventional RT-PCR assay. As a rapid detection method, ultra-rapid real-time PCR (URRT-PCR) was carried out with a GenSpector TMC-1000 silicon-glass chip-based thermal cycler, which has a 6μl micro-chamber volume and a fast outstandingly heating/cooling rate. Using this method, 10(3)copies of pBX-SBV3.8 clone were detected within 17 min after 40 PCR cycles, including melting point analysis. To reduce the detection time for SBV, synthesis of the cDNA of the SBV genome from a honeybee sample was attempted for different reaction times and the cDNA was used as the template for URRT-PCR assays. The results indicated that a 5 min reaction time was sufficient to synthesize cDNA as the template for the SBV URRT-PCR assay. This study described a novel PCR-based method that is able to detect an RNA virus in environmental samples within 22 min, including reverse transcription, PCR detection and melting point analysis in real-time. PMID:22079620

  18. Near-Real-Time Analysis of Publicly Communicated Disaster Response Information

    NASA Astrophysics Data System (ADS)

    Girard, Trevor

    2015-04-01

    During a disaster situation the public will need to make critical actions regarding what to do, where to go, how to get there, and so on. The more informed the public is, the better actions they are able to make, resulting in reduced disaster impacts. The criteria for what information to provide the public needs to change depending on the specific needs of the disaster affected population. The method of dissemination also needs to match the communication channels that the public typically uses in disaster situations. This research project investigates the dynamic information needs of disaster affected populations and how information leads to actions. The purpose of the research project is to identify key indicators for measuring how well informed the public is during disasters. The indicators are limited to those which can be observed as communication is happening (i.e., in near-real-time). By doing so, the indicators can be analyzed as disaster situations unfold, deficiencies can be identified, and recommendations can be made to potentially improve communication while the response is still underway. The end goal of the research is to improve the ability of communicators to inform disaster affected communities. A classification scheme has been developed to categorize the information provided to the public during disasters. Under each category is a set of typical questions that the information should answer. These questions are the result of a best observed practice review of the information available during 11 disasters. For example, under the category 'Life Saving Response', the questions which should be answered are who is doing what (Evacuation, SAR), where and when, and the amount of the affected communities' needs being covered by these actions. Review of what questions remain unanswered acts as the first indicator, referred to as an 'Information Gap Analysis'. Comparative analysis of the information within categories, between categories, and between similar

  19. Model-based assessment of erlotinib effect in vitro measured by real-time cell analysis.

    PubMed

    Benay, Stephan; Meille, Christophe; Kustermann, Stefan; Walter, Isabelle; Walz, Antje; Gonsard, P Alexis; Pietilae, Elina; Kratochwil, Nicole; Iliadis, Athanassios; Roth, Adrian; Lave, Thierry

    2015-06-01

    Real time cell analysis (RTCA) is an impedance-based technology which tracks various living cell characteristics over time, such as their number, morphology or adhesion to the extra cellular matrix. However, there is no consensus about how RTCA data should be used to quantitatively evaluate pharmacodynamic parameters which describe drug efficacy or toxicity. The purpose of this work was to determine how RTCA data can be analyzed with mathematical modeling to explore and quantify drug effect in vitro. The pharmacokinetic-pharmacodynamic erlotinib concentration profile predicted by the model and its effect on the human epidermoïd carcinoma cell line A431 in vitro was measured through RTCA output, designated as cell index. A population approach was used to estimate model parameter values, considering a plate well as the statistical unit. The model related the cell index to the number of cells by means of a proportionality factor. Cell growth was described by an exponential model. A delay between erlotinib pharmacokinetics and cell killing was described by a transit compartment model, and the effect potency, by an E max function of erlotinib concentration. The modeling analysis performed on RTCA data distinguished drug effects in vitro on cell number from other effects likely to modify the relationship between cell index and cell number. It also revealed a time-dependent decrease of erlotinib concentration over time, described by a mono-exponential pharmacokinetic model with nonspecific binding. PMID:25822652

  20. Real-time analysis, visualization, and steering of microtomography experiments at photon sources

    SciTech Connect

    von Laszeski, G.; Insley, J. A.; Foster, I.; Bresnahan, J.; Kesselman, C.; Su, M.; Thiebaux, M.; Rivers, M. L.; Wang, S.; Tieman, B., McNulty, I.

    2000-02-29

    A new generation of specialized scientific instruments called synchrotron light sources allow the imaging of materials at very fine scales. However, in contrast to a traditional microscope, interactive use has not previously been possible because of the large amounts of data generated and the considerable computation required translating this data into a useful image. The authors describe a new software architecture that uses high-speed networks and supercomputers to enable quasi-real-time and hence interactive analysis of synchrotron light source data. This architecture uses technologies provided by the Globus computational grid toolkit to allow dynamic creation of a reconstruction pipeline that transfers data from a synchrotron source beamline to a preprocessing station, next to a parallel reconstruction system, and then to multiple visualization stations. Collaborative analysis tools allow multiple users to control data visualization. As a result, local and remote scientists can see and discuss preliminary results just minutes after data collection starts. The implications for more efficient use of this scarce resource and for more effective science appear tremendous.

  1. A real-time multiple-cell tracking platform for dielectrophoresis (DEP)-based cellular analysis

    NASA Astrophysics Data System (ADS)

    Prasad, Brinda; Du, Shan; Badawy, Wael; Kaler, Karan V. I. S.

    2005-04-01

    There is an increasing demand from biosciences to develop new and efficient techniques to assist in the preparation and analysis of biological samples such as cells in suspension. A dielectrophoresis (DEP)-based characterization and measurement technique on biological cells opens up a broader perspective for early diagnosis of diseases. An efficient real-time multiple-cell tracking platform coupled with DEP to capture and quantify the dynamics of cell motion and obtain cell viability information is presented. The procedure for tracking a single DEP-levitated Canola plant protoplast, using the motion-based segmentation algorithm hierarchical adaptive merge split mesh-based technique (HAMSM) for cell identification, has been enhanced for identifying and tracking multiple cells. The tracking technique relies on the deformation of mesh topology that is generated according to the movement of biological cells in a sequence of images that allows the simultaneous extraction of the biological cell from the image and the associated motion characteristics. Preliminary tests were conducted with yeast cells and then applied to a cancerous cell line subjected to DEP fields. Characteristics, such as cell count, velocity and size, were individually extracted from the tracked results of the cell sample. Tests were limited to eight yeast cells and two cancer cells. A performance analysis to assess tracking accuracy, computational effort and processing time was also conducted. The tracking technique employed on model intact cells in DEP fields proved to be accurate, reliable and robust.

  2. Spectral analysis method and sample generation for real time visualization of speech

    NASA Astrophysics Data System (ADS)

    Hobohm, Klaus

    A method for translating speech signals into optical models, characterized by high sound discrimination and learnability and designed to provide to deaf persons a feedback towards control of their way of speaking, is presented. Important properties of speech production and perception processes and organs involved in these mechanisms are recalled in order to define requirements for speech visualization. It is established that the spectral representation of time, frequency and amplitude resolution of hearing must be fair and continuous variations of acoustic parameters of speech signal must be depicted by a continuous variation of images. A color table was developed for dynamic illustration and sonograms were generated with five spectral analysis methods such as Fourier transformations and linear prediction coding. For evaluating sonogram quality, test persons had to recognize consonant/vocal/consonant words and an optimized analysis method was achieved with a fast Fourier transformation and a postprocessor. A hardware concept of a real time speech visualization system, based on multiprocessor technology in a personal computer, is presented.

  3. Direct analysis in real time--a critical review on DART-MS.

    PubMed

    Gross, Jürgen H

    2014-01-01

    Direct analysis in real time mass spectrometry (DART-MS) has become an established technique for rapid mass spectral analysis of a large variety of samples. DART-MS is capable of analyzing the sample at atmospheric pressure, essentially in the open laboratory environment. DART-MS can be applied to compounds that have been deposited or adsorbed on to surfaces or that are being desorbed therefrom into the atmosphere. This makes DART-MS suitable and well-known for analysis of ingredients of plant materials, pesticide monitoring on vegetables, forensic and safety applications such as screening for traces of explosives, warfare agents, or illicit drugs on luggage, clothes, or bank notes, etc. DART can also be used for analysis of either solid or liquid bulk materials, as may be required in quality control, or to quickly investigate the identity of a compound from chemical synthesis. Even living organisms can be subjected to DART-MS. Driven by different needs in analytical practice, the combination of the DART ionization source and interface can be configured in multiple geometries and with various accessories to adapt the setup as required. Analysis by DART-MS relies on some sort of gas-phase ionization mechanism. In DART, initial generation of the ionizing species is by use of a corona discharge in a pure helium atmosphere which delivers excited helium atoms that, upon their release into the atmosphere, will initiate a cascade of gas-phase reactions. In the end, this results in reagent ions created from atmospheric water or (solvent) vapor in the vicinity of the surface subject to analysis where they effect a chemical ionization process. DART ionization processes may generate positive or negative ions, predominantly even-electron species, but odd-electron species do also occur. The prevailing process of analyte ion formation from a given sample is highly dependent on analyte properties. PMID:24036523

  4. DETECTION OF HUMAN ENTERIC VIRUSES IN STREAM WATER WITH RT-PCR AND CELL CULTURE

    EPA Science Inventory

    A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison to traditional cell culture and Escherich...

  5. Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

    ERIC Educational Resources Information Center

    Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

    2015-01-01

    A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

  6. Real-Time Polymerase Chain Reaction as a Tool for Evaluation of Magnetic Poly(Glycidyl methacrylate)-Based Microspheres in Molecular Diagnostics.

    PubMed

    Trachtová, Stepánka; Spanová, Alena; Horák, Daniel; Kozáková, Hana; Rittich, Bohuslav

    2016-01-01

    DNA amplification by real-time polymerase chain reaction (RT-PCR) was used for the evaluation of efficiency of polymer coating of magnetic hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) and poly(glycidyl methacrylate) (PGMA) microspheres with/without carboxyl groups. The inhibition effect of magnetic microspheres on real-time polymerase chain reaction (RT-PCR) course was evaluated by regression analysis after the addition of different concentrations of tested microspheres to PCR mixtures. Microspheres mostly did not interfere in RT-PCR till the concentration 50 µg/25 µl PCR mixture. No relationship between Fe content (and microsphere diameter) and inhibition effect was found. Microspheres containing carboxyl groups extinguished the fluorescence at lower concentrations (10-20 µg/25 µl PCR mixture) without inhibition of DNA amplification as PCR products were detected using agarose gel electrophoresis. Negative effect of maghemite on PCR course was partially reduced by coating of magnetic core by silica or polymers. Two inhibition mechanisms of DNA amplification were discussed in this work. PMID:26708828

  7. Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-β-glucanase.

    PubMed

    Keeratijarut, Angsana; Lohnoo, Tassanee; Yingyong, Wanta; Rujirawat, Thidarat; Srichunrusami, Chutatip; Onpeaw, Pornpit; Chongtrakool, Piriyaporn; Brandhorst, T Tristan; Krajaejun, Theerapong

    2015-09-01

    Pythiosis is a life-threatening infectious disease caused by Pythium insidiosum. Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of P. insidiosum. Failure to detect P. insidiosum by PCR amplification using the rDNA-specific primers has been reported. PinsEXO1, encoding an exo-1,3-β-glucanase, is an alternative, novel and efficient target for identification of P. insidiosum by conventional PCR. In this study, we aimed to develop a real-time (RT)-PCR approach targeting PinsEXO1 and compare its performance with conventional PCR for the detection of P. insidiosum. Both conventional and RT-PCR assays were positive for all 35 P. insidiosum strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1 × 10(-4) ng, respectively. In conclusion, the RT-PCR assay retained 100% sensitivity and 100% specificity for detection of P. insidiosum. It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of P. insidiosum. PMID:26296566

  8. Real-time inverse-model analysis and control on data collection

    NASA Astrophysics Data System (ADS)

    Vesselinov, V. V.; Robinson, B. A.; Vrugt, J. A.; Zyvoloski, G. A.

    2005-12-01

    Sophisticated numerical models are commonly used to simulate fluid and chemical flow in the subsurface. The science of flow in porous media is composed of general physical principles (transferable knowledge) and site-specific details. All sites are unique, so even if the physics is well understood, we need detailed, site-specific information to develop a model for each site (subsurface heterogeneity, initial and boundary conditions, etc.). In this respect, at each new site, we "start over". The most time- and resource-consuming step in reducing predictive uncertainty bounds in subsurface systems is the process of uncovering the site-specific details. The current paradigm is to perform a lengthy reconnaissance phase to understand the site, followed by additional data collection and modeling to synthesize the information. Model development methods are slow and labor-intensive for complex sites; therefore, model results generally lag behind the data collection by a considerable length of time. This delay limits the usefulness of the model as a tool to guide data collection: any given iteration of the model is out of date by the time it is completed. The whole process is unacceptably protracted in an era in which, for example, in the U.S. alone we may ultimately need hundreds of sites to implement CO2 geologic sequestration. Our technical capabilities for efficiently collecting and organizing subsurface data have progressed recently with the advent of modern data collection and transmission systems. However, our ability to process this information in the form of numerical models has lagged behind. We propose a new paradigm for the development of complex subsurface flow and transport models in which the inverse analysis is performed in real time, simultaneously with the data collection. Furthermore, we propose to use the inverse model to control the data collection or the operating conditions of an extraction system in real time. This is extremely important because post

  9. Real-time myoelectric control of a multi-fingered hand prosthesis using principal components analysis

    PubMed Central

    2012-01-01

    Background In spite of the advances made in the design of dexterous anthropomorphic hand prostheses, these sophisticated devices still lack adequate control interfaces which could allow amputees to operate them in an intuitive and close-to-natural way. In this study, an anthropomorphic five-fingered robotic hand, actuated by six motors, was used as a prosthetic hand emulator to assess the feasibility of a control approach based on Principal Components Analysis (PCA), specifically conceived to address this problem. Since it was demonstrated elsewhere that the first two principal components (PCs) can describe the whole hand configuration space sufficiently well, the controller here employed reverted the PCA algorithm and allowed to drive a multi-DoF hand by combining a two-differential channels EMG input with these two PCs. Hence, the novelty of this approach stood in the PCA application for solving the challenging problem of best mapping the EMG inputs into the degrees of freedom (DoFs) of the prosthesis. Methods A clinically viable two DoFs myoelectric controller, exploiting two differential channels, was developed and twelve able-bodied participants, divided in two groups, volunteered to control the hand in simple grasp trials, using forearm myoelectric signals. Task completion rates and times were measured. The first objective (assessed through one group of subjects) was to understand the effectiveness of the approach; i.e., whether it is possible to drive the hand in real-time, with reasonable performance, in different grasps, also taking advantage of the direct visual feedback of the moving hand. The second objective (assessed through a different group) was to investigate the intuitiveness, and therefore to assess statistical differences in the performance throughout three consecutive days. Results Subjects performed several grasp, transport and release trials with differently shaped objects, by operating the hand with the myoelectric PCA-based controller

  10. Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

    PubMed

    Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

    2016-08-28

    The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR. PMID:27197668

  11. Real-time estimation of Arctic sea ice thickness through maximum covariance analysis

    NASA Astrophysics Data System (ADS)

    Dirkson, Arlan; Merryfield, William J.; Monahan, Adam

    2015-06-01

    A challenge for model-based seasonal predictions of sea ice is an accurate representation of sea ice initial conditions, particularly sparsely observed sea ice thickness (SIT). The Canadian Seasonal to Interannual Prediction System (CanSIPS) currently initializes SIT by nudging simulated values toward a model-based climatology. To improve on this, we use sea ice data from Pan-Arctic Ice Ocean Modeling and Assimilation System to investigate how accurately SIT can be estimated in real time using better observed and physically relevant predictors. We (1) test the skill of several predictors using maximum covariance analysis (MCA), (2) apply an approach which blends sea ice concentration and lagged (4 month averaged) sea level pressure, and (3) compare this method against the current CanSIPS initialization scheme over 1981-2012. The MCA-based statistical model reduces SIT areal mean and temporal mean absolute errors by 48% relative to the current CanSIPS initialization and shows consistent skill estimating ice volume in all months (r = 0.95).

  12. Real-time sweat analysis via alternating current conductivity of artificial and human sweat

    NASA Astrophysics Data System (ADS)

    Liu, Gengchen; Alomari, Mahmoud; Sahin, Bunyamin; Snelgrove, Samuel E.; Edwards, Jeffrey; Mellinger, Axel; Kaya, Tolga

    2015-03-01

    Dehydration is one of the most profound physiological challenges that significantly affects athletes and soldiers if not detected early. Recently, a few groups have focused on dehydration detection using sweat as the main biomarker. Although there are some proposed devices, the electrical and chemical characteristics of sweat have yet to be incorporated into the validations. In this work, we have developed a simple test setup to analyze artificial sweat that is comprised the main components of human sweat. We provide theoretical and experimental details on the electrical and chemical behavior of the artificial sweat for various concentration values within a temperature range of 5 °C to 50 °C. We have also developed an efficient sweat collecting and detection system based on 3D printing. Human studies were conducted and this particular protocol has shown that dehydration starts to take effect as early as 40 min into the physical activity if there is no fluid intake during the exercise. We believe that our device will lead to developing viable real-time sweat analysis systems.

  13. Real-Time and Post-Reaction Microscopic Structural Analysis of Biomass Undergoing Pyrolysis

    SciTech Connect

    Haas, T. J.; Nimlos, M. R.; Donohoe, B. S.

    2009-01-01

    The structural complexity of unprocessed plant tissues used for thermochemical conversion of biomass to fuels and energy impedes heat and mass transfer and may increase the occurrence of tar-forming secondary chemical reactions. At industrial scales, gas and liquid products trapped within large biomass particles may reduce net fuel yields and increase tars, impacting industrial operations and increasing overall costs. Real-time microscopic analysis of poplar (Populus sp.) wood samples undergoing anoxic, pyrolytic heat treatment has revealed a pattern of tissue and macropore expansion and collapse. Post-reaction structural analyses of biomass char (biochar) by light and transmission electron microscopy have provided direct structural evidence of pyrolysis product mass-transfer issues, including trapped pyrolysis products and cell wall compression, and have demonstrated the impact of heat-transfer problems on biomass particles. Finally, microscopic imaging has revealed that pyrolyzed/gasified biochars recovered from a fluidized bed reactor retain a similar pre-reaction basic plant tissue structure as the samples used in this study, suggesting that the phenomena observed here are representative of those that occur in larger scale reactors.

  14. MALDI mass spectrometry imaging analysis of pituitary adenomas for near-real-time tumor delineation

    PubMed Central

    Calligaris, David; Feldman, Daniel R.; Norton, Isaiah; Olubiyi, Olutayo; Changelian, Armen N.; Machaidze, Revaz; Vestal, Matthew L.; Laws, Edward R.; Dunn, Ian F.; Santagata, Sandro; Agar, Nathalie Y. R.

    2015-01-01

    We present a proof of concept study designed to support the clinical development of mass spectrometry imaging (MSI) for the detection of pituitary tumors during surgery. We analyzed by matrix-assisted laser desorption/ionization (MALDI) MSI six nonpathological (NP) human pituitary glands and 45 hormone secreting and nonsecreting (NS) human pituitary adenomas. We show that the distribution of pituitary hormones such as prolactin (PRL), growth hormone (GH), adrenocorticotropic hormone (ACTH), and thyroid stimulating hormone (TSH) in both normal and tumor tissues can be assessed by using this approach. The presence of most of the pituitary hormones was confirmed by using MS/MS and pseudo-MS/MS methods, and subtyping of pituitary adenomas was performed by using principal component analysis (PCA) and support vector machine (SVM). Our proof of concept study demonstrates that MALDI MSI could be used to directly detect excessive hormonal production from functional pituitary adenomas and generally classify pituitary adenomas by using statistical and machine learning analyses. The tissue characterization can be completed in fewer than 30 min and could therefore be applied for the near-real-time detection and delineation of pituitary tumors for intraoperative surgical decision-making. PMID:26216958

  15. Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

    PubMed Central

    Ritzefeld, Markus; Sewald, Norbert

    2012-01-01

    Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR. PMID:22500214

  16. In Situ Chemical Characterization of Organic Aerosol Surfaces using Direct Analysis in Real Time

    NASA Astrophysics Data System (ADS)

    Chan, M.; Nah, T.; Wilson, K. R.

    2012-12-01

    Obtaining in situ information on the molecular composition of atmospheric aerosol is important for understanding the sources, formation mechanisms, aging and physiochemical properties of atmospheric aerosol. Most recently, we have used Direct Analysis in Real Time (DART), which is a "soft" atmospheric pressure ionization technique, for in situ chemical characterization of a variety of laboratory generated organic aerosol and heterogeneous processing oleic acid aerosol. A stream of aerosol particles is crossed with a thermal flow of metastable He atoms (produced by the DART source) in front of an inlet of a mass spectrometer. The thermally desorbed analytes are subsequently ionized with minimal fragmentation by reactive species in the DART ionization source (e.g., metastable He atoms). The ion signal scales with the aerosol surface area rather than aerosol volume, suggesting that aerosol particles are not completely vaporized in the ionization region. The DART can thus measure the chemical composition as a function of aerosol depth. Probing aerosol depth is determined by the thermal desorption rates of aerosol particles. Here, we investigate how the experimental parameters (e.g., DART gas temperature and residence time) and the physiochemical properties of aerosol particles (e.g., enthalpy of vaporization) affect the probing aerosol depth and the desorption-ionization mechanism of aerosol particles in the DART using a series of model organic compounds. We also demonstrate the potential application of DART for in situ chemically analyzing wet aerosol particles undergoing oxidation reactions.

  17. MALDI mass spectrometry imaging analysis of pituitary adenomas for near-real-time tumor delineation.

    PubMed

    Calligaris, David; Feldman, Daniel R; Norton, Isaiah; Olubiyi, Olutayo; Changelian, Armen N; Machaidze, Revaz; Vestal, Matthew L; Laws, Edward R; Dunn, Ian F; Santagata, Sandro; Agar, Nathalie Y R

    2015-08-11

    We present a proof of concept study designed to support the clinical development of mass spectrometry imaging (MSI) for the detection of pituitary tumors during surgery. We analyzed by matrix-assisted laser desorption/ionization (MALDI) MSI six nonpathological (NP) human pituitary glands and 45 hormone secreting and nonsecreting (NS) human pituitary adenomas. We show that the distribution of pituitary hormones such as prolactin (PRL), growth hormone (GH), adrenocorticotropic hormone (ACTH), and thyroid stimulating hormone (TSH) in both normal and tumor tissues can be assessed by using this approach. The presence of most of the pituitary hormones was confirmed by using MS/MS and pseudo-MS/MS methods, and subtyping of pituitary adenomas was performed by using principal component analysis (PCA) and support vector machine (SVM). Our proof of concept study demonstrates that MALDI MSI could be used to directly detect excessive hormonal production from functional pituitary adenomas and generally classify pituitary adenomas by using statistical and machine learning analyses. The tissue characterization can be completed in fewer than 30 min and could therefore be applied for the near-real-time detection and delineation of pituitary tumors for intraoperative surgical decision-making. PMID:26216958

  18. The U-tube sampling methodology and real-time analysis of geofluids

    SciTech Connect

    Freifeld, Barry; Perkins, Ernie; Underschultz, James; Boreham, Chris

    2009-03-01

    The U-tube geochemical sampling methodology, an extension of the porous cup technique proposed by Wood [1973], provides minimally contaminated aliquots of multiphase fluids from deep reservoirs and allows for accurate determination of dissolved gas composition. The initial deployment of the U-tube during the Frio Brine Pilot CO{sub 2} storage experiment, Liberty County, Texas, obtained representative samples of brine and supercritical CO{sub 2} from a depth of 1.5 km. A quadrupole mass spectrometer provided real-time analysis of dissolved gas composition. Since the initial demonstration, the U-tube has been deployed for (1) sampling of fluids down gradient of the proposed Yucca Mountain High-Level Waste Repository, Armagosa Valley, Nevada (2) acquiring fluid samples beneath permafrost in Nunuvut Territory, Canada, and (3) at a CO{sub 2} storage demonstration project within a depleted gas reservoir, Otway Basin, Victoria, Australia. The addition of in-line high-pressure pH and EC sensors allows for continuous monitoring of fluid during sample collection. Difficulties have arisen during U-tube sampling, such as blockage of sample lines from naturally occurring waxes or from freezing conditions; however, workarounds such as solvent flushing or heating have been used to address these problems. The U-tube methodology has proven to be robust, and with careful consideration of the constraints and limitations, can provide high quality geochemical samples.

  19. Interface for Online Coupling of Surface Plasmon Resonance to Direct Analysis in Real Time Mass Spectrometry.

    PubMed

    Zhang, Yiding; Li, Xianjiang; Nie, Honggang; Yang, Li; Li, Ze; Bai, Yu; Niu, Li; Song, Daqian; Liu, Huwei

    2015-07-01

    The online coupling of surface plasmon resonance (SPR) with mass spectrometry (MS) has been highly desired for the complementary information provided by each of the two techniques. In this work, a novel interface for direct and online coupling of SPR to direct analysis in real time (DART) MS was developed. A spray tip connected with the outlet of the SPR flow solution was conducted as the sampling part of the DART-MS, with which the online coupling interface of SPR-MS was realized. Four model samples, acetaminophen, metronidazole, quinine, and hippuric acid, dissolved in three kinds of common buffers were used in the SPR-DART-MS experiments for performance evaluation of the interface and the optimization of DART conditions. The results showed consistent signal changes and high tolerance of nonvolatile salts of this SPR-MS system, demonstrating the feasibility of the interface for online coupling of SPR with MS and the potential application in the characterization of interaction under physiological conditions. PMID:26067340

  20. LINEBACkER: Bio-inspired Data Reduction Toward Real Time Network Traffic Analysis

    SciTech Connect

    Teuton, Jeremy R.; Peterson, Elena S.; Nordwall, Douglas J.; Akyol, Bora A.; Oehmen, Christopher S.

    2013-09-28

    Abstract—One essential component of resilient cyber applications is the ability to detect adversaries and protect systems with the same flexibility adversaries will use to achieve their goals. Current detection techniques do not enable this degree of flexibility because most existing applications are built using exact or regular-expression matching to libraries of rule sets. Further, network traffic defies traditional cyber security approaches that focus on limiting access based on the use of passwords and examination of lists of installed or downloaded programs. These approaches do not readily apply to network traffic occurring beyond the access control point, and when the data in question are combined control and payload data of ever increasing speed and volume. Manual analysis of network traffic is not normally possible because of the magnitude of the data that is being exchanged and the length of time that this analysis takes. At the same time, using an exact matching scheme to identify malicious traffic in real time often fails because the lists against which such searches must operate grow too large. In this work, we introduce an alternative method for cyber network detection based on similarity-measuring algorithms for gene sequence analysis. These methods are ideal because they were designed to identify similar but nonidentical sequences. We demonstrate that our method is generally applicable to the problem of network traffic analysis by illustrating its use in two different areas both based on different attributes of network traffic. Our approach provides a logical framework for organizing large collections of network data, prioritizing traffic of interest to human analysts, and makes it possible to discover traffic signatures without the bias introduced by expert-directed signature generation. Pattern recognition on reduced representations of network traffic offers a fast, efficient, and more robust way to detect anomalies.

  1. Real-time PCR for the detection and quantification of geodermatophilaceae from stone samples and identification of new members of the genus blastococcus.

    PubMed

    Salazar, Oscar; Valverde, Aranzazu; Genilloud, Olga

    2006-01-01

    Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus. PMID:16391063

  2. Real-Time PCR for the Detection and Quantification of Geodermatophilaceae from Stone Samples and Identification of New Members of the Genus Blastococcus†

    PubMed Central

    Salazar, Oscar; Valverde, Aranzazu; Genilloud, Olga

    2006-01-01

    Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus. PMID:16391063

  3. Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus.

    PubMed

    Zhu, Xiangdong; Wang, Yufei; Chen, Jianfei; Zhang, Xin; Shi, Hongyan; Shi, Da; Gao, Jing; Feng, Li

    2016-08-01

    Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV. PMID:26912233

  4. Determination of cut-off cycle threshold values in routine RT-PCR assays to assist differential diagnosis of norovirus in children hospitalized for acute gastroenteritis.

    PubMed

    Trang, N V; Choisy, M; Nakagomi, T; Chinh, N T M; Doan, Y H; Yamashiro, T; Bryant, J E; Nakagomi, O; Anh, D D

    2015-11-01

    Norovirus (NV) is an important cause of acute gastroenteritis in children, but is also frequently detected in asymptomatic children, which complicates the interpretation of NV detection results in both the clinical setting and population prevalence studies. A total of 807 faecal samples from children aged <5 years hospitalized for acute gastroenteritis were collected in Thai Binh, Vietnam, from January 2011 to September 2012. Real-time RT-PCR was used to detect and quantify NV-RNA in clinical samples. A bimodal distribution of cycle threshold (Ct) values was observed in which the lower peak was assumed to represent cases for which NV was the causal agent of diarrhoea, whereas the higher peak was assumed to represent cases involving an alternative pathogen other than NV. Under these assumptions, we applied finite-mixture modelling to estimate a threshold of Ct <21·36 (95% confidence interval 20·29-22·46) to distinguish NV-positive patients for which NV was the likely cause of diarrhoea. We evaluated the validity of the threshold through comparisons with NV antigen ELISA results, and comparisons of Ct values in patients co-infected with rotavirus. We conclude that the use of an appropriate cut-off value in the interpretation of NV real-time RT-PCR results may improve differential diagnosis of enteric infections, and could contribute to improved estimates of the burden of NV disease. PMID:26418350

  5. Towards Real-Time High Performance Computing For Power Grid Analysis

    SciTech Connect

    Hui, Peter SY; Lee, Barry; Chikkagoudar, Satish

    2012-11-16

    Real-time computing has traditionally been considered largely in the context of single-processor and embedded systems, and indeed, the terms real-time computing, embedded systems, and control systems are often mentioned in closely related contexts. However, real-time computing in the context of multinode systems, specifically high-performance, cluster-computing systems, remains relatively unexplored. Imposing real-time constraints on a parallel (cluster) computing environment introduces a variety of challenges with respect to the formal verification of the system's timing properties. In this paper, we give a motivating example to demonstrate the need for such a system--- an application to estimate the electromechanical states of the power grid--- and we introduce a formal method for performing verification of certain temporal properties within a system of parallel processes. We describe our work towards a full real-time implementation of the target application--- namely, our progress towards extracting a key mathematical kernel from the application, the formal process by which we analyze the intricate timing behavior of the processes on the cluster, as well as timing measurements taken on our test cluster to demonstrate use of these concepts.

  6. An integrated real-time diagnostic concept using expert systems, qualitative reasoning and quantitative analysis

    SciTech Connect

    Edwards, R.M.; Lee, K.Y.; Kumara, S.; Levine, S.H.

    1989-01-01

    An approach for an integrated real-time diagnostic system is being developed for inclusion as an integral part of a power plant automatic control system. In order to participate in control decisions and automatic closed loop operation, the diagnostic system must operate in real-time. Thus far, an expert system with real-time capabilities has been developed and installed on a subsystem at the Experimental Breeder Reactor (EBR-II) in Idaho, USA. Real-time simulation testing of advanced power plant concepts at the Pennsylvania State University has been developed and was used to support the expert system development and installation at EBR-II. Recently, the US National Science Foundation (NSF) and the US Department of Energy (DOE) have funded a Penn State research program to further enhance application of real-time diagnostic systems by pursuing implementation in a distributed power plant computer system including microprocessor based controllers. This paper summarizes past, current, planned, and possible future approaches to power plant diagnostic systems research at Penn State. 34 refs., 9 figs.

  7. A performance analysis method for distributed real-time robotic systems: A case study of remote teleoperation

    NASA Technical Reports Server (NTRS)

    Lefebvre, D. R.; Sanderson, A. C.

    1994-01-01

    Robot coordination and control systems for remote teleoperation applications are by necessity implemented on distributed computers. Modeling and performance analysis of these distributed robotic systems is difficult, but important for economic system design. Performance analysis methods originally developed for conventional distributed computer systems are often unsatisfactory for evaluating real-time systems. The paper introduces a formal model of distributed robotic control systems; and a performance analysis method, based on scheduling theory, which can handle concurrent hard-real-time response specifications. Use of the method is illustrated by a case of remote teleoperation which assesses the effect of communication delays and the allocation of robot control functions on control system hardware requirements.

  8. Detection of Banana mild mosaic virus and Banana virus X by polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR).

    PubMed

    Teycheney, Pierre-Yves; Acina, Isabelle; Lockhart, Benham E L; Candresse, Thierry

    2007-06-01

    Viruses are important constraints to the movement and propagation of plant germplasm, especially for vegetatively propagated crops such as banana and plantain. Their control relies primarily on the use of virus-free plant material, whose production and certification requires sensitive and reliable detection methods. An existing polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR) assay was adapted to the detection of Banana mild mosaic virus (BanMMV) and Banana virus X, two Flexiviridae infecting Musa spp. PDO inosine-containing primers were found to be well suited to the detection of BanMMV, despite its high molecular diversity, but not to that of the highly conserved BVX, for which species-specific primers were designed. Sampling and sample processing steps were optimized in order to avoid nucleic acid purification prior to the reverse transcription step. A polyclonal anti-BanMMV antiserum was raised and successfully used for the immunocapture (IC) of BanMMV viral particles from leaf extracts, leading to the development of a PDO-IC-RT-nested PCR assay. Although the anti-BanMMV antiserum could to some extent recognize BVX viral particles, direct binding (DB) was shown to be a more efficient method for processing BVX-infected samples and a PDO-DB-RT-nested PCR assay was developed for the detection of BVX from leaf extracts. PMID:17280722

  9. Identification of individual herbal drugs in tea mixtures using restriction analysis of ITS DNA and real-time PCR.

    PubMed

    Slanc, P; Ravnikar, M; Strukelj, B

    2006-11-01

    We have studied a sedative tea made of Valerianae radix (Valeriana officinalis L.), Lupuli strobuli (Humulus lupulus L.), Melissae folium (Melissa officinalis L.) and Menthae piperitae folium (Mentha piperita L.). In order to identify the constituent drugs a method was established involving amplification of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA on the basis of restriction analysis and real-time PCR. ITS regions of individual drugs were amplified and sequenced. Restriction analysis was performed with selected restriction endonucleases Nae I, PshA I and Xcm I. Real-time PCR was carried out, using primers specifically designed for each individual herbal drug. Real-time PCR proved to be a method for identifying individual herbal drugs in a tea mixture with a single DNA extraction in a single PCR run, since its limit of detection is lower than that for restriction analysis. PMID:17152982

  10. Improving practices in nanomedicine through near real-time pharmacokinetic analysis

    NASA Astrophysics Data System (ADS)

    Magafia, Isidro B.

    More than a decade into the development of gold nanoparticles, with multiple clinical trials underway, ongoing pre-clinical research continues towards better understanding in vivo interactions. The goal is treatment optimization through improved best practices. In an effort to collect information for healthcare providers enabling informed decisions in a relevant time frame, instrumentation for real-time plasma concentration (multi-wavelength photoplethysmography) and protocols for rapid elemental analysis (energy dispersive X-Ray fluorescence) of biopsied tumor tissue have been developed in a murine model. An initial analysis, designed to demonstrate the robust nature and utility of the techniques, revealed that area under the bioavailability curve (AUC) alone does not currently inform tumor accumulation with a high degree of accuracy (R2=0.56), marginally better than injected dose (R2=0.46). This finding suggests that the control of additional experimental and physiological variables (chosen through modeling efforts) may yield more predictable tumor accumulation. Subject core temperature, blood pressure, and tumor perfusion are evaluated relative to particle uptake in a murine tumor model. New research efforts are also focused on adjuvant therapies that are employed to modify circulation parameters, including the AUC, of nanorods and gold nanoshells. Preliminary studies demonstrated a greater than 300% increase in average AUC using a reticuloendothelial blockade agent versus control groups. Given a better understanding of the relative importance of the physiological factors that influence rates of tumor accumulation, a set of experimental best practices is presented. This dissertation outlines the experimental protocols conducted, and discusses the real-world needs discovered and how these needs became specifications of developed protocols.

  11. Real-time face and gesture analysis for human-robot interaction

    NASA Astrophysics Data System (ADS)

    Wallhoff, Frank; Rehrl, Tobias; Mayer, Christoph; Radig, Bernd

    2010-05-01

    Human communication relies on a large number of different communication mechanisms like spoken language, facial expressions, or gestures. Facial expressions and gestures are one of the main nonverbal communication mechanisms and pass large amounts of information between human dialog partners. Therefore, to allow for intuitive human-machine interaction, a real-time capable processing and recognition of facial expressions, hand and head gestures are of great importance. We present a system that is tackling these challenges. The input features for the dynamic head gestures and facial expressions are obtained from a sophisticated three-dimensional model, which is fitted to the user in a real-time capable manner. Applying this model different kinds of information are extracted from the image data and afterwards handed over to a real-time capable data-transferring framework, the so-called Real-Time DataBase (RTDB). In addition to the head and facial-related features, also low-level image features regarding the human hand - optical flow, Hu-moments are stored into the RTDB for the evaluation process of hand gestures. In general, the input of a single camera is sufficient for the parallel evaluation of the different gestures and facial expressions. The real-time capable recognition of the dynamic hand and head gestures are performed via different Hidden Markov Models, which have proven to be a quick and real-time capable classification method. On the other hand, for the facial expressions classical decision trees or more sophisticated support vector machines are used for the classification process. These obtained results of the classification processes are again handed over to the RTDB, where other processes (like a Dialog Management Unit) can easily access them without any blocking effects. In addition, an adjustable amount of history can be stored by the RTDB buffer unit.

  12. Error analysis of real time and post processed or bit determination of GFO using GPS tracking

    NASA Technical Reports Server (NTRS)

    Schreiner, William S.

    1991-01-01

    The goal of the Navy's GEOSAT Follow-On (GFO) mission is to map the topography of the world's oceans in both real time (operational) and post processed modes. Currently, the best candidate for supplying the required orbit accuracy is the Global Positioning System (GPS). The purpose of this fellowship was to determine the expected orbit accuracy for GFO in both the real time and post-processed modes when using GPS tracking. This report presents the work completed through the ending date of the fellowship.

  13. Inhibitor-free DNA for real-time PCR analysis of synovial fluid from horses, cattle and pigs.

    PubMed

    Schneeweiss, Wilfried; Stanek, Christian; Wagner, Martin; Hein, Ingeborg

    2007-03-31

    The potential of five different commercial DNA isolation methods to remove real-time PCR inhibitors from the synovial fluid of horses, cattle and pigs was investigated. All kits with the exception of one included a silica column-based purification of the DNA. With the fifth kit, DNA purification is achieved by removing contaminating macromolecules by a desalting process. We used a recently developed method based on comparison of the real-time PCR signal of an artificial target incorporated into each PCR reaction in the presence of the isolated DNA from the sample, and in control samples containing water instead of isolated DNA. This was followed by statistical analysis of the data. Inhibition and subsequent reduction of the endpoint fluorescence in the real-time PCR reaction was encountered in many cases. Less frequently, the target copy number in the samples was underestimated. However, we found no experimental evidence of a negative influence of the reduced endpoint fluorescence signal on the detection limit of the real-time PCR assay. All kits tested were useful for analyzing pelleted synovial fluid from horses, cattle and pigs. When analyzing non-pelleted synovial fluid, three kits - two based on silica columns and one employing a desalting process - yielded inhibitor-free DNA for real-time PCR analysis. PMID:17222992

  14. Analysis of real-time Earth magnetosphere simulation for space weather using space weather cloud computing system

    NASA Astrophysics Data System (ADS)

    Watari, S.; Tsubouchi, K.; Kato, H.; Tanaka, T.; Shinagawa, H.; Murata, K. T.

    2011-12-01

    The Earth magnetosphere simulation is continuously running in real-time for space weather in the National Institute of Information and Communications Technology (NICT). Code of this simulation was originally developed by Tanaka (JGR, 1995) and was implemented as one of the NICT real-time space weather simulations by Den et al. (Space Weather, 2006). The space weather cloud computing system has a distributed large storage system and a data analysis system and has been constructed in the NICT. Using this space weather cloud computing system, it becomes possible to preserve the result of the real-time magnetosphere simulation. It enables to analyze the response of the magnetosphere for various solar wind conditions. There are several works on the real-time simulation using AE index (Kitamura et al., JGR, 2008), the polar cap potential (Kunitake et al, Journal of NICT, 2009), the plasma environment at gestational orbit (Nakamura et al., Journal of NICT, 2009). In this analysis, we focused magnetic variation at gestational orbit and location of magnetopause. At gestational orbit, there are continuous magnetic field observations by the GOES satellites. On magnetopause location, there is an empirical model called the Shue model, which takes account of dynamic pressure and south-ward IMF of solar wind. We compared the result of the real-time simulation with magnetic field variations observed by the GOES satellites and magnetopause location calculated by the Shue model. We will report the result of this study.

  15. Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera

    PubMed Central

    Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, Sara

    2014-01-01

    Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies. PMID:25340748

  16. The short-time spectrum analysis of real-time sampling speech with DSP TMS320VC5416 chip

    NASA Astrophysics Data System (ADS)

    Fan, Qinru; Ren, Wen-hua

    2013-07-01

    For automatic speech recognition (ASR), the research centers mainly on algorithm of improving robust, researchers put less emphasis on realization and application of better speech algorithm. Real-time proceeding of speech recognition directly influence on its application, so real-time proceeding of speech recognition is as important as study of algorithm. Speech transform domain method is a necessary technique of speech recognition, so real-time analysis of transform domain method is necessary. In transform domain methods, the short-time spectrum analysis is simple and easy to realize, especially the short-time FFT algorithm is applied to the short-time spectrum analysis. FFT algorithm reduces multiplications greatly. For the purpose, this paper presents short-time spectrum analysis of real-time sampling speech based on FFT algorithm. We use DSP TMS320VC5416 chip and speech codec ASIC TLV320AIC23 as hardware, the real-time speech signal is acquired by ASIC TLV320AIC23. When working frequency of TMS320VC5416 is set 160 MHz and sampling frequency is 44.1 kHz, the short-time FFT is radix-2 DIF-FFT algorithm and the length of short-time window is 128, the simulation waves and data show that the short-time FFT algorithm analysis based on TMS320VC5416 chip can meet real-time of system. For estimation of proceeding error, we make a calculation of radix- 2 DIT-IFFT. Comparing the result of DIT-IFFT and sampling speech data, error is less than 10-3.

  17. Determination of T-2 and HT-2 toxins from maize by direct analysis in real time mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct analysis in real time (DART) ionization coupled to mass spectrometry (MS) was used for the rapid quantitative analysis of T-2 toxin, and the related HT-2 toxin, extracted from corn. Sample preparation procedures and instrument parameters were optimized to obtain sensitive and accurate determi...

  18. RUSHMAPS: Real-Time Uploadable Spherical Harmonic Moment Analysis for Particle Spectrometers

    NASA Technical Reports Server (NTRS)

    Figueroa-Vinas, Adolfo

    2013-01-01

    RUSHMAPS is a new onboard data reduction scheme that gives real-time access to key science parameters (e.g. moments) of a class of heliophysics science and/or solar system exploration investigation that includes plasma particle spectrometers (PPS), but requires moments reporting (density, bulk-velocity, temperature, pressure, etc.) of higher-level quality, and tolerates a lowpass (variable quality) spectral representation of the corresponding particle velocity distributions, such that telemetry use is minimized. The proposed methodology trades access to the full-resolution velocity distribution data, saving on telemetry, for real-time access to both the moments and an adjustable-quality (increasing quality increases volume) spectral representation of distribution functions. Traditional onboard data storage and downlink bandwidth constraints severely limit PPS system functionality and drive cost, which, as a consequence, drives a limited data collection and lower angular energy and time resolution. This prototypical system exploit, using high-performance processing technology at GSFC (Goddard Space Flight Center), uses a SpaceCube and/or Maestro-type platform for processing. These processing platforms are currently being used on the International Space Station as a technology demonstration, and work is currently ongoing in a new onboard computation system for the Earth Science missions, but they have never been implemented in heliospheric science or solar system exploration missions. Preliminary analysis confirms that the targeted processor platforms possess the processing resources required for realtime application of these algorithms to the spectrometer data. SpaceCube platforms demonstrate that the target architecture possesses the sort of compact, low-mass/power, radiation-tolerant characteristics needed for flight. These high-performing hybrid systems embed unprecedented amounts of onboard processing power in the CPU (central processing unit), FPGAs (field

  19. Real-time analysis for intensive care: development and deployment of the artemis analytic system.

    PubMed

    Blount, Marion; Ebling, Maria R; Eklund, J Mikael; James, Andrew G; McGregor, Carolyn; Percival, Nathan; Smith, Kathleen P; Sow, Daby

    2010-01-01

    The lives of many thousands of children born premature or ill at term around the world have been saved by those who work within neonatal intensive care units (NICUs). Modern-day neonatologists, together with nursing staff and other specialists within this domain, enjoy modern technologies for activities such as financial transactions, online purchasing, music, and video on demand. Yet, when they move into their workspace, in many cases, they are supported by nearly the same technology they used 20 years ago. Medical devices provide visual displays of vital signs through physiological streams such as electrocardiogram (ECG), heart rate, blood oxygen saturation (SpO(2)), and respiratory rate. Electronic health record initiatives around the world provide an environment for the electronic management of medical records, but they fail to support the high-frequency interpretation of streaming physiological data. We have taken a collaborative research approach to address this need to provide a flexible platform for the real-time online analysis of patients' data streams to detect medically significant conditions that precede the onset of medical complications. The platform supports automated or clinician-driven knowledge discovery to discover new relationships between physiological data stream events and latent medical conditions as well as to refine existing analytics. Patients benefit from the system because earlier detection of signs of the medical conditions may lead to earlier intervention that may potentially lead to improved patient outcomes and reduced length of stays. The clinician benefits from a decision support tool that provides insight into multiple streams of data that are too voluminous to assess with traditional methods. The remainder of this article summarizes the strengths of our research collaboration and the resulting environment known as Artemis, which is currently being piloted within the NICU of The Hospital for Sick Children (SickKids) in Toronto

  20. Decision Consequence Model (DCM): Integrating environmental data and analysis into real time decision making

    SciTech Connect

    Cimorelli, A.J.; Stahl, C.H.; Chow, A.H.; Fernandez, C.

    1999-07-01

    A critical evaluation of the many environmental issues facing EPA Region 3 has established five major priorities: (1) ozone pollution (and its precursors); (2) impacts of acidification (acid deposition and acid mine drainage); (3) eutrophication of the Chesapeake Bay from atmospheric nitrogen deposition; (4) Cities/Urban Environment (ozone, particulate matter (PM), air toxics are some of the air components); and (5) Climate Change. Recognizing the complex nature of the systems controlling these issues, Region III's Air Protection Division (APD) is developing a decision support tool, i.e., the Decision Consequence Model (DCM), that will integrate and automate the analysis of environmental impacts in a manner that allows them to holistically address these regional priorities. Using this tool the authors intend to consider the interdependency of pollutants and their environmental impacts in order to support real-time decision making. The purpose of this paper is to outline the basic concept of the DCM and to present an example set of environmental indicators to illustrate how the DCM will be used to evaluate environmental impacts. The authors will discuss their process of indicator development, and present an example suite of indicators to provide a concrete example of the concepts presented above and, to illustrate the utility of the DCM to simultaneously evaluate multiple effects of a single pollutant. They will discuss the type of indicators chosen for this example as well as the general criteria the DCM indicators must satisfy. The framework that was developed to construct the indicators is discussed and used to calculate the example indicators. The yearly magnitudes of these example indicators are calculated for various multi-year periods to show their behavior over time.

  1. Sensitivity "Hot Spots" in the Direct Analysis in Real Time Mass Spectrometry of Nerve Agent Simulants

    NASA Astrophysics Data System (ADS)

    Harris, Glenn A.; Falcone, Caitlin E.; Fernández, Facundo M.

    2012-01-01

    Presented here are findings describing the spatial-dependence of sensitivity and ion suppression effects observed with direct analysis in real time (DART). Continuous liquid infusion of dimethyl methyl phosphonate (DMMP) revealed that ion yield "hot spots" did not always correspond with the highest temperature regions within the ionization space. For instance, at lower concentrations (50 and 100 μM), the highest sensitivities were in the middle of the ionization region at 200 °C where there was a shorter ion transport distance, and the heat available to thermally desorb neutrals was moderate. Conversely, at higher DMMP concentrations (500 μM), the highest ion yield was directly in front of the DART source at 200 °C where it was exposed to the highest temperature for thermal desorption. In matching experiments, differential analyte volatility was observed to play a smaller role in relative ion suppression than differences in proton affinity and the relative sampling positions of analytes. At equimolar concentrations sampled at the same position, suppression was as high as 26× between isoquinoline (proton affinity 952 kJ mol-1, boiling point 242 °C) and p-anisidine (proton affinity 900 kJ mol-1, boiling point 243 °C). This effect was exacerbated when sampling positions of the two analytes differed, reaching levels of relative suppression as high as 4543.0× ± 1406.0. To mitigate this level of relative ion suppression, sampling positions and molar ratios of the analytes were modified to create conditions in which ion suppression was negligible.

  2. Real-time assessment of fog-related crashes using airport weather data: a feasibility analysis.

    PubMed

    Ahmed, Mohamed M; Abdel-Aty, Mohamed; Lee, Jaeyoung; Yu, Rongjie

    2014-11-01

    The effect of reduction of visibility on crash occurrence has recently been a major concern. Although visibility detection systems can help to mitigate the increased hazard of limited-visibility, such systems are not widely implemented and many locations with no systems are experiencing considerable number of fatal crashes due to reduction in visibility caused by fog and inclement weather. On the other hand, airports' weather stations continuously monitor all climate parameters in real-time, and the gathered data may be utilized to mitigate the increased risk for the adjacent roadways. This study aims to examine the viability of using airport weather information in real-time road crash risk assessment in locations with recurrent fog problems. Bayesian logistic regression was utilized to link six years (2005-2010) of historical crash data to real-time weather information collected from eight airports in the State of Florida, roadway characteristics and aggregate traffic parameters. The results from this research indicate that real-time weather data collected from adjacent airports are good predictors to assess increased risk on highways. PMID:25108899

  3. Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

    EPA Science Inventory

    A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

  4. REAL TIME PCR ANALYSIS OF INDOOR MOLDS: PRINCIPLES, PROCEDURES AND APPLICATIONS

    EPA Science Inventory

    This presentation will endeavor to present an overview of the real time polymerase chain reaction method developed for indoor mold detection and quantification by the EPA. It will begin with a brief discussion of the PCR technology that provides the basis for this method and how ...

  5. Real-time dynamics of a hot Yang-Mills theory: a numerical analysis

    NASA Astrophysics Data System (ADS)

    Ambjørn, J.; Anagnostopoulos, K. N.; Krasnitz, A.

    2002-03-01

    We discuss recent results obtained from simulations of high temperature, classical, real time dynamics of SU(2) Yang-Mills theory at temperatures of the order of the electroweak scale. Measurements of gauge covariant and gauge invariant autocorrelations of the fields indicate that the ASY-Bödecker scenario is irrelevant at these temperatures.

  6. Real-time dynamics of a hot Yang-Mills theory: a numerical analysis

    NASA Astrophysics Data System (ADS)

    Ambjørn, J.; Anagnostopoulos, K. N.; Krasnitz, A.

    We discuss recent results obtained from simulations of high temperature, classical, real time dynamics of SU(2) Yang-Mills theory at temperatures of the order of the electroweak scale. Measurements of gauge covariant and gauge invariant autocorrelations of the fields indicate that the ASY-Bödecker scenario is irrelevant at these temperatures.

  7. Observer Interface Analysis for Standardization to a Cloud Based Real-Time Space Situational Awareness (SSA)

    NASA Astrophysics Data System (ADS)

    Eilers, J.

    2013-09-01

    The interface analysis from an observer of space objects makes a standard necessary. This standardized dataset serves as input for a cloud based service, which aimed for a near real-time Space Situational Awareness (SSA) system. The system contains all advantages of a cloud based solution, like redundancy, scalability and an easy way to distribute information. For the standard based on the interface analysis of the observer, the information can be separated in three parts. One part is the information about the observer e.g. a ground station. The next part is the information about the sensors that are used by the observer. And the last part is the data from the detected object. Backbone of the SSA System is the cloud based service which includes the consistency check for the observed objects, a database for the objects, the algorithms and analysis as well as the visualization of the results. This paper also provides an approximation of the needed computational power, data storage and a financial approach to deliver this service to a broad community. In this context cloud means, neither the user nor the observer has to think about the infrastructure of the calculation environment. The decision if the IT-infrastructure will be built by a conglomerate of different nations or rented on the marked should be based on an efficiency analysis. Also combinations are possible like starting on a rented cloud and then go to a private cloud owned by the government. One of the advantages of a cloud solution is the scalability. There are about 3000 satellites in space, 900 of them are active, and in total there are about ~17.000 detected space objects orbiting earth. But for the computation it is not a N(active) to N problem it is more N(active) to N(apo peri) quantity of N(all). Instead of 15.3 million possible collisions to calculate a computation of only approx. 2.3 million possible collisions must be done. In general, this Space Situational Awareness System can be used as a

  8. Real-Time QoS Routing Protocols in Wireless Multimedia Sensor Networks: Study and Analysis

    PubMed Central

    Alanazi, Adwan; Elleithy, Khaled

    2015-01-01

    Many routing protocols have been proposed for wireless sensor networks. These routing protocols are almost always based on energy efficiency. However, recent advances in complementary metal-oxide semiconductor (CMOS) cameras and small microphones have led to the development of Wireless Multimedia Sensor Networks (WMSN) as a class of wireless sensor networks which pose additional challenges. The transmission of imaging and video data needs routing protocols with both energy efficiency and Quality of Service (QoS) characteristics in order to guarantee the efficient use of the sensor nodes and effective access to the collected data. Also, with integration of real time applications in Wireless Senor Networks (WSNs), the use of QoS routing protocols is not only becoming a significant topic, but is also gaining the attention of researchers. In designing an efficient QoS routing protocol, the reliability and guarantee of end-to-end delay are critical events while conserving energy. Thus, considerable research has been focused on designing energy efficient and robust QoS routing protocols. In this paper, we present a state of the art research work based on real-time QoS routing protocols for WMSNs that have already been proposed. This paper categorizes the real-time QoS routing protocols into probabilistic and deterministic protocols. In addition, both categories are classified into soft and hard real time protocols by highlighting the QoS issues including the limitations and features of each protocol. Furthermore, we have compared the performance of mobility-aware query based real-time QoS routing protocols from each category using Network Simulator-2 (NS2). This paper also focuses on the design challenges and future research directions as well as highlights the characteristics of each QoS routing protocol. PMID:26364639

  9. A meta-analysis to evaluate the effectiveness of real-time PCR for diagnosing novel coronavirus infections.

    PubMed

    Lin, C; Ye, R; Xia, Y L

    2015-01-01

    Novel coronavirus (nCoV) belongs to the Coronaviridae family, which includes the virus that causes SARS, or severe acute respiratory syndrome. However, infection source, transmission route, and host of nCoV have not yet been thoroughly characterized. In some cases, nCoV presented a limited person-to-person transmission. Therefore, early diagnosis of nCoV may be of importance for reducing the spread of disease in public. Methods for nCoV diagnosis involve smear dyeing inspection, culture identification, and real-time PCR detection, all of which are proved highly effective. Here, we performed a meta-analysis to evaluate the effectiveness of real-time PCR for diagnosing nCoV infection. Fifteen articles conformed to the inclusion and exclusion criteria for further meta-analysis on the basis of a wide range of publications searched from databases involving PubMed, EMBASE, Web of Science, Medline, ISI. We analyzed the stability and publication bias as well as examined the heterogeneity inspection of real-time PCR detection in contrast to smear staining and culture identification. The fixed-effect model was adopted in our meta-analysis. Our result demonstrated that the combination of real-time PCR and smear diagnostics yielded an odds ratio (OR) = 1.91, 95% confidence interval (CI) = 1.51-2.41, Z = 5.43, P < 0.05, while the combination of real-time PCR and culture identification yielded OR = 2.44, 95%CI = 1.77-3.37, Z = 5.41, P < 0.05. Therefore, we propose real-time PCR as an efficient method that offers an auxiliary support for future nCoV diagnosis. PMID:26634531

  10. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR.

    PubMed

    Choi, Hoseong; Cho, Won Kyong; Yu, Jisuk; Lee, Jong-Seung; Kim, Kook-Hyung

    2013-03-01

    To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated